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Pathway mediates nitroglycerin-induced relief from angina pectoris


what kind of Pathway mediates nitroglycerin-induced relief from angina pectoris,please provide some idea…


The effect of nitroglycerin on the cardiac tissue is similar to nitric oxide (NO), which forms in the endothelium of blood vessels from arginin metabolism products.

So, the general scheme can look like this:

R-NO2 is converted to R-NO outside of the cell (glutathion-SH-dependent reaction). NO is lipophilic and rapidly penetrates into the cell where it activates guanylate cyclase. From this moment you can see the resulting influence on myosin and actin on this picture:


Nitric oxide synthetic pathway in patients with microvascular angina and its relations with oxidative stress.

NO is an important signaling molecule involved in the maintenance of vascular function. It promotes several beneficial effects in the vasculature by inducing vasorelaxation, inhibition of leukocyte-endothelium adhesion, smooth muscle cells migration and proliferation, and platelet aggregation [1,2]. A decreased NO bioavailability is well documented in several cardiovascular diseases, including hypertension, atherosclerosis, and ischemia-reperfusion injury. A reduction of circulating NO species (nitrite and nitrosylated compounds), which contribute to the total NO availability, is described in individuals with endothelial dysfunction. The decrease is correlated with increasing numbers of cardiovascular risk factors [3-5].

NO is synthesized by the enzymatic action of NO synthases (NOSs), catalyzing the oxidation of the amino acid L-arginine (Arg) to equimolar amounts of NO and Lcitrulline (Cit), in the presence of oxygen and cofactors. Although synthesis and release of NO are related to the substrate bioavailability [6], other potential causes of NO deficiency in disease settings have been proposed. Among these, the high circulating levels of endogenous methylarginines, that is, symmetric, asymmetric dimethylarginine (SDMA, ADMA) and monomethylarginine (MMA), act as NOsynthesis inhibitors [7, 8]. In addition, oxidative stress plays a pivotal role in determining NO bioavailability by the oxidation of the cofactors/the enzymes involved in NO metabolism or by the direct inactivation of NO.

Endothelial cells are considered the major source of NO in the vasculature however, it has been shown that also circulating cells may contribute to NO synthesis, that is, platelets, monocytes, and red blood cells (RBCs). RBCs express functional NOS [9,10], similar to the enzyme of endothelial cells [11], which serves as an intraluminal NO source and contributes to the regulation of systemic blood pressure [12]. In addition, the transporter for cationic amino acids [13] and all the enzymes involved in dimethylarginine metabolism (synthesis and catabolism) [14] have been identified in RBCs. Human RBCs also express the enzyme arginase that competes with NOS for their common substrate Arg to form Lornithine (Orn) [15]. Two different isoforms of arginase are expressed in human [16] and, recently, it has been shown that arginase I plays an essential role in the control of RBCNOS function and in the release of bioactive NO [17]. Indeed, in experimental models of atherosclerosis [18], myocardial ischemia [19], hypertension [20], and ageing [21], arginase activity has been reported to be upregulated at vascular level.

Microvascular angina (MVA) is a pathological condition characterized by the typical anginal pain, electrocardiographic (ECG) abnormalities at rest (ST-segment depression or T-wave inversion), all features that increase during exercise, in the presence of nonobstructed epicardial coronary arteries [22-24]. Even if the pathophysiology of MVA has not been disentangled yet, insulin resistance, abnormal autonomic control, enhanced sodium hydrogen exchange activity, abnormal cardiac sensitivity, and microvascular spasm have been proposed as potential causes [25]. In addition, increased concentrations of circulating C-reactive protein have been shown to correlate with vascular abnormalities in patients with MVA, suggesting a role of inflammation in this pathological condition [26].

Oxidative stress per se, either directly or through the reduction of NO bioavailability leading to an impairment of endothelium dependent vasodilation, has been involved in the pathophysiology of MVA [27, 28]. In particular, impaired endothelium-dependent vasodilatation of the coronary microvasculature [27] and its related impaired function, which limits coronary flow reserve [28-30], have been proposed to induce MVA syndrome.

Alterations in flow-mediated coronary dilation are a frequent finding in patients with MVA. In the microcirculation, blood flow is largely dependent on hemorheological properties, particularly RBC deformability, whose importance increases in capillaries compared with larger vessel [31]. A decreased RBC deformability has been shown in patients with CAD and diabetes mellitus [32] and it has been related to a decreased NO release [33]. In addition, due to the structural properties and blood flow in the microcirculatory bed, blood cells are in close contact with endothelium. As it has been shown that eNOS expression decreases in the microvasculature [34], it could be speculated that within capillaries RBCNOS may play a more decisive role [35].

Moreover, it has been recently shown that in patients with cardiac

syndrome X an increase of red cell distribution width (RDW), a measurement of size variability, and of erythrocytes, occurs [36]. Even if it has been reported that reduction of nitrate and nitrite, coupled to increases in ADMA and SDMA, occurs in plasma of MVA patients [37, 38], no information on the levels of the single components of the NO pathway in RBCs is available yet. Thus, in this study, we have characterized oxidative stress and the NO biosynthetic pathway in RBCs of MVA patients in comparison to patients with coronary artery disease (CAD) or healthy subjects (Ctrl).

2.1. Study Population. Patients with MVA (n = 25) characterized by stable effort angina or inducible ischaemia and reduction of the coronary flow reserve, documented by a positive stress test (at least 2.0 mm horizontal or downsloping ST-segment depression) or by a positive SPECT, despite the absence of angiographically documented coronary disease, were recruited. These patients were compared with angiographically documented CAD patients (n = 22) and with subjects deemed as healthy on the bases of the absence of clinical symptoms, the instrumental and laboratory examination (Ctrl = 20), and the negative stress test from a previously described cohort [10]. Exclusion criteria were considered as follows: a history of congestive heart failure, significant valvular diseases, hypertrophic cardiomyopathy, vasospastic angina, recent (<6 months) acute coronary syndrome, surgical or percutaneous revascularization, pacemaker dependency, and atrial fibrillation. Patients with renal insufficiency (serum creatinine concentration >1.4 mg/dL), hepatic disease, recent infection, recent major surgical interventions, immunological disorders, and chronic inflammatory or neoplastic diseases were also excluded. This observational study was carried out in accordance with the Declaration of Helsinki and approvedbythe local ethics research committee of Centro Cardiologico Monzino (number S1687/610). Written informed consent to participate was obtained from all subjects.

2.2. Blood Collection. EDTA-anticoagulated blood was drawn from the antecubital vein of subjects while fasting to obtain whole blood, plasma, and erythrocyte samples. After centrifugation (1,200 g for 10 min at 4[degrees]C), plasma was separated and aliquots were stored at -80[degrees]C until analyses. Aliquots of packed red cells were lysed by cold deionized water to obtain lysed RBCs and stored at -80[degrees]C until analyses.

2.3. Arg/NO Metabolic Pathway. We simultaneously measured Arg, ADMA, SDMA, MMA, Cit, and Orn by liquid chromatography-tandem mass spectrometry (LC-MS/MS) [39]. The ratio Arg/(Orn + Cit), as index of global Arg availability [40, 41], and the ratio Orn/Cit, as indicator of the relative activity of arginase and NOS [19], were computed. All the determinations were performed both in plasma and in lysed RBCs.

2.4. Oxidative Stress. It was evaluated by the ratio between disulphide and reduced forms of glutathione (GSSG/GSH). GSH and GSSG were measured by LC-MS/MS method on whole blood, after proteins precipitation with trichloroacetic acid [42]. Levels of GSH and GSSG were expressed as [micro]mol/g Hb.

2.5. RBC-NOS and Arginase Expression. The RBC-NOS expression was performed by immunofluorescence analysis in a subgroup of subjects (n = 10 per group matched for age and sex). RBCs slides were prepared as previously described [10]. Briefly, after blocking of nonspecific reactive sites, RBCs were incubated overnight at 4[degrees]C with a monoclonal anti-eNOS (2.5 [micro]g/mL) (BD Biosciences, Milano, Italy) or polyclonal anti-arginase I or monoclonal anti-arginase II (4 [micro]g/mL, for both) (Santa Cruz Biotechnology, DBA Italia s.r.l., Milano, Italy) antibodies. After three washings, an antimouse or anti-rabbit AlexaFluor488 conjugated secondary antibody (Invitrogen, Life Technologies Italia, Monza, Italy) was added and the immune complexes were visualized by laser scanning confocal microscope (LSM710, Carl Zeiss, Milano, Italy) using a 63x/1.3 oil immersion objective lens. Images were captured and the fluorescence intensity (densitometric sum of grey) was quantified. Data are expressed as the mean level of fluorescence intensity, subtracted of negative control value obtained on the same slide in the absence of primary antibody. Multiple fields of view (at least three randomly selected areas) were captured for each slide.

2.6. Statistics and Scores Development. Numerical variables were summarized as mean and standard deviation (SD), unless otherwise stated, and categorical variables were summarized as frequencies and percentages. A sample size of 20 subjects per group allowed a statistical power of 90% to deem as significant a between-group difference in any analyte approximately equal to one standard deviation, with an alpha error of 0.05. Variables were compared between MVA and CAD or Ctrl by f-test or by covariance analysis, adjusting for age and sex. Variables with skewed distribution were log-transformed before analysis. Immunofluorescence intensity was compared between groups by repeated measures covariance analysis, taking into account replicate measures for each subject. All analyses were performed by SAS v. 9.2 (SAS Institute Inc., Cary, NC, USA).

In order to provide a global indicator of all the variables related to NO pathway and to contain inflation of alpha error due to multiple testing, we developed a score similar to the OXY-SCORE, devised by our group few years ago [43]. First, to account for different measurement ranges and units, all the variables were standardized that is, the mean was subtracted from individual values and the result was divided by the standard deviation. Second, the standardized values of the variables generally accepted as positively associated with endothelial function (Arg and Cit) were added, whereas standardized values of the variables negatively associated with endothelial function (ADMA, SDMA, MMA, and Orn) were subtracted. It is important to note that these associations were intended as "a priori" and were not inferred from the present study. We created a first score using variables measured in plasma (NO plasma score) and another score using variables measured in the RBCs (NO RBC score). Similarly, we created oxidative stress score, a simplified version of the OXY-SCORE including GSSG (with a plus sign) and GSH (with a minus sign).

3.1. Population. The principal demographic and clinical characteristics of the two patient groups and of healthy subjects analyzed in this study are depicted in Table 1. No significant differences were found among groups except for age (P = 0.01 MVA versus CAD) that was considered as a confounder for group comparisons.

3.2. Biochemical Determinations of Metabolites Involved in Arg/NO Pathway and Oxidative Stress Status. In order to evaluate the potential impairment of Arg/NO pathway in MVA patients, we simultaneously measured the principal metabolites involved in this pathway, both in plasma and in the RBC compartment, and we compared them to the levels measured in CAD and in Ctrl (Table 2). In plasma, MVA patients showed Arg, Cit, and Orn levels similar to those of CAD patients and Ctrl. ADMA levels, instead, were higher in both MVA and CAD patients compared to Ctrl. SDMA and MMA levels did not differ among the three groups studied. In accordance to these findings, the Arg bioavailability (Arg/Orn + Cit ratio) was lower in MVA than in Ctrl and similar to CAD. In addition, the MVA Orn/Cit ratio, an index of activities of the Arg metabolic enzymes arginase and NOS, showed levels intermediate between those of CAD and Ctrl (Table 2).

In the RBC compartment, the levels of NO inhibitors ADMA and SDMA in MVA and CAD patients were higher than in Ctrl (Table 2). Interestingly, MMA levels were the highest in MVA. Arg bioavailability was similar in the three groups of subjects, whereas the Orn/Cit ratio was significantly lower in MVA than in CAD group but similar to Ctrl (Table 2).

Patients with MVA had higher levels of oxidative stress with respect to Ctrl, but lesser than those determined in CAD patients, as documented by the GSSG/GSH ratio measured in whole blood (Figure 1(a)). Specifically, both groups of patients showed lower levels of GSH and higher levels of GSSG with respect to Ctrl (Figure 1(b)).

Figure 2 shows the distribution of the analytes measured in plasma or RBCs of MVA and CAD patients expressed as fold change over Ctrl. In general, the analytes of the NO pathway behaved similarly in MVA and CAD and they were moderately elevated with respect to Ctrl, both in plasma and in RBCs. A special case is represented by MMA in RBCs, whose levels were higher in MVA with respect to Ctrl and CAD patients. As expected, the oxidative stress, in particular the oxidized form of glutathione, was higher in both MVA and CAD patients with respect to Ctrl.

3.3. Arginine Metabolic Enzymes: RBC-NOS and Arginase. The expression of RBC-NOS, visualized by immunofluorescence staining, revealed strong quantitative differences between both patient groups and Ctrl. RBCs of MVA and CAD patients had significantly lower RBC-NOS fluorescence, localized in the membrane and into the cytosol, with respect to Ctrl (Figure 3(a)).

The expression of both isoforms of arginase was also evaluated. RBCs of MVA patients and of Ctrl expressed lesser levels of arginase I than CAD patients (P = 0.02) Figure 3(b)). In contrast, the expression of arginase II was not detectable in RBCs of Ctrl and of MVA or CAD patients (data not shown).

3.4. Summary Scores of NO Pathway and Oxidative Stress. The analytes, that is, substrate, inhibitors, and enzymatic products involved in NO synthesis, were combined into appropriate scores (see Section 2) in order to summarize the Arg/NO pathway in the examined clinical settings. In Figure 4, the Cartesian plane was defined by the NO plasma score (x-axis) and the NO RBC score (y-axis) the intersection of the axes identifies the midpoint of the entire sample, and the units are expressed in terms of standard deviations. The Ctrl group was placed in the first quadrant (positive values for both scores), whereas the two patient groups were placed in the third quadrant (negative values for both scores). To be noticed, the MVA group was located in a more negative position, along the NO RBC score axis, compared with the CAD group however, the difference did not reach statistical significance.

Figure 5 shows the Cartesian plane defined by the oxidative stress score (x-axis) and the NO plasma score (y-axis). In this graph, the control group was placed in the quadrant characterized by a negative oxidative score and by a positive NO score. In contrast, both groups of patients were placed in the quadrant relative to a positive oxidative score and a negative NO score, with the CAD group located in a more extreme position with respect to MVA (although this difference did not reach statistical significance: P = 0.08 for multivariate ANOVA).

The study described above shows for the first time that RBCs of patients with MVA contain higher levels of inhibitors of the NO synthesis than Ctrl and that these levels do not markedly differ from those found in CAD patients. A similar picture is found in plasma, as previously described by others [37, 38]. Finally, NOS expression in RBCs was found markedly reduced in both MVA and CAD patients. In addition, oxidative stress was found increased in both patient groups, mostly in CAD.

The pathophysiology of MVA is not completely understood yet, even if the several metabolic, haemodynamic, and vasospastic alterations have been linked to this syndrome. Recently, it has been reported that RDW values are significantly higher in both MVA and CAD patients compared to healthy subjects [36]. However, as documented by the absence of modifications in RDW values (data not shown), in our study, the impairment of NO pathway in RBCs of MVA patients is not associated with changes in the size of circulating RBCs. The RBCs of MVA patients, however, showed higher levels of NO synthesis inhibitors and this finding parallels the data found in plasma. As a consequence, in a Cartesian plane, defined by NO scores, the MVA group was located in a negative position along the NO RBC score axis with respect to Ctrl, thus suggesting a possible alteration in NO production, more pronounced in MVA with respect to CAD.

The limitation of our study might be the calculation of the NO scores without measuring NO itself. This highly reactive molecule and its active metabolites are influenced by several factors, including dietary nitrate intake and renal function, particularly in the plasma compartment. Thus, we cannot exclude that other NOS independent factors may add additional information for an overall picture of this metabolic pathway in MVA.

Of interest is the observation that, similar to what previously described for CAD patients [10], we found a marked reduction in NOS expression in RBCs of MVA patients. This finding is of particular relevance because RBCs have a systemic impact in terms of NO production and may represent an important compartment, whose alteration participates to the reduction in the overall NO production.

Arg is the substrate for the NOS enzymes, including RBCNOS, and it has been shown that an increase of substrate availability in the stenotic lesion induced dilation of the coronary artery segment [44]. Arg is also substrate for the arginase enzyme, whose activity is increased in different pathological conditions associated with a reduction of NO [17, 45].

Two different isoforms of arginase are identified in human and arginase I, which is the only arginase so far described in RBCs, accounts for about 98% of total blood arginase activity [46]. In our condition, greater amounts of arginase I in CAD patients, but not in MVA patients, were found. Since it has been reported that erythroid progenitor cells express both arginase I and arginase II [15], we measured also this enzyme in RBCs. According to the literature [17], we failed to detect measurable amounts of arginase II in Ctrl or in patients.

Increased erythrocyte arginase activity associated with lowered NO plasma levels and with impairment in erythrocytes has been reported in sickle cell disease patients [40]. Interestingly, the consumption of cocoa flavanols reduced the erythrocyte arginase activity, suggesting a possible therapeutic intervention by the regulation of Arg and NO bio availability [47].

An important condition able to affect NO bioavailability is oxidative stress. Of relevance is the observation that the ratio between oxidized and reduced glutathione was almost doubled in whole blood of MVA patients, suggesting an increased oxidative stress in this condition. A role of oxidative stress in lowering NO bioavailability has been previously highlighted, but the information in MVA is still scanty [27, 48, 49]. We found a marked increase of GSSG/GSH, based on the increase of oxidized glutathione, which was even more pronounced in CAD patients and on a decrease of GSH in MVA patients. This observation is in accordance with data reported by Dhawan et al. [50], who showed a positive correlation between GSH levels and coronary flow velocity reserve, thus predicting impaired microvascular function.

Finally, the concomitant assessment of oxidative stress and NO pathway in patients indicates that both MVA and CAD patients are placed in the Cartesian plane quadrant relative to a positive oxidative score and a negative NO score, with the CAD group located in a more extreme position with respect to MVA.

Thus, as previously suggested by Rassaf and collaborators [51], a multiple-level approach by assessing biochemical, structural, and functional changes in the vasculature may be important for an early diagnosis of cardiovascular diseases and for a better characterization of this multifactorial disease.

Our study shows that changes in the Arg/NO metabolic profile, coupled to increases in oxidative stress, occur in MVA with a trend toward an impairment similar to that of CAD patients. In particular we have described for the first time alterations in the capacity of RBCs to produce NO in a pathological condition characterized mostly by alterations at the microvascular bed with no significant coronary stenosis.

The authors declare that there is no conflict of interests regarding the publication of this paper.

Benedetta Porro and Sonia Eligini contributed equally to this paper as first authors.

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[50] S. S. Dhawan, P. Eshtehardi, M. C. McDaniel et al., "The role of plasma aminothiols in the prediction of coronary microvascular dysfunction and plaque vulnerability," Atherosclerosis, vol. 219, no. 1, pp. 266-272, 2011.

[51] T. Rassaf, P. Kleinbongard, and M. Kelm, "The L-arginine nitric oxide pathway: avenue for a multiple-level approach to assess vascular function," Biological Chemistry, vol. 387, no. 10-11, pp. 1347-1349, 2006.

Benedetta Porro, (1) Sonia Eligini, (1) Fabrizio Veglia, (1) Alessandro Lualdi, (1,2) Isabella Squellerio, (1) Susanna Fiorelli, (1) Marta Giovannardi, (1) Elisa Chiorino, (1) Alessia Dalla Cia, (1) Mauro Crisci, (1) Jose Pablo Werba, (1) Elena Tremoli, (1,3) and Viviana Cavalca (1,2)

(1) Centro Cardiologico Monzino, I.R.C.C.S., 20138 Milan, Italy

(2) Dipartimento di Scienze Cliniche e di Comunita, Universita degli Studi di Milano, 20138 Milan, Italy

(3) Dipartimento di Scienze Farmacologiche e Biomolecolari, Universita degli Studi di Milano, 20133 Milan, Italy

Correspondence should be addressed to Viviana Cavalca [email protected]

Received 27 February 2014 Revised 28 March 2014 Accepted 29 March 2014 Published 22 April 2014


GLP-1 Receptor Agonists and Cardiovascular Prevention: Insights from the LEADER Trial

The primary goals of type 2 diabetes treatment are prevention of microvascular, macrovascular, and other complications while maintaining quality of life. Strategies to control hyperglycemia are known to prevent microvascular complications such as retinopathy, neuropathy and nephropathy, but the impact on macrovascular complications has been limited, presumably as a consequence of the relatively small contribution of hyperglycemia to the pathogenesis of atherosclerosis. Although glycemic control is an essential component of treatment of type 2 diabetes, efforts to prevent macrovascular complications such as myocardial infarction, stroke, and peripheral vascular disease necessitate treatment of dyslipidemia, smoking cessation, blood pressure control, implementation of heart healthy dietary habits and regular physical activity, and possible treatment with aspirin and other medications. Although there may be an expectation that treatments for hyperglycemia should also prevent macrovascular complications in type 2 diabetes, it is important to recognize that microvascular complications are most impacted by glycemic control, whereas macrovascular complications are most impacted by control of traditional cardiovascular risk factors.

GLP-1 Biology

Glucagon-like peptide-1 (GLP-1) is peptide hormone that is secreted by enteroendocrine L-cells primarily in the distal small intestine and colon, alpha cells in pancreatic islets, and neurons in the central nervous system. 1 GLP-1 secreted in response to nutrient ingestion serves as an incretin hormone that mediates several beneficial regulatory effects on glucose assimilation and homeostasis. Among these physiologic effects, GLP-1 stimulates glucose-dependent insulin secretion from pancreatic islet cells (attenuated in the presence of a low plasma glucose concentration), suppresses secretion of the glucose-raising hormone glucagon from pancreatic islet alpha cells, delays gastric emptying, and suppresses appetite through mechanisms that may include binding to GLP-1 receptors in the arcuate nucleus, paraventricular nucleus, and dorsomedial nucleus of the hypothalamus. 2,3 GLP-1 receptors are also expressed in vascular endothelial and smooth muscle cells, an observation that has enhanced interest in potential cardiovascular effects of GLP-1 receptor agonists. The results of a recent study demonstrated that a variant in the GLP-1 receptor gene (rs10305492) was associated with lower fasting glucose concentrations, decreased risk of developing type 2 diabetes (OR 0.83 95% confidence interval [CI] 0.76-0.91), and decreased risk of coronary heart disease (OR 0.93 95% confidence interval [CI] 0.87-0.98) on the basis of data from up to 11,806 individuals assessed by targeted exome sequencing and follow-up in 39,979 individuals by targeted genotyping. 4 The results of this study provided support for the notion that treatment with GLP-1 receptor agonists may produce cardiovascular benefits. Five GLP-1 receptor agonists are currently available for clinical use (Table 1).

Table 1: GLP-1 Receptor Agonists Currently Available for Treatment of Type 2 Diabetes in the United States

5-10 &mug SQ BID
ER formulation: 2 mg SQ weekly

1.2 – 1.8 mg SQ daily
3 mg SQ daily for weight loss

FDA Requirements for New Diabetes Medications

On the basis of controversial data suggesting that rosiglitazone may increase the risk of cardiovascular events, the US Food and Drug Administration (FDA) established a standard nearly 10 years ago that drugs developed for treatment of diabetes needed to be proven to not increase the risk of cardiovascular events. Rosiglitazone was later exonerated, but the requirement to prove non-inferiority to placebo for cardiovascular risk for new diabetes medications has persisted. 5

LEADER Trial

The Liraglutide Effect and Action in Diabetes: Evaluation of Cardiovascular Outcome Results (LEADER) trial began in 2010 and involved 9,340 adults with increased cardiovascular risk and type 2 diabetes who were treated with liraglutide or placebo for a median treatment exposure of 3.5 years and median follow-up duration of 3.8 years. 6 The purpose of the trial was to demonstrate non-inferiority of liraglutide compared to placebo for cardiovascular risk, in accordance with FDA guidelines. The inclusion criteria included a diagnosis of type 2 diabetes with HgbA1c > 7.0%, age > 50 years with at least one coexisting cardiovascular condition at study entry (coronary heart disease, cerebrovascular disease, peripheral vascular disease, chronic kidney disease of stage 3 or greater, or chronic heart failure of New York Heart Association class II or III) or age > 60 years with at least one cardiovascular risk factor as determined by the investigator (microalbuminuria or proteinuria, hypertension and left ventricular hypertrophy, left ventricular systolic or diastolic dysfunction, or an ankle-brachial index of < 0.9). The mean age was 64 +/- 7 years, the mean duration of diabetes was 12.8 +/- 8.1 years, the mean HgbA1c concentration was 8.7 +/- 1.5 %, and the mean body mass index was 32.5 ± 6.3 at study entry. Established cardiovascular disease was present in 81.3% and CKD (> stage 3) was present in 24.7%. Accordingly, the study population had a high risk of cardiovascular events. The primary composite outcome in the time-to-event analysis was the first occurrence of death from cardiovascular causes, nonfatal myocardial infarction, or nonfatal stroke.

The median dose of liraglutide was 1.78 mg daily, which reflected the target dose of 1.8 mg daily or the highest tolerable dose. The HgbA1c concentration decreased by 0.4 (95% CI ל.45 to ל.34) at 36 months, body weight modestly decreased by 2.3 kg (95% CI מ.5 to מ.0), and systolic blood pressure decreased by ם.2 mm Hg (95% CI ם.9 to ל.5) during treatment with liraglutide compared to placebo, all P < 0.05.

The hazard ratio for the primary cardiovascular outcome was significantly reduced in the liraglutide group compared to placebo, with hazard ratio 0.87 (95% CI 0.78 to 0.97, P = 0.01). The expanded composite cardiovascular outcome (consisting of death from cardiovascular causes, nonfatal myocardial infarction, or nonfatal stroke, coronary revascularization, or hospitalization for unstable angina pectoris or heart failure) was also significantly reduced, with a hazard ratio of 0.88 (95% CI 0.81 to 0.96, P = 0.005). The rate of cardiovascular mortality was significantly lower in the liraglutide group, with hazard ratio 0.78 (95% CI 0.66 to 0.93, P=0.007), and total mortality (death from any cause) was also significantly lower in the liraglutide group, with hazard ratio 0.85 (95% CI 0.74 to 0.97, P = 0.02). The individual rates of nonfatal myocardial infarction, nonfatal stroke, and hospitalization for heart failure were nonsignificantly lower in the liraglutide group. The rate of incident nephropathy was significantly reduced by liraglutide compared to placebo (HR 0.78, 95% CI 0.67 to 0.92, P = 0.003), but the incidence of retinopathy was unaffected (HR 1.15, 95% CI 0.87 to 1.53, P = 0.33).

Severe hypoglycemia was less frequent in the liraglutide group (2.4 vs 3.3%, P = 0.02). Side-effects that lead to discontinuation of treatment were significantly more common in the liraglutide group (9.5 vs 7.3%, P < 0.001) and included nausea (1.6 vs. 0.4%), vomiting (0.7 vs. < 0.1%), diarrhea (0.6 vs. 0.1%), abdominal pain (0.2 vs. 0.1%), decreased appetite (0.2 vs. < 0.1%), and abdominal discomfort (0.2 vs. 0%), as anticipated. The incidence of acute gallstone disease was 3.1 versus 1.9% (P < 0.001). Despite the prior suggestion that GLP-1 receptor agonists may increase the risk of pancreatitis, the rates of acute and chronic pancreatitis were not significantly different in the liraglutide treatment group compared to placebo (0.4 vs. 0.5%, P = 0.44 and 0 vs. 0.04%, P = 0.16, respectively). A subsequent analysis of these data confirmed these findings, as well as demonstrating that the incidence of pancreatitis among a subgroup of 267 individuals with a previous history of pancreatitis was not increased during treatment with liraglutide compared to placebo. 7 Although the difference did not reach statistical significance, the rate of pancreatic cancer was 0.3% in the liraglutide group compared to 0.1% in the placebo group (P = 0.06). In general, liraglutide was well tolerated with a placebo-controlled excess discontinuation rate of only 2.2%.

It is interesting to speculate why the primary composite outcome was driven primarily by cardiovascular and total mortality (P = 0.02 and 0.007, respectively), but the rates of many nonfatal events were not significantly different between the liraglutide and placebo groups. The incidence of total myocardial infarctions (fatal, nonfatal, and silent) was only borderline significantly different for liraglutide treatment compared to placebo (P = 0.046). It is somewhat surprising that none of the individual rates of fatal, nonfatal, and silent myocardial infarction, total stroke, nonfatal stroke, fatal stroke, transient ischemic attack, coronary revascularization, and hospitalization for unstable angina or heart failure were significantly different between treatment groups. These findings are still consistent with an antiatherosclerotic mechanism of cardiovascular event reduction during treatment with liraglutide, but the predominant effect on reduction in cardiovascular mortality suggest that other mechanisms may be responsible liraglutide mediated reduction in the primary and extended composite outcomes. A purely anti-atherosclerotic mechanism would not be anticipated to predominantly reduce cardiovascular mortality without significantly decreasing nonfatal events. The 20% reduction in the occurrence of confirmed hypoglycemia and 31% reduction in severe hypoglycemia are examples of nonatherosclerotic mechanisms by which treatment with liraglutide may have reduced cardiovascular mortality, but further analysis of the LEADER data and additional studies are needed to delineate the mechanisms by which liraglutide reduced the incidence of primary and extended composite outcomes in this study. An increased understanding of these mechanisms may shed light on the reasons why cardiovascular benefit has not been demonstrated with some other GLP-1 receptor agonists, as described below.

The subgroup analyses suggested that patients with established cardiovascular disease (>50 years old) and those with a glomerular filtration rate (GFR) < 60 ml/min/1.72 m2 may derive greater cardiovascular benefit from treatment with liraglutide compared to other subgroups. It is unclear why these two subgroups may have achieved more significant reductions in cardiovascular events, but subjects with established CVD or renal insufficiency both have increased risk of CHD events and therefore would be anticipated to be more likely to experience significant reductions in the incidence of cardiovascular. In contrast to the results for these two subgroups, the primary composite event rates were not significantly different when subjects were stratified for sex, age, geographic area, race or ethnicity, body mass index, HgbA1c concentration, duration of diabetes, heart failure, or number and type of anti-diabetic medications.

To summarize the results of the LEADER trial, the data demonstrated significant improvements in glycemic control, decreased incidence of severe hypoglycemia, modest weight loss, modest lowering of systolic blood pressure, decreased incidence of nephropathy, and a reduction in composite cardiovascular endpoints as well as total and cardiovascular mortality in high risk patients with type 2 diabetes treated with liraglutide compared to placebo. The rates of any adverse event (62.3 vs. 60.8%) or severe adverse event (32.2 vs. 32.8%) were not significantly different between the liraglutide and placebo treatment groups.

Results From Studies of Other GLP-1 Receptor Agonists

Since the effects of many medications on cardiovascular event rates are attributable to a class effect, and all GLP-1 receptor agonists reportedly have the same mechanism of action, it is reasonable to hypothesize that other GLP-1 receptor agonists will also prevent cardiovascular events. The limited data that are available from cardiovascular outcome trials using other GLP-1 receptor agonists have yielded inconsistent findings, with some trials showing no cardiovascular benefit.

The exploratory results from a large uncontrolled study population of 39,275 patients suggested that patients treated with exenatide twice daily had a significantly lower rate of cardiovascular events and hospitalizations compared to patients treated with other glucose-lowering medications. 8 Despite these positive findings, the results from the subsequent randomized placebo-controlled EXenatide Study of Cardiovascular Event Lowering (EXSCEL) trial failed to demonstrate cardiovascular benefit in 14,752 high risk subjects with type 2 diabetes who were treated with long-acting exenatide 2 mg SQ weekly compared to placebo. 9,10 Fewer cardiovascular events occurred in the exenatide group, but the results were not significantly different from the placebo group. Full details from the study are still unavailable.

The results of another cardiovascular outcomes trial using lixisenatide also did not show cardiovascular benefit. In the Evaluation of Cardiovascular Outcomes in Patients With Type 2 Diabetes Mellitus After Acute Coronary Syndrome During Treatment With Lixisenatide (ELIXA) trial, 6068 patients with type 2 diabetes and cardiovascular disease were treated with lixisenatide for a median duration of 2.1 years. 11 The subjects in this trial had a mean age of 60.3 +/- 9.6 years and HgbA1c 7.6 +/- 1.3%. The study demonstrated that lixisenatide was non-inferior to placebo for cardiovascular outcomes, but the primary composite cardiovascular outcome was not significantly reduced in the lixisenatide group (HR 1.02 95% CI 0.89 to 1.17).

In contrast, the experimental GLP-1 receptor agonist, semaglutide, has been demonstrated to have cardiovascular benefit. The randomized placebo-controlled Trial to Evaluate Cardiovascular and Other Long-term Outcomes with Semaglutide in Subjects with Type 2 Diabetes (SUSTAIN-6) was designed to demonstrate the noninferiority of semaglutide compared to placebo for cardiovascular safety in 3297 patients with type 2 diabetes. 12 The study population consisted of 3297 subjects with type 2 diabetes and a mean age of 64.6 +/- 7.4, baseline HgbA1c 8.7 +/- 1.5, and baseline cardiovascular disease in 83% of the subjects. The results demonstrated a significant reduction in cardiovascular events in patients treated with semaglutide 0.5 or 1.0 mg subcutaneously weekly (hazard ratio 0.74 95% confidence interval [CI] 0.58 to 0.95). Rates of nephropathy were lower, but complications of retinopathy were higher with semaglutide compared to placebo. Semaglutide is not yet FDA approved for clinical use in the United States.

Summary and Conclusions

In summary, GLP-1 has important postprandial physiological effects that include stimulation of insulin secretion in a glucose-dependent manner, suppression of glucagon secretion, decreased rate of gastric emptying, and suppression of appetite. Multiple GLP-1 receptor agonists have been developed for treatment of type 2 diabetes. All of the five currently clinically available drugs produce significant improvement in glycemic control in association with modest weight loss, but so far only liraglutide 1.2 to 1.8 mg SQ daily has been demonstrated to reduce the risk of cardiovascular events. The Endocrinologic and Metabolic Drugs Advisory Committee (EMDAC) of the FDA recently voted in favor of adding information from the LEADER trial to the labeling for liraglutide. Liraglutide is also FDA approved at the higher dose of 3 mg SQ daily for the purpose of weight loss. Long-acting exenatide and lixisenatide were non-inferior to placebo in relation to cardiovascular events, but neither demonstrated a significant reduction in cardiovascular events. Cardiovascular outcomes data are not yet available for albiglutide and dulaglutide, but the experimental GLP-1 agonist semaglutide has been shown to reduce the risk of cardiovascular events compared to placebo. It is possible that the differences in results from the various cardiovascular outcomes trials are related to differences in study populations or study design, but these features do not provide a clear explanation for the absence of a significant reduction in cardiovascular events with lixisenatide and extended release exenatide. Further studies are needed to clarify whether cardiovascular event reduction in response to treatment with GLP-1 receptor agonists is a class effect and to elucidate the mechanisms by which liraglutide and semaglutide reduce cardiovascular events. In the meantime, this class of drugs provides an effective option for treatment of hyperglycemia and mediating modest weight loss in type 2 diabetes.

  1. Drucker DJ. The biology of incretin hormones. Cell Metab 20063:153-65.
  2. Campbell JE, Drucker DJ. Pharmacology, physiology, and mechanisms of incretin hormone action. Cell Metab 201317:819-37.
  3. Flint A, Raben A, Astrup A, Holst JJ. Glucagon-like peptide 1 promotes satiety and suppresses energy intake in humans. J Clin Invest 1998101:515-20.
  4. Scott RA, Freitag DF, Li L, et al. A genomic approach to therapeutic target validation identnifies a glucose-lowering GLP1R variant protective for coronary heart disease. Sci Transl Med 20168:341ra76.
  5. US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER). Guidance for Industry: Diabetes Mellitus - Evaluating Cardiovascular Risk in Antidiabetic Therapies to Treat Type 2 Diabetes. Silver Spring: FDA, 2008.
  6. Marso SP, Daniels GH, Brown-Frandsen K, et al. Liraglutide and cardiovascular outcomes in type 2 diabetes. N Engl J Med 2016375:311-22.
  7. Steinberg WM, Buse JB, Ghorbani MLM, et al. Amylase, lipase, and acute pancreatitis in people with type 2 diabetes treated with liraglutide: results from the LEADER randomized trial. Diabetes Care 201740:966-72.
  8. Best JH, Hoogwerf BJ, Herman WH, et al. Risk of cardiovascular disease events with type 2 diabetes prescribed the glucagon-like peptide 1 (GLP-1) receptor agonist exenatide twice daily or other glucose-lowering therapies: a retrospective analysis of the LifeLink database. Diabetes Care 201134:90-5.
  9. Holman RR Share via:

Keywords: Glucagon-Like Peptide 1, Primary Prevention, Secondary Prevention, Diabetes Mellitus, Diabetes Mellitus, Type 2, Blood Pressure, Hypertrophy, Left Ventricular, Cardiovascular Diseases, Body Mass Index, Risk Factors, Angina, Unstable, Myocardial Infarction, Heart Failure, Hypoglycemia, Hypertension, Peripheral Vascular Diseases, Renal Insufficiency, Chronic, Metabolic Syndrome X


Results

Target screening of acupuncture in treating COVID-19

The application of STITCH and SwissTargetPrediction database gave rise to genes correlated with the effects of active ingredients after acupuncture. COVID-19- associated genes were collected from GeneCard database. In total, human targets were matched following normalization of gene names in the original files. Subsequently, 204 ingredient acupuncture-associated target (Supplementary Table S1 and S2), and 11 300 COVID-19 targets (Supplementary Table S2 and S3) were utilized for plotting a Venn diagram, which yielded to coincident targets ( Figure 1 ). Collectively, our analysis showed that acupuncture generated 180 potential therapeutic targets of COVID-19.

The intersection generated between acupuncture-associated target and COVID-19-associated genes. Using online databases, we identified 180 shared biotargets of acupuncture against COVID-19.

Establishment and analysis of PPI network

PPI network was constructed based on STRING database and visualized by Cytoscape after topological analysis, showing 180 nodes. �gree’ is defined as the number of connections of a node in the network graph, reflecting the interaction information between nodes. The larger value indicates the greater importance of the node [2]. The degree of targets was shown in Supplementary Table S4. BDKRB1 had the greatest degree (41), followed by ADCY5 (34), CXCR4 (31), FPR1 (31) and C3AR1 (28). Top 20 core targets sorted by the value of degree were shown in Table 1 , which were identified as the key targets of acupuncture in COVID-19.

Table 1

Target nameDegreeTarget nameDegree
BDKRB141.0OPRK125.0
ADCY534.0OPRL125.0
CXCR431.0MTNR1B25.0
FPR131.0DRD325.0
C3AR128.0DRD425.0
OPRM127.0C5AR125.0
OPRD127.0ADORA325.0
HTR1A26.0ADRA2A25.0
DRD225.0HTR1B25.0
PDYN25.0HRH325.0

GO and KEGG enrichment analysis

GO analysis can be used to reflect target functions from three aspects, including cellular components (CC), molecular functions (MF) and biological processes (BP) [1]. In our study, 522 GO entries were enriched (P <𠂐.05) (Supplementary Table S5). The top 10 pathways with the most enriched GO analysis were shown in Figure 2 . Top 10 BP of acupuncture against COVID-19 was shown in Table 2 . The phospholipase C-activating G-protein coupled receptor signaling pathway, positive regulation of cytosolic calcium ion concentration and response to drug were the top three GO terms in BP with low P-adjust value. Plasma membrane, integral component of plasma membrane and integrin complex were the top three GO terms in CC with low P-adjust value. And drug binding, enzyme binding and virus receptor activity were the top three GO terms in MF with low P-adjust value.

GO enrichment analysis (P-adjust value < 0.05). GO: Gene Ontology BP: biological processes CC: cellular components MF: molecular functions.

Table 2

Top 10 biological processes of acupuncture against COVID-19

IDDescription P-value P-adjustNumber of gene
GO:0007200Phospholipase C-activating G-protein coupled receptor signaling pathway5.12E-219.38E-1819
GO:0042493Response to drug1.20E-191.10E-1630
GO:0007204Positive regulation of cytosolic calcium ion concentration2.11E-161.36E-1320
GO:0007268Chemical synaptic transmission1.02E-144.68E-1223
GO:0045907Positive regulation of vasoconstriction1.48E-145.41E-1212
GO:0071880Adenylate cyclase-activating adrenergic receptor signaling pathway1.16E-133.55E-1110
GO:0008284Positive regulation of cell proliferation6.11E-131.60E-1028
GO:0050900Leukocyte migration3.04E-126.96E-1016
GO:0007187G-protein coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger4.07E-118.30E-0911
GO:0001975Response to amphetamine8.72E-101.60E-079

In terms of KEGG analysis, 61 most enriched signal pathways were acquired (P <𠂐.05) (Supplementary Table S6). The top 20 pathways with high significance were selected and presented in Figure 3 , including neuroactive ligand–receptor interaction, serotonergic synapse, calcium signaling pathway, focal adhesion, alcoholism, pathways in cancer, cocaine addiction, cGMP−PKG signaling pathway, viral carcinogenesis, etc. Top 10 KEGG pathways of acupuncture against COVID-19 were displayed in Table 3 .

KEGG pathways enrichment analysis (P-adjust value < 0.05). KEGG: Kyoto Encyclopedia of Genes and Genomes.

Table 3

Top 10 KEGG pathway of acupuncture against COVID-19

IDDescription P-value P-adjustNumber of gene
hsa04080Neuroactive ligand–receptor interaction5.03E-351.00E-3254
hsa04726Serotonergic synapse8.14E-138.10E-1121
hsa04020Calcium signaling pathway1.50E-109.93E-0923
hsa04510Focal adhesion6.63E-083.30E-0621
hsa05034Alcoholism1.56E-076.23E-0619
hsa05200Pathways in cancer1.47E-064.87E-0527
hsa05030Cocaine addiction1.72E-064.89E-0510
hsa04022cGMP-PKG signaling pathway4.28E-069.46E-0516
hsa05414Dilated cardiomyopathy3.93E-069.78E-0512
hsa05203Viral carcinogenesis6.13E-061.22E-0418

The construction of compound-target and compound-disease-target network

The compound-target and compound-disease-target network of acupuncture on COVID-19 was shown in Figures 4 and ​ and5, 5 , respectively. The degree of dopamine and β-Endorphin were 93 and 96, respectively. Among the keywords of COVID-19, the degree of heat was the highest (161), followed by cough (140), viral pneumonia (109) and coronavirus (18).

Component-target network of acupuncture for COVID-19. In the network, there are two active components have interactions with 180 protein targets. Red nodes represent protein targets, light blue nodes represent active components.

Compound-disease-target network of acupuncture for COVID-19. Red nodes represent targets, light blue nodes represent active components, dark blue nodes represent the key words of COVID-19.


Atherosclerotic lesions typically develop where arteries branch. In response to disturbed blood flow that naturally occurs in these forked regions, blood vessels thicken. When this happens, the endothelial cells lining the interior vascular surface become activated and exhibit increased permeability, making it easier for LDL to enter into the intima – the innermost layer of the vessel wall. Individuals with high cholesterol have abnormally elevated levels of LDL in the blood, which increases the number of LDLs accessing and colonising the vascular wall. A major problem here is that when LDLs are exposed to the new environment, they undergo oxidative modifications that transform them into pro-inflammatory molecules, which release factors from the endothelium that initiate and maintain inflammation in the vessel wall.

Several cells of the immune system respond to these pro-inflammatory signals. One particular type, called monocytes, migrate from the blood to these inflammatory sites in the vessel wall and transform into larger, mature macrophages. Macrophages are armed with the necessary machinery to recognise and remove oxidised LDL from the vascular space. Proteins located along the macrophage membrane, called scavenger receptors, recognise oxidised LDL and bring it inside to be used or eliminated by the cell. However, accumulated LDL in these diseased areas increases the internalisation rate, which transforms these highly mobile removal workers into fat-laden and less mobile ‘foam’ cells. The lipids engulfed by the macrophage give it a ‘foamy’ appearance (red colour on the right). Paralysed, these foam cells die and become part of the expanding fatty plaque they were previously trying to prevent. Over time this plaque expands, narrowing the vessel lumen and reducing the volume of blood that can pass through. If and when this plaque ruptures, a blood clot will emerge and obstruct the vessel entirely, starving the organ it serves of its oxygen supply. If it serves the heart, it can cause a heart attack, while if it occurs in the brain, it is called stroke.

While progress has been made in terms of diagnosis, prevention and treatment, atherosclerosis and its cardiovascular consequences continue to cause high rates of morbidity and mortality worldwide. Therefore, it is important to improve our understanding of the biological mechanisms involved, so that better therapies can be developed.

Pinocytosis: An Unexplored Target in Atherosclerosis

Dr Gábor Csányi and his group have contributed to a better understanding of how macrophages ingest bad cholesterol and transform into lipid-laden foam cells. They have shown that LDLs do not actually need to be oxidised to be internalised, and also, that another mechanism of uptake is involved in macrophage foam cell formation. Essentially, in addition to relying on the selective uptake of modified LDLs by scavenger receptors, macrophages use pinocytosis to ingest unmodified LDLs. Pinocytosis, often referred to as ‘cell drinking’, is the engulfing of extracellular fluid – liquid that surrounds the cell. This process is nonspecific, because the cell swallows everything in the fluid, including random dissolved particles. The rate of pinocytosis and pericellular solute concentration are the primary factors that determine the extent of solute internalisation.

So why do cells risk allowing entry to everything? Immune cells, like macrophages, use pinocytosis to survey

Lipid-laden macrophage or foam cell

the surrounding environment for antigens. They bring samples inside and evaluate them to establish whether there are foreign invaders that might be potentially harmful and need to be eliminated. Additionally, the cells use pinocytosis to internalise extracellular nutrients to support their growth and metabolic needs. Macrophages use two forms of pinocytosis to internalise fluid and associated solutes – macropinocytosis and micropinocytosis. As their names suggest, they ingest large and small volumes of extracellular fluid, respectively. While both types of pinocytosis may participate in the ingestion of unmodified LDLs, Dr Csányi and his group are specifically interested in the role that macropinocytosis plays in this process.

Macropinocytosis starts when extracellular signalling molecules interact with specific protein receptors housed on the cell membrane. When the cell receives the initiating signal, membrane ‘ruffles’ form, creating folds and protrusions that extend into the surrounding environment. These protrusions move back towards the plasma membrane, much like a swimmer doing the breast stroke, capturing fluid and solutes in their wake. Membrane protrusions can also transition into curved ruffles and form ice cone like macropinocytotic cups, which close on the top, capturing extracellular fluid and solutes. Following this, the newly formed fluid sacks pinch off from the plasma membrane to form irregular shaped and sized vesicles called macropinosomes, which travel into the cytoplasm – the cell’s interior.

A ‘thirsty’ macrophage develops membrane ruffles to drink extracellular fluid

Dr Csányi’s research group has delved deeper into the mechanisms by which macropinocytosis of lipids are regulated. ‘My research focuses on a better understanding of the mechanisms leading to atherosclerosis and applying this understanding to develop and implement new strategies for its prevention and treatment,’ he explains. ‘Utilising a broad range of innovative technologies and strategies, we investigate the mechanisms by which cardiovascular risk factors, inflammatory mediators, matrix proteins, and genes promote the transition from a healthy “normal” vessel wall to a “diseased” vasculature.’

Dr Csányi and his team devised several intricate experiments to investigate what molecules and pathways regulate macropinocytosis. The team has identified a new mechanism regulating macropinocytosis of unmodified LDL by macrophages.

The role of Nox2 in Macropinocytosis

In their research, Dr Csányi and his team demonstrated that Nox2 participates in macropinocytosis. Belonging to the NADPH oxidase (Nox) family of protein oxidases, Nox2 functions in generating reactive oxygen species (ROS) and mediating intracellular redox reactions. Briefly, this enzyme complex is responsible for transferring electrons to molecular oxygen to produce superoxide anions – the precursor radicals of ROS. Low levels of ROS regulate signalling pathways that are important for maintaining healthy cellular physiology, while increased ROS generation leads to oxidative stress and contributes to vascular and other diseases.

Internalised fluid (arrows)

Dr Csányi chose Nox2 not only because of its importance in ROS production in macrophages, but also because it exists downstream in a signalling chain of molecules, namely Protein Kinase C (PKC) and Ras-1, known to function in macropinocytosis. Therefore, he proposed that these other proteins may send signals to Nox2 to help them initiate macropinocytosis. Dr Csányi and his team conducted their initial experiments using macrophages in the lab. They used 4ß-PMA, a molecule known to induce macropinocytosis in macrophages, to test whether Nox2 participates. A clever way to explore whether a particular molecule is important in a biological process is to disrupt its function. Firstly, they deactivated the Nox2 protein and found that 4ß-PMAinduced macropinocytosis and the amount of lipid ingested was significantly reduced. To clarify these findings, they silenced the Nox2 gene before it was able to be fully translated into functional protein. Again, macropinocytosis was inhibited.

While evaluating the biological processes in cells on their own was informative, it was important to test whether similar results translated to animal models of atherosclerosis. To test this, the team extracted macrophages from wild-type mice, whose genetics have not been manipulated, and Nox2 knockout mice, where Nox2 expression is genetically removed. These macrophages were treated with 4ß-PMA to induce macropinocytosis and subsequently injected into the peritoneal cavities of ApoEˉ/ˉ mice–bred to develop high cholesterol similar to that seen in atherosclerotic patients. A day later, Dr Csányi and his colleagues extracted and analysed the macrophages and found that macropinocytosis and lipid uptake was significantly diminished in those taken from the Nox2 knockout mice. Cumulatively, these experiments highlighted Nox2 as a principal player in the induction of macropinocytosis. Next, Dr Csányi’s laboratory sought to identify the signalling mechanisms downstream of Nox2 involved in macropinocytosis.

In further investigations, Dr Csányi and his team found that intracellular Nox2 signalling activates cofilin (an actin-binding protein) to trigger membrane ruffling – the first stage of macropinocytosis. It does this by stimulating a secondary signalling pathway, called the phosphoinositide 3 kinase/Akt pathway (PI3K/Akt). For the first time, Dr Csányi and his colleagues demonstrated that Nox2-mediated intracellular redox signalling plays a pivotal role in a mechanism that initiates macrophage LDL macropinocytosis.

CD47 and Nox1: Partners in Crime

Dr Csányi, Dr Pagano and their colleagues defined a physiologically relevant pathway that mediates macrophage macropinocytosis of unmodified LDL. The extracellular matrix surrounding a cell’s exterior provides structural and biochemical stability. It is composed of different molecules, including a group of matricellular proteins, which help to regulate pathways that are important for normal cell maintenance. One type of matricellular protein, called thrombospondin-1 (TSP1), is elevated above healthy levels in the intimal layer of the vessel wall in individuals with atherosclerosis. Therefore, Dr Csányi proposed that TSP1 may act as a physiological stimulator of LDL macropinocytosis in macrophages.

The research team found that incubating macrophages with physiologically relevant concentrations of TSP1 leads to extensive plasma membrane ruffling, macropinosome formation and increased uptake of unmodified LDL compared to untreated controls. Subsequently, they demonstrated that TSP1 binds to a specific protein receptor, CD47, to carry out this process.

While these findings were new and exciting, the team sought to uncover the remaining parts of the chain involved in transmitting the instructions to initiate macropinocytosis. Dr Csányi and colleagues genetically blocked the activity of different molecules in the pathway to see whether they are involved in TSP1-CD47-induced membrane ruffling and macropinocytosis. It has previously been shown that TSP1 signals through Nox1, another isoform of the NADPH oxidase family, to induce superoxide anion production in the vascular smooth muscle. Based on these findings, Dr Csányi proposed that TSP1 may use the same mechanism in macrophages to stimulate macropinocytosis. Indeed, they measured increased levels of superoxide anions inside macrophages upon TSP1 treatment. Additionally, macrophages exhibited reduced membrane ruffling, macropinocytosis activity and lipid uptake in response to TSP1 when Nox1 was inhibited. The in vivo importance of this pathway was recapitulated in a mouse model of atherosclerosis.

However, the final piece of this signalling puzzle was still missing. How does the TSP1/CD47/Nox1 axis trigger membrane ruffling and macropinocytosis? Similar to the previous study, the link was cofilin. TSP1 was found to induce cofilin activation when CD47 and Nox1 were functioning in wild-type macrophages but failed to do so when CD47 and Nox1 had been genetically blocked. The study by Dr Csányi and his team demonstrated for the first time that a signalling pathway involving CD47, Nox1 and cofilin contributes to TSP1-induced macropinocytic uptake of unmodified LDL by macrophages.

What Does It All Mean?

Dr Csányi’s work has given us important new insights into the specific mechanisms involved in macrophage uptake of LDL. In addition, it has clarified that macrophages also use macropinocytosis to internalise unmodified forms of bad cholesterol, contributing to the formation of foam cells that become part of atherosclerotic lesions. Dr Csányi states that, ‘these findings support a new paradigm in which redox signaling-mediated lipid macropinocytosis independent of extracellular lipid oxidation propagates initiation and development of vascular inflammatory disease.’ Finally, and perhaps most importantly, this research has highlighted specific mechanistic components that could be targeted for atherosclerosis prevention and treatment.

Meet the researcher


Dr Gábor Csányi
Vascular Biology Center Department of Pharmacology & Toxicology
Augusta University
Georgia, USA

Dr Gábor Csányi graduated from the University of Szeged in Hungary with a Masters of Pharmacology in 2003. He completed his PhD in Medicine at the University of Szeged and Jagiellonian University in Kraków, Poland in 2008. During his PhD, much of his work was focused on characterising functional alterations of endothelium-derived vasodilators that occur in several cardiovascular pathologies, including atherosclerosis, ischemic heart failure, dilated cardiomyopathy and diabetes mellitus. Following his PhD, Dr Csányi moved to the USA to carry out his postdoctoral training in the laboratory of Dr Patrick Pagano at the University of Pittsburgh in Pennsylvania. In his second year, Dr Csányi received an American Heart Association (AHA) Postdoctoral Fellowship. At present, he is an Assistant Professor in the Vascular Biology Center at Augusta University in Georgia, USA and his research is funded by the NIH Pathway to Independence Award (K99/ R00).

KEY COLLABORATORS

Patrick Pagano, PhD, Vascular Medicine Institute, University of Pittsburgh, USA
Stefan Chlopicki, PhD, Jagiellonian Centre for Experimental Therapeutics, Krakow, Poland
David Fulton, PhD, Vascular Biology Center, Augusta University, USA

National Institutes of Health (K99HL114648, 4R00HL114648-03, R01HL079207 and R01HL112914).

Gabor Csanyi, PhD
Pushpankur Ghoshal, PhD
Bhupesh Singla, PhD Huiping
Lin Madison Carpenter

G Csányi, DM Feck, P Ghoshal, B Singla, H Lin, S Nagarajan, DN Meijles, IA Ghouleh, N Cantu-Medellin, EE Kelley, L Mateuszuk, JS Isenberg, S Watkins and PJ Pagano, CD47 and Nox1 mediate dynamic fluid-phase macropinocytosis of native LDL, Antioxidants & Redox Signaling, 2017, 1–16.

P Ghoshal, B Singla, H Lin, DM Feck, N Cantu-Medellin, EE Kelley, S Haigh, D Fulton and G Csányi, Nox2-mediated PI3K and cofilin activation convers alternate redox control of macrophage pinocytosis, Antioxidants & Redox Signaling, 2016, 1–15.


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Monoamines (also known as "biogenic amines") include three classes of neurotransmitters:

  • Catecholamines
    • Dopamine (DA), norepinephrine (NE, also called noradrenaline) and epinephrine (E, also called adrenaline) make up a class of neurotransmitters named on the basis of the hydroxylated phenol ring termed a catechol nucleus.
    • Serotonin (5-hydroxytryptamine 5-HT) is the principal member of this group of compounds. The name serotonin is derived from the fact that this substance was first isolated from the serum based on its ability to cause an increase in blood pressure. Melatonin, a second indolamine, is restricted to the pineal and is released into the blood stream in a manner that is regulated by the diurnal cycle. Melatonin will not be covered further in this chapter.
    • Histamine has been recognized as a neurotransmitter in the CNS only within the past fifteen years.

    The structure of the monoamine neurotransmitters is shown in Figure 12.1.

    Figure 12.1
    Structure of the monoamine neurotransmitters.

    12.2 Anatomy of Catecholamines

    Catecholamines are neurotransmitters in a sympathetic limb of the autonomic nervous system and in the CNS.

    12.3 Autonomic Nervous Systems

    As shown in Figure 12.2, norepinephrine is a neurotransmitter in postganglionic sympathetic neurons where it acts on smooth muscle to cause either contraction or relaxation, depending on the types of receptors present (see below). DA is a neurotransmitter in autonomic ganglia where it modulates cholinergic transmission and in the kidney, where it produces renal vasodilation and inhibits Na + and H2O reabsorption. Epinephrine and norepinephrine are neurohumoral agents released into the circulation by the adrenal medulla. The ratio of E to NE released is 4 to 1.

    Figure 12.2
    Location of NE and Epi at sympathetic nerve endings and in the adrenal medulla.

    12.4 Central Nervous System

    Generally, the cell bodies of catecholamine neurons are found in clusters in the brain stem or midbrain and project to other regions of the brain and spinal cord. NE, for example, projects to almost every area of the brain. In contrast, DA has a more restricted projection. Epinephrine, which will not be covered in this chapter, has the most restricted distribution.

    Figure 12.3
    Location of DA in the rat CNS

    The major site of DA cell bodies is the midbrain. These clusters of cells give rise to four DA systems shown in Figure 12.3:

    1. Mesostriatal system (blue and red in Figure 12.3),
    2. Mesolimbocortical system (purple in Figure 12.3),
    3. Periventricular system (orange in Figure 12.3), and
    4. Tuberohypophyseal system. (green in Figure 12.3).

    Mesostriatal DA System - The mesostriatal DA system, referred to as the nigrostriatal pathway, is composed of two components, a dorsal mesostriatal pathway and a ventral mesostriatal pathway. These two pathways are important for movement control and reward mechanisms.

    The dorsal mesostriatal pathway (blue) originates in the substantia nigra par compacta and ascends to innervate the corpus striatum (caudate, putamen, and globus pallidus), where it modulates the output of the corpus striatum. The destruction of the nigrostriatal cells in Parkinson's disease produces marked motor deficits.

    The ventral mesostriatal pathway (red) also originates in the substantia nigra and ventral tegmental area, and innervates the nucleus accumbens, olfactory tubercles and medial caudate-putamen. The ventral nigrostriatal pathway plays an important role in positive incentive characteristics of rewarding behaviors and the psychostimulants, as will be discussed further in Chapter 12, Part 10.

    Mesolimbocortical DA System - The Mesolimbocortical DA system (purple) originates in the midbrain and projects to limbic structures (septum, amygdala, hippocampus, olfactory nucleus, and limbic cortex). This DA system is believed to participate in schizophrenia. (See Chapter 12, Part 11) The current hypothesis is that an increase in DA function in the mesolimbic system and a decreased function in the mesocortical DA systems occur in schizophrenia.

    Periventricular DA System - The periventricular DA system (orange in Fig. 12.3) coordinates motivated behavior. These DA cells originate in the periventricular region of hypothalamus and send short axons to several thalamic and hypothalamic nuclei. Collaterals also descend to the intermediolateral cell column of the spinal cord to synapse with sympathetic preganglionic neurons. This dual innervation of hypothalamic and sympathetic preganglionic neurons is believed to integrate the central and autonomic components of motivated behaviors, including behaviors such as sex, thirst and appetite.

    Tuberohypophyseal DA System - The tuberohypophyseal DA system mediates the control of milk production during lactation. The DA cells (green) originate in the periventricular and arcuate nuclei of the hypothalamus and project to the median eminence of the hypothalamus where they release DA into the capillary plexus of the hypophyseal-portal system. DA travels to the anterior pituitary where it inhibits the release of prolactin, the hormone that stimulates milk production in lactating animals.

    12.6 Norepinephrine - Anatomy

    Figure 12.4
    Location of NE in the rat CNS.

    The major site of NE cell bodies is the medulla and pons. The NE cells consist of three main groups shown in Figure 12.4:

    1. locus coeruleus complex (purple and red in Figure 12.4),
    2. lateral tegmental system (blue in Figure 12.4), and
    3. dorsal medullary system (green in Figure 12.4).

    In all three cases the neurons project diffusely to broad regions of the brain where their nerve terminals lack conventional synaptic junctions. Release of transmitter from these cells is described as volume transmission, because NE, once released, is thought to diffuse and influence a number of adjacent cells.

    Locus Coeruleus System - The locus coeruleus (LC-purple and red) is considered the most influential of the cell groups even though it consists of less than 2,000 cells on either side of the midline. This importance is because LC axons project rostrally via the dorsal noradrenergic bundle to innervate nearly the entire telencephalon and diencephalon, as well as dorsally to innervate the cerebellum and caudally to innervate the spinal cord. The nerve fibers are so highly ramified in the terminal fields such that each axon may branch as many as 100,000 times. This pattern of innervation enables the LC to synchronously modulate cellular activity across wide expanses of the cortex.

    Lateral Tegmental System - The axons of the lateral tegmental system (blue) project caudally to the intermediolateral cell column of the spinal cord where they inhibit sympathetic preganglionic cells, and ventrally to the hypothalamus. The joint innervation of the hypothalamus and the intermediolateral column cells is believed to be the basis for NE integration of central and peripheral sympathetic autonomic function.

    Dorsal Medullary System - As a complement to the lateral tegmental system, the dorsal medullary system (green) projects to the nucleus solitarius, as well as to the brain stem nuclei that control cranial parasympathetic function (glossopharyngeal, facial, and trigeminal- nuclei and the dorsal vagal nuclear complex). These NE systems are believed to provide control of the cranial parasympathetic system in a manner analogous to the lateral tegmental system's control of the sympathetic system.

    (Note: there is no figure for epinephrine anatomy)

    Two clusters of epinephrine (E) cells are located in the medullary reticular formation. One cluster of cells in the ventrolateral medulla sends ascending projections to innervate the periaqueductal gray and several hypothalamic and olfactory nuclei. This cluster also sends a descending projection to innervate the sympathetic preganglionic cells of the intermediolateral column in a manner analogous to the NE lateral tegmental system. The second group of cells, located in the dorsomedial medulla near the floor of the fourth ventricle, project to several parasympathetic cranial nerve nuclei (similar to the dorsal medullary NE system, described above). These adrenergic cells are believed to coordinate eating and various visceral functions including the regulation of blood pressure.

    Figure 12.5
    5-HT neuronal pathways in the rat CNS are in two clusters of cells, one in the caudal brain stem (B1-B4) and the other in the rostral brainstem (B5-B9)

    As shown in Figure 12.5, serotonin cells are located in two clusters:

    1. a caudal system in the medulla (B1-B4, green in Figure 12.5)
    2. a rostral system in the midbrain (B5-B9, purple and blue in Figure 12.5).

    Both project widely throughout the CNS.

    Caudal System: The caudal cluster of 5-HT cells (B1-B4) is located close to the midline and project caudally to the spinal cord dorsal and ventral horns as well as the intermediolateral cell column. These pathways are believed to mediate the role of 5-HT in sensory, motor and autonomic functions, respectively.

    Rostral System: The rostral midbrain cluster of cells (B5-B9), (raphe nuclei) are distributed throughout the midbrain. A cluster of cells located medially and another located dorsally provide over 80% of the 5-HT innervation of the forebrain. These cells project to the diencephalon, basal ganglia, limbic system, cortex, mesencephalic gray and inferior and superior colliculi. Some evidence supports the conclusion that the innervation of forebrain structures by serotonergic processes is complementary to that of NE. Another important aspect of 5-HT microanatomy is that two distinct patterns of innervation exist for these medial and dorsal systems. The dorsal system is similar in its anatomy to that of catecholamine neurons with thin diffusely branching axons lacking classic synaptic contacts (volume neurotransmission). The medial system, in contrast, appears to have classical synapses and is characterized by the presence of thick axons with large round nerve endings that make extensive synaptic contacts. These differences imply a marked difference in the physiological function of these two 5-HT systems.

    Other Systems: In addition to the above two pathways, another 5-HT pathway projects partially from one of the rostral nuclei (B5) and partially from two caudal nuclei (B2 & B3, dark green in Figure 12.5) to innervate the cerebellar cortex and deep cerebellar nuclei. There is also a widespread 5-HT projection to structures within the brainstem, including the locus coeruleus, several cranial nuclei, inferior olivary nucleus, and nucleus solitarius.

    Figure 12.6
    Histamine neuronal pathways mapped in the rat CNS using histidine decarboxylase immunoreactivity.

    Histamine cells (HA) are located exclusively in the basal posterior hypothalamus. These cells project extensively throughout the neural axis in a manner analogous to the NE and 5-HT systems. Although HA has not been investigated extensively, based on its diffuse innervation of the CNS and lack of classic synaptic contacts, it is likely that histamine has a broad behavioral and physiological function.

    Histamine is also the major active substance released from mast cells. The presence of mast cells in the blood in the CNS has hindered the analysis of the role of histamine as a neurotransmitter.

    12.10 Introduction to Cell Biology

    The monoamines will be considered as a group in discussing the cell biology of their 1) synthesis, 2) storage and 3) release. Monoamine receptors and termination of action of each monoamine will be considered separately.

    12.11 Cell Biology - Biosynthesis of Monoamines

    All monoamine (MA) neurotransmitters are synthesized from amino acids through a series of enzyme catalyzed reactions in which hydroxylation, decarboxylation and/or methylation convert the precursor amino acid into the active monoamine neurotransmitter.

    Biosynthesis of Dopamine (DA), Norepinephrine (NE), and 5-hydroxytryptamine (5-HT)

    Biosynthesis of all monoamines occurs primarily in the nerve terminal. As shown in Figure 12.7, the first step in the synthesis of catecholamines (DA and NE, as well as E, not shown) is the hydroxylation of the tyrosine to form DOPA. An analogous reaction, the hydroxylation of tryptophan to 5 hydroxytryptophane (5-HTP) is the first step in the biosynthesis of 5-HT. Both tyrosine hydroxylase and tryptophan hydroxylase are the rate-limiting steps in the biosynthetic pathway of the respective monoamines. Both enzymes are mixed function mono-oxygenases requiring molecular oxygen, iron and the cofactor, tetrahydrobiopterin (BH4) for activity. BH4 is converted to BH2 during the hydroxylation and must be regenerated to BH4 in order for monoamine biosynthesis to continue. As shown in Figure 12.7, the enzyme pteridine reductase regenerates the active cofactor. Pteridine reductase is therefore also an essential enzyme in the synthesis of catecholamines. The next step in the biosynthesis of monoamines is the decarboxylation by aromatic amino acid decarboxylase (AADC) to form the corresponding monoamine (Dopamine and 5 hydroxytryptamine 5-HT, respectively). NE is then formed from dopamine through an additional reaction, the hydroxylation of the 2 nd carbon of the DA side chain. This last hydroxylation step occurs within the monoamine storage vesicle (see Figure 12.9a) and is catalyzed by dopamine β hydroxylase.

    Biosynthesis of the DA and NE precursor L-DOPA and the 5-HT precursor 5-HTP through hydroxylation using tyrosine hydroxylase (TH) and tryptophan hydroxylase (TryH.) These intermediates are then decarboxylated by a nonspecific decarboxylase, aromatic amino acid decarboxylase (AADC) to form the respective monoamines. Pteridine reductase regenerates the cofactor BH4.Biosynthesis of the DA and NE precursor L-DOPA and the 5-HT precursor 5-HTP through hydroxylation using tyrosine hydroxylase (TH) and tryptophan hydroxylase (TryH.) These intermediates are then decarboxylated by a nonspecific decarboxylase, aromatic amino acid decarboxylase (AADC) to form the respective monoamines. Pteridine reductase regenerates the cofactor BH4.

    Two additional cofactors are required for the synthesis of monoamines vitamin B6 is necessary as a cofactor for AADC catalyzed decarboxylation. Vitamin C is required as a cofactor for DA conversion to NE in the storage vesicle (see Figure 12.9a).

    Biosynthesis of Epinephrine (E)

    Epinephrine is synthesized in adrenal medulla and CNS by methylation of NE on the amino-terminus (not shown). The enzyme that catalyzes this reaction is phenyl ethanolamine N methyl transferase (PNMT). This enzyme uses S-adenosyl methionine as the methyl donor to methylate norepinephrine to form epinephrine (the nor refers to the lack of the methyl group). PNMT's localization outside the storage vesicle requires that norepinephrine shuttle out of the vesicle to be converted to epinephrine and then back into the storage vesicle for storage and release.

    Biosynthesis of Histamine (HA)

    Figure 12.8
    Biosynthesis of histamine from histidine.

    As shown in Figure 12.8, in contrast to the catecholamines and 5-HT, the biosynthesis of histamine does not require hydroxylation. Histamine is the product of the decarboxylation of the amino acid, histidine, to form the monoamine, histamine, in a single step that is analogous to the decarboxylation of DOPA and 5-HTP. A different enzyme is used to decarboxylate histidine, histidine decarboxylase, as shown in Figure 12.8. This enzyme, like AADC, requires vitamin B6.

    Regulation of Catecholamine Biosynthesis

    The concentration of catecholamines in nerve terminals remains relatively constant despite frequent fluctuations in neuronal activity. This homeostasis is achieved through the regulation of TH activity. TH is phosphorylated and activated by both calcium and cAMP dependent protein kinases. A longer-term regulation of CA synthesis also occurs. This regulation is mediated through altered transcription of TH mRNA and altered TH mRNA stability. Both mechanisms lead to increased levels of TH protein.

    Regulation of Serotonin Biosynthesis

    The level of serotonin is regulated principally by the amount of tryptophan available to serotonergic neurons. This has two important implications for the level of serotonin in the brain. First, because tryptophan is not synthesized in mammals, the level of tryptophan available for serotonin biosynthesis is dependent on diet. Thus, diets high in tryptophan can markedly elevate serotonin levels. Second, because tryptophan is transported across the blood brain barrier by a transport system which also transports certain other amino acids, diets high in these amino acids can reduce the level of serotonin in the brain by competing with tryptophan for transport into the CNS. As will be discussed later, altered serotonin level in the CNS can have marked consequences on behavior.

    Regulation of Histamine Biosynthesis

    Thus far the mechanism for the regulation of histamine biosynthesis is unknown.

    12.12 Storage of Monoamines

    Monoamine neurotransmitters are stored in vesicles that appear dark at the EM level and are thus referred to as dense core vesicles. MA neurotransmitters are stored at a high concentration and are complexed with ATP and several proteins called chromogranins. One of these chromogranins is the enzyme, dopamine β hydroxylase (D β H), that converts DA to NE. As shown in Figure 12.9, MA neurotransmitters are taken into the vesicles by an exchange of H + for the MA. In NE cells DA is taken up and converted to NE by D β H. As described above in the synthesis section, D β H hydroxylates the amino side chain. The uptake of MA neurotransmitters into storage vesicles is inhibited by the drug reserpine.

    An antiporter that exchanges protons for monoamines (MA) mediates storage of monoamines in dense core vesicles. Left: In NE cells, DA is taken up then converted to NE within the vesicle by the enzyme DBH. Right: All other monoamine cells merely store the MA neurotransmitters.

    12.13 Release of Monoamines

    Figure 12.10
    MA release and interaction with both presynaptic and postsynaptic receptors.

    Neuronal activation elicits the release of MA neurotransmitters by a calcium-dependent exocytosis, as described in Lecture 10, under Secretory Mechanism. The vesicular contents are released from the nerve terminal into the extracellular space during secretion. Because there is no classic postsynaptic specialization associated with the majority of MA nerve endings, the released MA neurotransmitters diffuse to postsynaptic cells in the vicinity where they stimulate MA receptors (volume neurotransmission).

    MA neurotransmitters also act on the presynaptic cell, as shown in Figure 12.10 to influence their cell biology in a feed back manner. The interaction with the presynaptic receptors (termed autoreceptors) can both stimulate MA biosynthesis and inhibit the further release of neurotransmitter. Both the pre- and postsynaptic MA receptors are G protein linked, seven trans-membrane receptors. Their structure is similar to the muscarinic receptors discussed in the Lecture 11, Cholinergic Neurotransmission.

    12.14 Properties of Monoamine Receptors

    The vast majority of the MA receptors are seven transmembrane, G-protein coupled receptors (GPCR) that mediate MA action through one of a few mechanisms. These are the same mechanisms employed by other GPCR, such as the muscarinic receptors (Chapter 12, Part 5) and GPC-glutamate receptors (Chapter 13, Part 3). These mechanisms are:

    1. Stimulation or inhibition of adenylyl cyclase (Click here to see mechanism),
    2. Stimulation of PLC β or PLA (Click here to see mechanism), and
    3. Direct action on ion channel (Click here to see mechanism).

    As will be described below, one type of MA receptor, 5-HT3, is unusual in that it is NOT LINKED TO G PROTEIN LINKED RECEPTORS. Instead, 5-HT3 receptors are ligand gated ion channels, similar in structure and function to ionotropic nicotinic cholinergic receptors and glutamate receptors.

    The receptors for NE and E were originally classified based on the observation that some physiological actions were mimicked by the catecholamine analog, isoproterenol, whereas others were not. This observation led to the convention that actions that could be mimicked by isoproterenol were classified as mediated by β -receptors. Those actions that were not mimicked by isoproterenol were classified as mediated by α -receptors.

    12.16 Relationship Between Peripheral NE and E Receptor Type, Location and Effector Mechanism

    This classification has since been extended to include subclasses of α and β receptors based on the capacity of drugs to selectively activate (or block) specific physiological responses to NE and E. The molecular cloning of mRNAs for distinct subclasses of NE and E receptors also aided in the classification of receptors. Tables I, II and III summarize autonomic and CNS NE and E receptor types, their location and their physiological action. Noteworthy is the fact that most α receptor responses are excitatory, while most β responses are inhibitory (although some exceptions exist, e.g. cardiac muscle). Also, the α receptor is invariably linked to IP3 production, whereas the β receptor is associated with increased levels of cAMP.

    Table I
    Relationship Between Peripheral NE and E Receptor Type, Location, and Effector Mechanism
    Class Location Synaptic Action Linked to:
    α Uterine muscle Contraction IP3 production
    α Blood vessels Constriction IP3 production
    α Bladder Contraction IP3 production
    α Spleen Contraction IP3 production
    α Iris Pupil dilation ?
    β 1 Heart Increased rate and force of contraction Increased cAMP
    β 2 Blood vessels Relaxation Increased cAMP
    β 2 Bronchial muscle Relaxation Increased cAMP
    β 2 Bladder Relaxation Increased cAMP
    β 2 Spleen Relaxation Increased cAMP
    β 3 Fat cells Lipolysis Increased cAMP

    12.17 Relationship Between CNS NE Receptor Type and Effector Mechanism

    The distribution of NE receptors in the CNS is complex and not yet well resolved. Generally, both α and β receptors are believed to be modulators of the actions of other neurotransmitters. As summarized in Table II, α 1 receptors are often excitatory, acting via IP3. In contrast, α 2 receptors are inhibitory acting via decreased levels of cAMP. β receptors are inhibitory and act through increased levels of cAMP (TABLE II). The anatomical location of the specific receptor subtypes is not yet clearly delineated.

    Table II
    Relationship Between CNS NE Receptor Type and Effector Mechanism
    Class Synaptic Action Signaling Mechanism
    α 1 Slow depolarization IP3 production
    α 2 Slow hyperpolarization Decreased cAMP
    β 1 Decreased excitability Increased cAMP
    β 2 Decreased excitability Increased cAMP

    In the CNS, dopamine receptors, designated by the letter D, are grouped into two large families based on cDNA-derived structural similarities, synaptic action and signaling mechanism (TABLE III). The D1 family (D1 and D5) increases cAMP level, and has a positive influence on the excitability of its target cell. The D2 family (D2, D3, and D4) decreases cAMP level and decreases the excitability of the target cell. As shown in Table III the two families of receptors appear to have similar anatomical distributions. However, this may be misleading. Future research will probably show that the location of the receptors is on distinct postsynaptic cells or on presynaptic versus postsynaptic sites.

    12.19 Relationship Between CNS Dopamine Receptor Type, Location, and Effector Mechanism

    Table III
    Class Location Synaptic Action Signaling Mechanism
    D1 family
    (D1, D5)
    Caudate -putamen, nucleus accumbens, olfactory tubercles, hippocampus, hypothalamus Increased excitability Increased cAMP
    D2 family
    (D2, D3, D4)
    Caudate -putamen, nucleus accumbens, olfactory tubercles, frontal cortex, diencephalon, brain stem Decreased excitability Decreased cAMP

    All but one of the 5-HT receptors belongs to the G protein coupled receptor superfamily. The one exception is the 5-HT3 receptor, which is a ligand gated ion channel. As is apparent from the summary in Table IV, 5-HT mediated actions occur through the same types of second messenger mechanisms as cholinergic and catecholamine G protein linked receptors.

    Two classes of 5-HT receptors, 5-HT1B and 5-HT1D, appear to predominantly act as autoreceptors to modulate the synthesis and release of 5-HT from the presynaptic terminal of serotonergic neurons. Other receptor types lead to an increase in the excitability of the target cell (5-HT2 and 5-HT4), while still others (5-HT1) decrease excitability. Interestingly, receptors that mediate increased excitability do so through at least three mechanisms, PLC β stimulation, stimulation of adenylyl cyclase or the direct interaction of 5-HT with the ion channel to depolarize the membrane.

    12.21 Relationship Between CNS 5-HT Receptor Type and Effector Mechanism

    Table IV
    Class Receptor Type Synaptic Action Signaling Mechanism
    5-HT1A G protein linked
    Decreased excitability (increased K + conductance)
    1) Decreased cAMP
    2) direct K + channel opening by G proteins
    5-HT1B G protein linked Autoreceptor-mediated decreased 5-HT release Decreased cAMP
    5-HT1E 5-HT1F G protein linked ? Decreased cAMP
    5-HT1D G protein linked Autoreceptor-mediated decreased 5-HT release Decreased cAMP
    5-HT2 G protein linked Increased excitability
    (decreased K + conductance)
    IP3 production
    5-HT4 G protein linked Increased excitability
    (decreased K + conductance)
    Increased cAMP followed by phosphorylation of K + channels
    5-HT3 Ligand gated pentameric cation channel Ligand gated pentameric cation channel Rapid depolarization Increased Na + , K + and Ca 2+ conductance

    Three subtypes of histamine receptors have been identified. All three are G protein linked and all three are present in the CNS as well as the periphery. Thus far, only peripheral H receptors have been characterized (See Table V).

    12.23 Relationship Between CNS and Peripheral Histamine Receptor Type, Location and Effector Mechanism

    H1 receptors mediate the well-known physiological responses to histamine that occur in response to histamine liberation from mast cells. A large number of prescription and over the counter drugs, antihistamines, act by blocking H1 receptors. Because most H1 blockers also have a sedative effect and cause drowsiness, it appears likely that H1 receptors are also present in the CNS. Recently developed H1 blockers that do not cross that blood brain barrier have circumvented the sedative problem.

    The mechanism of action of H1 receptors is the activation of PLC β

    H2 receptors are responsible for the peripheral actions of histamine that are not blocked by H1 antagonists. These receptors are coupled to stimulation of cAMP and are responsible for histamine's stimulation of gastric acid secretion. Recently developed specific H2 receptor blockers, Tagamet and Zantac, are effective clinically for excess secretion of gastric acid. Because these drugs do not cross the blood brain barrier, they have no effects on the CNS.

    H3 histamine receptors are found on histamine nerve terminals where they regulate the release of histamine. There is evidence for these receptors on the terminals of other neurotransmitter types as well, indicating that histamine may regulate the synthesis and secretion of other neurotransmitters. When presynaptic receptors are located on cells other than their own neurotransmitter type they are called heteroreceptors.

    1. Peripherally - contraction of smooth muscle, fluid secretion from respiratory passage cells, increased release of catecholamines from adrenal medulla
    2. CNS, wide spread, especially hypothalamus actions unknown
    1. Peripherally - contraction of smooth muscle and gastric acid secretion
    2. CNS, wide spread, especially the striatum, actions unknown
    1. CNS, wide spread on nerve endings - mediates decreased neurotransmitter release

    12.24 Inactivation of MA Neurotransmitters by Reuptake and Metabolism

    The major mechanism for the inactivation of secreted MA is the reuptake into the nerve terminal from which the MA was released. Under conditions of very high neuronal activity, the MA will also be taken up by neighboring glial cells and will overflow into the capillaries perfusing the CNS. Under all three situations, a portion of the MA will be metabolized by enzymes that inactivate the MA, converting them to inactive products. As described below, measurement of these metabolites is used clinically and in research to monitor the activity of the MA systems.

    12.25 Reuptake of MA Neurotransmitters

    High affinity transport (reuptake) into axon terminals is the main process of inactivation of released monoamines. Reuptake requires sodium ions and a source of energy (e.g., ATP) and is mediated by a protein carrier located on the plasma membrane of the monamine neurons. Tricyclic antidepressants and cocaine inhibit the transporters for DA, NE and 5-HT. Within the past ten years the structure of several MA transporters has been determined and shown to consist of a twelve transmembrane protein with both the N and C terminal ends residing within the cytoplasm (Figure 12.11). The powerful addictive drugs cocaine and amphetamine increase the level of MA neurotransmitters in the extracellular space. Cocaine acts by blocking the transport of MA (Figure 12.11) neurotransmitters into the terminal and as a consequence increases MA in the extracellular space. In contrast, amphetamine reverses the transport direction (Figure 12.11), transporting MA neurotransmitters out of the nerve terminal.

    Figure 12.11
    Reuptake of MA neurotransmitters by a transporter with a twelve transmembrane structure.

    A low affinity uptake of monoamines into surrounding glial cells also inactivates released monoamines. Because this process acts only at very high concentrations of monoamines, it is believed to only come into play when the concentration of released neurotransmitter is very high.

    A portion of released catecholamines diffuse to the extracellular space where monoamine oxidase (MAO) and/or catechol-0-methyl-transferase (COMT) eventually catabolize it. This route of inactivation is more prominent following extremely high levels of catecholaminergic neuronal activity.

    12.26 Metabolism of MA Neurotransmitters

    Catecholamines and 5-HT : The enzymatic metabolism of MA neurotransmitters is carried out by MAO, COMT and histamine methyl transferase. These enzymes are widely distributed in tissues.

    Monoamine Oxidase (MAO) : This metabolic enzyme is located on the outer membrane of the mitochondrion and metabolizes DA, NE and 5-HT by oxidative deamination of (see Figure 12.12) to the corresponding aldehyde (DHPA, DHPGA and 5HIAA, respectively). DHPA and PHPGA are aldehyde intermediates that must be further metabolized by aldehyde reductase or dehydrogenase to alcohols and acids, respectively. These metabolites are excreted (see Table VI below), or further metabolized by methylation through the action of catechol-O-methyltransferase and then excreted (see below). Pargyline, an irreversible inhibitor of MAO, blocks monoamine degradation.


    S. miltiorrhiza for the Treatment of Clinical Cardiovascular Diseases

    TsIIA is currently used in China for the treatment of patients with CHD and ischemic stroke, but TsIIA is not easily absorbed by the intestinal pathway, and then STS injection has been developed to improve the bioavailability of the herbal medicine (Yu et al., 2018). Danshen, Danhong, salvianolate, STS injections, and other SM preparations are widely used in China to treat stable AP (SAP) caused by CHD (Zhang et al., 2018a). Salvianolate injections are composed of water-soluble extract of SM (Han et al., 2011 Li et al., 2019). Danhong injection is a modern patented Chinese medicine extracted from SM and Flos Carthami (Zou et al., 2018 Feng et al., 2019). It was approved by the China Food and Drug Administration (FDA) in 2002 (Zou et al., 2018). Compound Danshen dripping pills (CDDP) are a modern Chinese medicine preparation consisting of SM, Panax notoginseng, and borneol (Jia et al., 2018). It is the first TCM approved by the American FDA for the treatment of CVDs in Phase II clinical trials (Luo et al., 2013 Zhang et al., 2018b). Therefore, the clinical preparations of SM are mainly divided into three categories: simple monomer preparation, such as STS injection water-soluble complex, such as salvianolate and Danshen injection and compound preparation of SM, such as CDDP also the form of SM preparation includes tablets, injections, capsules, formulations, and drop pills (Liu et al., 2007). This article summarizes the scientific literature that reported the effects of SM on clinical CVDs like CHD, hyperlipidemia, and hypertension (Table 3).

    Table 3 Clinical trials of S. miltiorrhiza preparations for controlling cardiovascular diseases.

    SAP and UAP

    AP can be divided into SAP and UAP. UAP is a common coronary syndrome between SAP and acute MI, which can easily lead to MI or sudden death (Tan et al., 2018). Chronic SAP accounts for about 50% of all patients with coronary artery disease (CAD) (Chen et al., 2017a). Symptoms of chronic SAP are highly associated with the development of atherosclerotic plaque, which blocks at least one large epicardial coronary artery and triggers an imbalance between myocardial oxygen supply and demand (Chen et al., 2017a).

    In a randomized, single-blinded, placebo-controlled, adaptive clinical trial, 156 patients with SAP were randomized into either the placebo (glucose) group or the SM extract (salvianolate injection and Danshen drop pills) group in a 1:1 ratio (Chen et al., 2017a). Participants were treated with glucose or salvianolate injection (200 mg/250 ml 0.9% saline injection, IV drip, qd) for 10 days during hospitalization, followed by the open-label Danshen drop spill (30 pills/day) in the SM extract group for 60 days after discharge (Chen et al., 2017a). Using assessment tools, including the Seattle Angina Questionnaire (SAQ), frequency of AP, angina grade, consumption of short-acting nitrates, and so forth, it was demonstrated that SM extract is beneficial for SAP (Chen et al., 2017a). In addition, previous study reported that five SM-based preparations were effective in the treatment of SAP with clinical improvement rate of 72.4% to 91.6% and electrocardiogram (ECG) improvement rate of 54.5% to 71.6% (Zhang et al., 2018a). The order of five SM-based preparations was as follows: Danhong injection > salvianolate injection > STS injection > Danshen injection of bioactive compounds > Danshen injection (Zhang et al., 2018a).

    In another randomized controlled trial (RCT), 100 UAP patients were randomized into two groups that received STS injection (60 mg/250 ml 0.9% sodium chloride injection, qd, 4 weeks) combined with a loading dose of 300 mg aspirin and a maintenance dose of 100 mg of aspirin plus baseline therapy, or 250 ml 0.9% sodium chloride injection (qd, 4 weeks) combined with the same doses of aspirin and baseline therapy (Yan et al., 2009). The severity of AP ameliorated in 94 patients who completed the treatment, with a significant amelioration in total effective rate in the trial groups (Yan et al., 2009). STS can significantly reduce the AP attacks in patients with UAP, which may be associated with decreased levels of fibrinogen (FIB) (Yan et al., 2009). Moreover, in 17 RCTs involving 1,372 patients, the meta-analysis showed that the combination of STS injection and Western medicine for the treatment of UAP significantly improved the total effective rate and the total effective rate of ECG and reduced the level of CRP, FIB, and whole blood high shear viscosity (Tan et al., 2018). In 22 RCTs involving 2,050 patients, the meta-analysis showed that combination of salvianolate injection and Western medicine in the treatment of UAP improved the total effective rate and the total effective rate of ECG, and increased the serum NO lever (Zhang et al., 2016). Therefore, the combined use of STS injection and salvianolate injection was more effective than Western medicine (Zhang et al., 2016 Tan et al., 2018).

    Myocardial Infarction

    MI, also known as acute MI (AMI), is the most severe manifestation of CAD, which causes more than 4 million deaths in northern Asia and Europe, and more than 2 to 4 million deaths in the United States (Nichols et al., 2014 Gao et al., 2017 Wang et al., 2018b). Atherosclerotic plaque rupture is the cause of approximately 70% MI (Benjamin et al., 2017). Patients who survive from AMI may subsequently suffer HF, manifested as fibrotic scar tissue, thinning of the ventricular wall, and reduced systolic function (Opie et al., 2006 Wang et al., 2018b).

    Fifty-two patients with non-ST elevation MI (NSTEMI) undergoing PCI were randomized into two groups that received the conventional therapy (n = 26) or the conventional therapy plus SM (n = 26, 1 g each time, three times per day for 1 month after PCI) (Zhang et al., 2014b). Elevated levels of asymmetric dimethylarginine (ADMA) in serum are associated with cardiovascular events and are one of the important biomarkers for predicting adverse events and patient mortality after PCI (Lu et al., 2003 Derkacz et al., 2011). The plasma ADMA level in the two groups was significantly decreased at day 30 after PCI with statistical difference, but the reduction in the SM treatment group was more obvious (Zhang et al., 2014b). The improvement of prognosis after the application of SM in patients with PCI may be related to the negative regulation of ADMA by SM (Zhang et al., 2014b). One hundred eight patients with AMI undergoing PCI were randomized into two groups that received the routine treatment (n = 46) or the routine treatment plus intravenous infusion of salvianolate injection (n = 62, 200 mg administered once at 24 h before surgery, once a day after surgery, 1 week) (Zhang et al., 2017a). The changes of oxidative stress indexes, hemodynamic indexes, cardiac function indexes, and related biochemical indicators were analyzed in the two groups at 24 h before surgery and the 8th day after surgery (Zhang et al., 2017a). It was found that salvianolate injection can effectively improve oxidative stress, enhance myocardial perfusion volume, and promote cardiac function recovery in the perioperative period of PCI (Zhang et al., 2017a). In a double-blind RCT, 35 patients with STEMI undergoing PCI were eligible for qi-yin deficiency syndrome, and blood stasis syndromes were randomized into two groups that received Western medicine (n = 18) or Western medicine plus American ginseng and SM preparations (n = 17) for 3 months (Qiu et al., 2009). At the state of dobutamine stress, the left ventricular ejection fraction (LVEF) in the treatment group was higher than that in the control group, and the symptoms of TCM were improved (Qiu et al., 2009). Therefore, TCM treatment can improve the clinical symptoms and quality of life of AMI patients undergoing PCI, and is conducive to myocardial microcirculation (Qiu et al., 2009). A statistical study has shown that the mortality of SM preparation plus conventional care AMI patients is approximately halved compared to conventional care alone (Peto odds ratio, 0.46 95% confidence interval, 0.28𠄰.75) (Wu et al., 2008).

    Hypertension

    Hypertension is a complex disease involving multiple organ systems, a primary modifiable risk factor for heart disease (Ramirez and Sullivan, 2018), and one of the most common non-communicable diseases in the world, with an increasing incidence rate in developing countries (Gupta et al., 2016 Anupama et al., 2017 Miao et al., 2018). Hypertension is often termed the “silent killer” because many hypertensive patients do not know they have the disease before the onset (Ramirez and Sullivan, 2018). Uncontrolled hypertension causes many complications including but not limited to HF, heart attacks, kidney failure, aneurysms, strokes, and dementia (Ramirez and Sullivan, 2018). The other symptoms include aging (Thawornchaisit et al., 2013), overweight or obesity (Shihab et al., 2012 Tsujimoto et al., 2012), dyslipidemia (Laaksonen et al., 2008), resting heart rate (RHR) (Aladin et al., 2016), hyperuricemia (Krishnan et al., 2007), impaired glucose regulation (Morio et al., 2013 Talaei et al., 2014), and estimated glomerular filtration rate (eGFR) (Takase et al., 2012) these are considered independent risk factors for the development of hypertension (Huang et al., 2018a).

    In a double-blind, placebo-controlled, randomized, single-center clinical trial, 55 patients with uncontrolled mild to moderate dose for hypertension were randomized into two groups that received Fufang Danshen capsule (formula mixture, 1,000 mg, twice daily, n = 30) or placebo capsules (n = 25) for 12 weeks (Yang et al., 2012b). The results showed that the Fufang Danshen extract had reduced systolic blood pressure and pulse rate also it was found that it was well tolerated in patients with hypertension, and no significant difference in adverse effects between the two groups was found (Yang et al., 2012b).

    Pulmonary Heart Disease

    Cor pulmonale [pulmonary heart disease (PHD)] is a chronic progressive complicated disease that requires continuous treatment and imposes a huge financial burden on individuals and society (Liu et al., 2014a). PHD is defined as right ventricular failure secondary to pulmonary hypertension (PH), which is mainly caused by various lung diseases, such as chronic obstructive pulmonary disease (COPD) or pulmonary vascular disease (Han et al., 2007 Weitzenblum and Chaouat, 2009 Huang et al., 2018b). PH caused by respiratory system diseases and/or chronic hypoxemia is the main pathological mechanism of chronic PHD (Shujaat et al., 2007 Shi et al., 2015). Antibiotics, diuretics, oxygen therapy, vasodilators, and anticoagulants are currently used medicines for the treatment of PHD also, some studies have shown that the safety and effectiveness of TCM combined with conventional treatment is useful in the treatment of these diseases (Shi et al., 2015).

    The results of many clinical trials have indicated that SM and compound Danshen injection may be alternatives to PHD (Liu et al., 2014a). A systematic review of the efficacy and safety of SM and compound Danshen injection in PHD patients involved 2,715 patients identified in 35 RCTs (Liu et al., 2014a). Meta-analysis used I 2 test for heterogeneity, and randomized or fixed models were selected based on the heterogeneity of the included studies (Liu et al., 2014a). SM and compound Danshen injection have reached favorable conclusions in reducing blood viscosity, plasma viscosity, hematocrit, and mean pulmonary artery pressure (mPAP) by improving blood partial pressure of oxygen (PaO2) (Liu et al., 2014a). In a study enrolled in five hospitalized inpatients, these patients were suffering from various types of serious PH and did not receive the sufficient benefits from sildenafil treatment for at least 3 months (Wang et al., 2013b). After 8 weeks of STS infusion, the patient’s exercise capacity improved, and the Borg dyspnea score was significantly reduced, demonstrating that STS alone or in combination with sildenafil for PH treatment showed significant effect (Wang et al., 2013b). In an RCT, 20 children with congenital heart defects and PH were randomly assigned to two groups that received placebo (n = 10) or SM (200 mg/kg, IV, after anesthesia induction, and at the time of rewarming, n = 10) before cardiac surgery (Xia et al., 2003). The outcome has indicated that SM helps to reduce the ET-1 response and is associated with increased hemodynamic stability after surgery, thereby exerting potent antioxidant therapeutic effect (Xia et al., 2003). Moreover, another clinical trial demonstrated that SM can significantly attenuate lipid peroxide reaction, regulate the imbalance of three antioxidant enzymes [RBC superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT)], and enhance the body’s defense ability against free radical-induced lipid peroxidation damage (Zhang and Chen, 1994).

    Hyperlipidemia

    Hyperlipidemia is a common disease caused by abnormal blood lipid metabolism, which is considered to be a highly independent risk factor for atherosclerotic cardiovascular and cerebrovascular diseases, such as CHD and stroke (Shenghua et al., 2018). Hyperlipidemia is the result of complex interactions between genetic and environmental factors, which can be treated by altering the diet and drugs that regulate lipid metabolism through many mechanisms (Chu et al., 2015). More than 50 TCM formulas have been used to treat hyperlipidemia, of which SM is thought to be beneficial to patients primarily by improving cardiovascular function (Xie et al., 2012). In an RCT, 81 hyperlipidemia patients with phlegm and blood stasis syndrome were randomized into two groups that received CDDP (n = 40) or simvastatin (n = 41) for 3 months (Zhang et al., 2007). The results of this study have shown that CDDP has the effective action for lowering the blood lipid levels without impairing liver function, and its protective liver function may be related to its role in improving antioxidant and reducing inflammation (Zhang et al., 2007).


    No ethical approval was required for this manuscript as this study did not involve human subjects or laboratory animals.

    Zhenzhen Han is a doctor at First Teaching Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin, China. She is also a researcher at National Clinical Research Center for Chinese Medicine Acupuncture and Moxibustion.

    Yang Zhang is a doctor of Tianjin Hospital of Integrated Traditional Chinese and Western Medicine, Tianjin, China.

    Pengqian Wang is a researcher at Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China.

    Qilin Tang is a researcher at Hebei University of Chinese Medicine, Hebei, China. Her main interests lie in creating and analyzing network data and figures constructed.

    Professor Kai Zhang is a researcher and doctor at Tianjin Gong An Hospital, Tianjin, China. His research is focused on traditional Chinese medicine, basic and clinical research of acupuncture, evidence-based medicine and bioinformatics.


    The Pathogenesis, Prevention, and Treatment of Atherosclerosis

    Atherosclerosis remains the major cause of death and premature disability in developed societies. Moreover, current predictions estimate that by the year 2020 cardiovascular diseases, notably atherosclerosis, will become the leading global cause of total disease burden. Although many generalized or systemic risk factors predispose to its development, atherosclerosis affects various regions of the circulation preferentially and has distinct clinical manifestations that depend on the particular circulatory bed affected. Atherosclerosis of the coronary arteries commonly causes myocardial infarction (MI) (Chap. 35) and angina pectoris (Chap. 33). Atherosclerosis of the arteries supplying the central nervous system frequently provokes strokes and transient cerebral ischemia. In the peripheral circulation, atherosclerosis causes intermittent claudication and gangrene and can jeopardize limb viability. Involvement of the splanchnic circulation can cause mesenteric ischemia. Atherosclerosis can affect the kidneys either directly (e.g., renal artery stenosis) or as a common site of atheroembolic disease (Chap. 38).

    Even within a particular arterial bed, stenoses due to atherosclerosis tend to occur focally, typically in certain predisposed regions. In the coronary circulation, for example, the proximal left anterior descending coronary artery exhibits a particular predilection for developing atherosclerotic disease. Similarly, atherosclerosis preferentially affects the proximal portions of the renal arteries and, in the extracranial circulation to the brain, the carotid bifurcation. Indeed, atherosclerotic lesions often form at branching points of arteries which are regions of disturbed blood flow. Not all manifestations of atherosclerosis result from stenotic, occlusive disease. Ectasia and the development of aneurysmal disease, for example, frequently occur in the aorta (Chap. 38). In addition to focal, flow-limiting stenoses, nonocclusive intimal atherosclerosis also occurs diffusely in affected arteries, as shown by intravascular ultrasound and postmortem studies.

    Atherogenesis in humans typically occurs over a period of many years, usually many decades. Growth of atherosclerotic plaques probably does not occur in a smooth, linear fashion but discontinuously, with periods of relative quiescence punctuated by periods of rapid evolution. After a generally prolonged “silent” period, atherosclerosis may become clinically manifest. The clinical expressions of atherosclerosis may be chronic , as in the development of stable, effort-induced angina pectoris or predictable and reproducible intermittent claudication. Alternatively, a dramatic acute clinical event such as MI, stroke, or sudden cardiac death may first herald the presence of atherosclerosis. Other individuals may never experience clinical manifestations of arterial disease despite the presence of widespread atherosclerosis demonstrated postmortem.

    INITIATION OF ATHEROSCLEROSIS

    An integrated view of experimental results in animals and studies of human atherosclerosis suggests that the “fatty streak” represents the initial lesion of atherosclerosis. These early lesions most often seem to arise from focal increases in the content of lipoproteins within regions of the intima. This accumulation of lipoprotein particles may not result simply from increased permeability, or “leakiness,” of the overlying endothelium ( Fig. 30-1 ) . Rather, the lipoproteins may collect in the intima of arteries because they bind to constituents of the extracellular matrix, increasing the residence time of the lipid-rich particles within the arterial wall. Lipoproteins that accumulate in the extracellular space of the intima of arteries often associate with glycosaminoglycans of the arterial extracellular matrix, an interaction that may slow the egress of these lipid-rich particles from the intima. Lipoprotein particles in the extracellular space of the intima, particularly those retained by binding to matrix macromolecules, may undergo oxidative modifications. Considerable evidence supports a pathogenic role for products of oxidized lipoproteins in atherogenesis. Lipoproteins sequestered from plasma antioxidants in the extracellular space of the intima become particularly susceptible to oxidative modification, giving rise to hydroperoxides, lysophospholipids, oxysterols, and aldehydic breakdown products of fatty acids and phospholipids. Modifications of the apoprotein moieties may include breaks in the peptide backbone as well as derivatization of certain amino acid residues. Local production of hypochlorous acid by myeloperoxidase associated with inflammatory cells within the plaque yields chlorinated species such as chlorotyrosyl moieties. High-density lipoprotein (HDL) particles modified by HOCl-mediated chlorination function poorly as cholesterol acceptors, a finding that links oxidative stress with impaired reverse cholesterol transport, which is one likely mechanism of the antiatherogenic action of HDL (see later). Considerable evidence supports the presence of such oxidation products in atherosclerotic lesions. A particular member of the phospholipase family, lipoprotein-associated phospholipase A 2 (LpPL A 2 ), can generate proinflammatory lipids, including lysophosphatidyl choline-bearing oxidized lipid moieties from oxidized phospholipids found in oxidized low-density lipoproteins (LDLs). An inhibitor of this enzyme is in clinical development.

    FIGURE 30-1
    Cross-sectional view of an artery depicting steps in development of an atheroma, from left to right. The upper panel shows a detail of the boxed area below. The endothelial monolayer overlying the intima contacts blood. Hypercholesterolemia promotes accumulation of LDL particles ( light spheres ) in the intima. The lipoprotein particles often associate with constituents of the extracellular matrix, notably proteoglycans. Sequestration within the intima separates lipoproteins from some plasma antioxidants and favors oxidative modification. Such modified lipoprotein particles ( darker spheres ) may trigger a local inflammatory response that signals subsequent steps in lesion formation. The augmented expression of various adhesion molecules for leukocytes recruits monocytes to the site of a nascent arterial lesion.

    Once adherent, some white blood cells migrate into the intima. The directed migration of leukocytes probably depends on chemoattractant factors, including modified lipoprotein particles themselves and chemoattractant cytokines (depicted by the smaller spheres), such as the chemokine macrophage chemoattractant protein-1 produced by vascular wall cells in response to modified lipoproteins. Leukocytes in the evolving fatty streak can divide and exhibit augmented expression of receptors for modified lipoproteins (scavenger receptors). These mononuclear phagocytes ingest lipids and become foam cells, represented by a cytoplasm filled with lipid droplets. As the fatty streak evolves into a more complicated atherosclerotic lesion, smooth-muscle cells migrate from the media ( bottom of lower panel hairline ) through the internal elastic membrane ( solid wavy line ) and accumulate within the expanding intima, where they lay down extracellular matrix that forms the bulk of the advanced lesion ( bottom panel , right side ).

    Leukocyte recruitment

    Accumulation of leukocytes characterizes the formation of early atherosclerotic lesions (Fig. 30-1). Thus, from its very inception, atherogenesis involves elements of inflammation, a process that now provides a unifying theme in the pathogenesis of this disease. The inflammatory cell types typically found in the evolving atheroma include monocyte-derived macrophages and lymphocytes. A number of adhesion molecules or receptors for leukocytes expressed on the surface of the arterial endothelial cell probably participate in the recruitment of leukocytes to the nascent atheroma. Constituents of oxidatively modified low-density lipoprotein can augment the expression of leukocyte adhesion molecules. This example illustrates how the accumulation of lipoproteins in the arterial intima may link mechanistically with leukocyte recruitment, a key event in lesion formation.

    Laminar shear forces such as those encountered in most regions of normal arteries also can suppress the expression of leukocyte adhesion molecules. Sites of predilection for atherosclerotic lesions (e.g., branch points) often have disturbed flow. Ordered, pulsatile laminar shear of normal blood flow augments the production of nitric oxide by endothelial cells. This molecule, in addition to its vasodilator properties, can act at the low levels constitutively produced by arterial endothelium as a local anti-inflammatory autacoid, e.g., limiting local adhesion molecule expression. Exposure of endothelial cells to laminar shear stress increases the transcription of Krüppel-like factor 2 (KLF2) and reduces the expression of a thioredoxin-interacting protein (Txnip) that inhibits the activity of the endogenous antioxidant thioredoxin. KLF2 augments the activity of endothelial nitric oxide synthase, and reduced Txnip levels boost the function of thioredoxin. Laminar shear stress also stimulates endothelial cells to produce super-oxide dismutase, an antioxidant enzyme. These examples indicate how hemodynamic forces may influence the cellular events that underlie atherosclerotic lesion initiation and potentially explain the favored localization of atherosclerotic lesions at sites that experience disturbance to laminar shear stress.

    Once captured on the surface of the arterial endothelial cell by adhesion receptors, the monocytes and lymphocytes penetrate the endothelial layer and take up residence in the intima. In addition to products of modified lipoproteins, cytokines (protein mediators of inflammation) can regulate the expression of adhesion molecules involved in leukocyte recruitment. For example, interleukin 1 (IL-1) or tumor necrosis factor α (TNF-α) induce or augment the expression of leukocyte adhesion molecules on endothelial cells. Because products of lipoprotein oxidation can induce cytokine release from vascular wall cells, this pathway may provide an additional link between arterial accumulation of lipoproteins and leukocyte recruitment. Chemoattractant cytokines such as monocyte chemoattractant protein 1 appear to direct the migration of leukocytes into the arterial wall.

    Foam-cell formation

    Once resident within the intima, the mononuclear phagocytes mature into macrophages and become lipid-laden foam cells, a conversion that requires the uptake of lipoprotein particles by receptor-mediated endocytosis. One might suppose that the well-recognized “classic” receptor for LDL mediates this lipid uptake however, humans or animals lacking effective LDL receptors due to genetic alterations (e.g., familial hypercholesterolemia) have abundant arterial lesions and extraarterial xanthomata rich in macrophage-derived foam cells. In addition, the exogenous cholesterol suppresses expression of the LDL receptor thus, the level of this cell-surface receptor for LDL decreases under conditions of cholesterol excess. Candidates for alternative receptors that can mediate lipid loading of foam cells include a growing number of macrophage “scavenger” receptors, which preferentially endocytose modified lipoproteins, and other receptors for oxidized LDL or very low-density lipoprotein (VLDL). Monocyte attachment to the endothelium, migration into the intima, and maturation to form lipid-laden macrophages thus represent key steps in the formation of the fatty streak, the precursor of fully formed atherosclerotic plaques.

    ATHEROMA EVOLUTION AND COMPLICATIONS

    Although the fatty streak commonly precedes the development of a more advanced atherosclerotic plaque, not all fatty streaks progress to form complex atheromata. By ingesting lipids from the extracellular space, the mono-nuclear phagocytes bearing such scavenger receptors may remove lipoproteins from the developing lesion. Some lipid-laden macrophages may leave the artery wall, exporting lipid in the process. Lipid accumulation, and hence the propensity to form an atheroma, ensues if the amount of lipid entering the artery wall exceeds that removed by mononuclear phagocytes or other pathways.

    Export by phagocytes may constitute one response to local lipid overload in the evolving lesion. Another mechanism, reverse cholesterol transport mediated by high-density lipoproteins, probably provides an independent pathway for lipid removal from atheroma. This transfer of cholesterol from the cell to the HDL particle involves specialized cell-surface molecules such as the ATP binding cassette (ABC) transporters. ABCA1 , the gene mutated in Tangier disease, a condition characterized by very low HDL levels, transfers cholesterol from cells to nascent HDL particles and ABCG1 to mature HDL particles. “Reverse cholesterol transport” mediated by these ABC transporters allows HDL loaded with cholesterol to deliver it to hepatocytes by binding to scavenger receptor B 1 or other receptors. The liver cell can metabolize the sterol to bile acids that can be excreted. This export pathway from macrophage foam cells to peripheral cells such as hepatocytes explains part of the antiatherogenic action of HDLs. (Anti-inflammatory and antioxidant properties also may contribute to the atheroprotective effects of HDLs.) Thus, macrophages may play a vital role in the dynamic economy of lipid accumulation in the arterial wall during atherogenesis.

    Some lipid-laden foam cells within the expanding intimal lesion perish. Some foam cells may die as a result of programmed cell death, or apoptosis . This death of mononuclear phagocytes results in the formation of the lipid-rich center, often called the necrotic core , in established atherosclerotic plaques. Macrophages loaded with modified lipoproteins may elaborate cytokines and growth factors that can further signal some of the cellular events in lesion complication. Whereas accumulation of lipid-laden macrophages characterizes the fatty streak, buildup of fibrous tissue formed by extracellular matrix typifies the more advanced atherosclerotic lesion. The smooth-muscle cell synthesizes the bulk of the extracellular matrix of the complex atherosclerotic lesion. A number of growth factors or cytokines elaborated by mononuclear phagocytes can stimulate smooth-muscle cell proliferation and production of extracellular matrix. Cytokines found in the plaque, including IL-1 and TNF-α, can induce local production of growth factors, including forms of platelet-derived growth factor (PDGF), fibroblast growth factors, and others, which may contribute to plaque evolution and complication. Other cytokines, notably interferon γ (IFN-γ) derived from activated T cells within lesions, can limit the synthesis of interstitial forms of collagen by smooth-muscle cells. These examples illustrate how atherogenesis involves a complex mix of mediators that in the balance determines the characteristics of particular lesions.

    The arrival of smooth-muscle cells and their elaboration of extracellular matrix probably provide a critical transition, yielding a fibrofatty lesion in place of a simple accumulation of macrophage-derived foam cells. For example, PDGF elaborated by activated platelets, macrophages, and endothelial cells can stimulate the migration of smooth-muscle cells normally resident in the tunica media into the intima. Such growth factors and cytokines produced locally can stimulate the proliferation of resident smooth-muscle cells in the intima as well as those that have migrated from the media. Transforming growth factor β (TGF-β), among other mediators, potently stimulates interstitial collagen production by smooth-muscle cells. These mediators may arise not only from neighboring vascular cells or leukocytes (a “paracrine” pathway), but also, in some instances, may arise from the same cell that responds to the factor (an “autocrine” pathway). Together, these alterations in smooth-muscle cells, signaled by these mediators acting at short distances, can hasten transformation of the fatty streak into a more fibrous smooth-muscle cell and extracellular matrix-rich lesion.

    In addition to locally produced mediators, products of blood coagulation and thrombosis likely contribute to atheroma evolution and complication. This involvement justifies the use of the term atherothrombosis to convey the inextricable links between atherosclerosis and thrombosis. Fatty streak formation begins beneath a morphologically intact endothelium. In advanced fatty streaks, however, microscopic breaches in endothelial integrity may occur. Microthrombi rich in platelets can form at such sites of limited endothelial denudation, owing to exposure of the thrombogenic extracellular matrix of the underlying basement membrane. Activated platelets release numerous factors that can promote the fibrotic response, including PDGF and TGF-β. Thrombin not only generates fibrin during coagulation, but also stimulates protease-activated receptors that can signal smooth-muscle migration, proliferation, and extracellular matrix production. Many arterial mural microthrombi resolve without clinical manifestation by a process of local fibrinolysis, resorption, and endothelial repair, yet can lead to lesion progression by stimulating these profibrotic functions of smooth-muscle cells ( Fig. 30-2 D ) .

    FIGURE 30-2
    Plaque rupture, thrombosis, and healing. A. Arterial remodeling during atherogenesis. During the initial part of the life history of an atheroma, growth is often outward, preserving the caliber of the lumen. This phenomenon of “compensatory enlargement” accounts in part for the tendency of coronary arteriography to underestimate the degree of atherosclerosis. B. Rupture of the plaque’s fibrous cap causes thrombosis. Physical disruption of the atherosclerotic plaque commonly causes arterial thrombosis by allowing blood coagulant factors to contact thrombogenic collagen found in the arterial extracellular matrix and tissue factor produced by macrophage-derived foam cells in the lipid core of lesions. In this manner, sites of plaque rupture form the nidus for thrombi. The normal artery wall has several fibrinolytic or antithrombotic mechanisms that tend to resist thrombosis and lyse clots that begin to form in situ. Such antithrombotic or thrombolytic molecules include thrombomodulin, tissue- and urokinase-type plasminogen activators, heparan sulfate proteoglycans, prostacyclin, and nitric oxide. C. When the clot overwhelms the endogenous fibrinolytic mechanisms, it may propagate and lead to arterial occlusion. The consequences of this occlusion depend on the degree of existing collateral vessels. In a patient with chronic multivessel occlusive coronary artery disease (CAD), collateral channels have often formed. In such circumstances, even a total arterial occlusion may not lead to myocardial infarction (MI), or it may produce an unexpectedly modest or a non-ST-segment elevation infarct because of collateral flow. In a patient with less advanced disease and without substantial stenotic lesions to provide a stimulus for collateral vessel formation, sudden plaque rupture and arterial occlusion commonly produces an ST-segment elevation infarction. These are the types of patients who may present with MI or sudden death as a first manifestation of coronary atherosclerosis. In some cases, the thrombus may lyse or organize into a mural thrombus without occluding the vessel. Such instances may be clinically silent. D. The subsequent thrombin-induced fibrosis and healing causes a fibroproliferative response that can lead to a more fibrous lesion that can produce an eccentric plaque that causes a hemodynamically significant stenosis. In this way, a nonocclusive mural thrombus, even if clinically silent or causing unstable angina rather than infarction, can provoke a healing response that can promote lesion fibrosis and luminal encroachment. Such a sequence of events may convert a “vulnerable” atheroma with a thin fibrous cap that is prone to rupture into a more “stable” fibrous plaque with a reinforced cap. Angioplasty of unstable coronary lesions may “stabilize” the lesions by a similar mechanism, producing a wound followed by healing.

    Microvessels

    As atherosclerotic lesions advance, abundant plexuses of microvessels develop in connection with the artery’s vasa vasorum. Newly developing microvascular networks may contribute to lesion complications in several ways. These blood vessels provide an abundant surface area for leukocyte trafficking and may serve as the portal for entry and exit of white blood cells from the established atheroma. Microvessels in the plaques may also furnish foci for intraplaque hemorrhage. Like the neovessels in the diabetic retina, microvessels in the atheroma may be friable and prone to rupture and can produce focal hemorrhage. Such a vascular leak can provoke thrombosis in situ, yielding local thrombin generation, which in turn can activate smooth-muscle and endothelial cells through ligation of protease-activated receptors. Atherosclerotic plaques often contain fibrin and hemosiderin, an indication that episodes of intraplaque hemorrhage contribute to plaque complications.

    Calcification

    As they advance, atherosclerotic plaques also accumulate calcium . Proteins usually found in bone also localize in atherosclerotic lesions (e.g., osteocalcin, osteopontin, and bone morphogenetic proteins). Mineralization of the atherosclerotic plaque recapitulates many aspects of bone formation, including the regulatory participation of transcription factors such as Runx2.

    Plaque evolution

    Although atherosclerosis research has focused much attention on proliferation of smooth-muscle cells, as in the case of macrophages, smooth-muscle cells also can undergo apoptosis in the atherosclerotic plaque. Indeed, complex atheromata often have a mostly fibrous character and lack the cellularity of less advanced lesions. This relative paucity of smooth-muscle cells in advanced atheromata may result from the predominance of cytostatic mediators such as TGF-β and IFN-γ (which can inhibit smooth-muscle cell proliferation), and also from smooth-muscle cell apoptosis. Some of the same proinflammatory cytokines that activate atherogenic functions of vascular wall cells can also sensitize these cells to undergo apoptosis.

    Thus, during the evolution of the atherosclerotic plaque, a complex balance between entry and egress of lipoproteins and leukocytes, cell proliferation and cell death, extracellular matrix production, and remodeling, as well as calcification and neovascularization, contribute to lesion formation. Multiple and often competing signals regulate these various cellular events. Many mediators related to atherogenic risk factors, including those derived from lipoproteins, cigarette smoking, and angiotensin II, provoke the production of proinflammatory cytokines and alter the behavior of the intrinsic vascular wall cells and infiltrating leukocytes that underlie the complex pathogenesis of these lesions. Thus, advances in vascular biology have led to increased understanding of the mechanisms that link risk factors to the pathogenesis of atherosclerosis and its complications.

    CLINICAL SYNDROMES OF ATHEROSCLEROSIS

    Atherosclerotic lesions occur ubiquitously in Western societies. Most atheromata produce no symptoms, and many never cause clinical manifestations. Numerous patients with diffuse atherosclerosis may succumb to unrelated illnesses without ever having experienced a clinically significant manifestation of atherosclerosis. What accounts for this variability in the clinical expression of atherosclerotic disease?

    Arterial remodeling during atheroma formation ( Fig. 30-2 A ) represents a frequently overlooked but clinically important feature of lesion evolution. During the initial phases of atheroma development, the plaque usually grows outward, in an abluminal direction. Vessels affected by atherogenesis tend to increase in diameter, a phenomenon known as compensatory enlargement , a type of vascular remodeling. The growing atheroma does not encroach on the arterial lumen until the burden of atherosclerotic plaque exceeds

    40% of the area encompassed by the internal elastic lamina. Thus, during much of its life history, an atheroma will not cause stenosis that can limit tissue perfusion.

    Flow-limiting stenoses commonly form later in the history of the plaque. Many such plaques cause stable syndromes such as demand-induced angina pectoris or intermittent claudication in the extremities. In the coronary circulation and other circulations, even total vascular occlusion by an atheroma does not invariably lead to infarction. The hypoxic stimulus of repeated bouts of ischemia characteristically induces formation of collateral vessels in the myocardium, mitigating the consequences of an acute occlusion of an epicardial coronary artery. By contrast, many lesions that cause acute or unstable atherosclerotic syndromes, particularly in the coronary circulation, may arise from atherosclerotic plaques that do not produce a flow-limiting stenosis. Such lesions may produce only minimal luminal irregularities on traditional angiograms and often do not meet the traditional criteria for “significance” by arteriography. Thrombi arising from such nonocclusive stenoses may explain the frequency of MI as an initial manifestation of coronary artery disease (CAD) (in at least one-third of cases) in patients who report no prior history of angina pectoris, a syndrome usually caused by flow-limiting stenoses.

    Plaque instability and rupture

    Postmortem studies afford considerable insight into the microanatomic substrate underlying the “instability” of plaques that do not cause critical stenoses. A superficial erosion of the endothelium or a frank plaque rupture or fissure usually produces the thrombus that causes episodes of unstable angina pectoris or the occlusive and relatively persistent thrombus that causes acute MI ( Fig. 30-2 B ) . In the case of carotid atheromata, a deeper ulceration that provides a nidus for the formation of platelet thrombi may cause transient cerebral ischemic attacks.

    Rupture of the plaque’s fibrous cap ( Fig. 30-2 C ) permits contact between coagulation factors in the blood and highly thrombogenic tissue factor expressed by macrophage foam cells in the plaque’s lipid-rich core. If the ensuing thrombus is nonocclusive or transient, the episode of plaque disruption may not cause symptoms or may result in episodic ischemic symptoms such as rest angina. Occlusive thrombi that endure often cause acute MI, particularly in the absence of a well-developed collateral circulation that supplies the affected territory. Repetitive episodes of plaque disruption and healing provide one likely mechanism of transition of the fatty streak to a more complex fibrous lesion (Fig. 30-2 D ). The healing process in arteries, as in skin wounds, involves the laying down of new extracellular matrix and fibrosis.

    Not all atheromata exhibit the same propensity to rupture. Pathologic studies of culprit lesions that have caused acute MI reveal several characteristic features. Plaques that have caused fatal thromboses tend to have thin fibrous caps, relatively large lipid cores, and a high content of macrophages. Morphometric studies of such culprit lesions show that at sites of plaque rupture, macrophages and T lymphocytes predominate and contain relatively few smooth-muscle cells. The cells that concentrate at sites of plaque rupture bear markers of inflammatory activation. In addition, patients with active atherosclerosis and acute coronary syndromes display signs of disseminated inflammation. For example, atherosclerotic plaques and even microvascular endothelial cells at sites remote from the “culprit” lesion of an acute coronary syndrome can exhibit markers of inflammatory activation.

    Inflammatory mediators regulate processes that govern the integrity of the plaque’s fibrous cap and, hence, its propensity to rupture. For example, the T cell-derived cytokine IFN-γ, which is found in atherosclerotic plaques, can inhibit growth and collagen synthesis of smooth-muscle cells, as noted earlier. Cytokines derived from activated macrophages and lesional T cells can boost production of proteolytic enzymes that can degrade the extracellular matrix of the plaque’s fibrous cap. Thus, inflammatory mediators can impair the collagen synthesis required for maintenance and repair of the fibrous cap and trigger degradation of extracellular matrix macromolecules, processes that weaken the plaque’s fibrous cap and enhance its susceptibility to rupture (so-called vulnerable plaques). In contrast to plaques with these features of vulnerability, those with a dense extracellular matrix and relatively thick fibrous cap without substantial tissue factor–rich lipid cores seem generally resistant to rupture and unlikely to provoke thrombosis.

    Features of the biology of the atheromatous plaque, in addition to its degree of luminal encroachment, influence the clinical manifestations of this disease. This enhanced understanding of plaque biology provides insight into the diverse ways in which atherosclerosis can present clinically and the reasons why the disease may remain silent or stable for prolonged periods, punctuated by acute complications at certain times. Increased understanding of atherogenesis provides new insight into the mechanisms linking it to the risk factors discussed later, indicates the ways in which current therapies may improve outcomes, and suggests new targets for future intervention.

    PREVENTION AND TREATMENT

    THE CONCEPT OF ATHEROSCLEROTIC RISK FACTORS

    The systematic study of risk factors for atherosclerosis emerged from a coalescence of experimental results, as well as from cross-sectional and ultimately longitudinal studies in humans. The prospective, community-based Framingham Heart Study provided rigorous support for the concept that hypercholesterolemia, hypertension, and other factors correlate with cardiovascular risk. Similar observational studies performed worldwide bolstered the concept of “risk factors” for cardiovascular disease.

    From a practical viewpoint, the cardiovascular risk factors that have emerged from such studies fall into two categories: those modifiable by lifestyle and/or pharmacotherapy, and those that are immutable, such as age and sex. The weight of evidence supporting various risk factors differs. For example, hypercholesterolemia and hypertension certainly predict coronary risk, but the magnitude of the contributions of other so-called nontraditional risk factors, such as levels of homocysteine, levels of lipoprotein (a) (Lp[a]), and infection, remains controversial. Moreover, some biomarkers that predict cardiovascular risk may not participate in the causal pathway for the disease or its complications. For example, recent genetic studies suggest that C-reactive protein (CRP) does not itself mediate atherogenesis, despite its ability to predict risk. Table 30-1 lists the risk factors recognized by the current National Cholesterol Education Project Adult Treatment Panel III (ATP III). The later sections will consider some of these risk factors and approaches to their modification.

    TABLE 30-1
    MAJOR RISK FACTORS (EXCLUSIVE OF LDL CHOLESTEROL) THAT MODIFY LDL GOALS


    Discussion

    We found that the orally administered CGRP antagonist, telcagepant, had no effect on the haemodynamic response of healthy volunteers to therapeutic doses of sublingual NTG. In addition, no vasoconstrictor effect was seen after a single 500 mg dose of telcagepant compared with placebo. These findings support a more favourable cardiovascular safety and tolerability profile of CGRP receptor antagonists over the triptans 10, 15 .

    Animal research, suggesting a role for CGRP in the vasodilatory effect of NTG, led us to investigate whether telcagepant attenuated NTG-induced vascular changes in healthy subjects 23 . NTG-induced vasodilation is associated with an endogenous CGRP release in both feline cerebral arterioles and rat aortas 17, 37 . These findings in animals suggest that CGRP antagonism might impede on the vasodilation induced by exogenous NO donors such as NTG. In humans, however, there is still much debate surrounding the interaction between NO and CGRP. In the resistance vessels of the human forearm, CGRP8-37, a CGRP receptor antagonist, has no effect on the vasodilation induced by the NO donor sodium nitroprusside 13 . The vasodilator mechanism of CGRP itself on the other hand seems to be partly mediated by the release of NO in the forearm vascular territory but not in the human skin 14, 38 . The interaction between NO and CGRP is thus different between vascular territories and between species. This clearly illustrates the need for sensitive techniques by which the drug-induced vascular effects of new compounds can be assessed at an early stage of clinical development.

    In this respect, pulse wave analyses and brachial diameter measurements present very suitable techniques. They provide a non-invasive and more sensitive assessment of drug-induced haemodynamic changes than those obtained by more conventional oscillometric blood pressure measurements 25, 26 . In addition, the effect of NTG on these parameters is well known. Oliver et al. conducted a study in which they established the dose-relationship of sublingual NTG to changes in BAD and radial and aortic AIx in healthy men 28 . The change in heart rate, brachial diastolic blood pressure, aortic and radial AIx and BAD following 0.4 mg of NTG in our study are largely in agreement with their data.

    Given the results, we conclude that telcagepant does not have a clinically significant effect on the decrease in aortic AIx following a single therapeutic dose of NTG in healthy men. Importantly, these data support the conclusion that NTG remains an effective treatment for ischaemia on the background of telcagepant therapy. This is crucial as one of the most important mechanisms of actions of NTG to relieve angina is to reduce the afterload of the heart, which is adequately reflected by the decrease in the aortic AIx 39 . More generally, this finding indicates that treatment with a potent CGRP-receptor antagonist does not interfere with the pharmacological effect of NTG, thus discarding a role for CGRP in NTG-induced vasodilation.

    The analysis of the BAD permits an online assessment of the vasoactive effects of a drug on the brachial artery 25 . Whether the effect of a drug on the BAD reflects coronary artery behaviour has never been determined and would require simultaneous acquisition of both peripheral arterial ultrasound measurements and invasive coronary angiography. It is nevertheless of interest that previous studies have linked impairment of brachial artery response to NTG with coronary artery disease, suggesting similarities between both vascular territories 40 . The results obtained in the present study indicate that telcagepant has no significant effect on brachial vasodilation induced by a single dose of NTG in healthy men.

    It was recently demonstrated, both in a dose finding phase II study and in a confirmatory phase III study, that a 300 mg dose of telcagepant has an efficacy comparable with that of the triptans for the treatment of acute migraine headache 10, 41 . The anticipated clinical dose of telcagepant (relative to the formulation used in this study) is a single 300 mg dose, with an optional second 300 mg dose 2 h later, if needed. A single 500 mg dose, as used in this study, achieves pharmacokinetic exposures similar to 2 × 300 mg doses, administered 2 h apart.

    We are confident that the single 500 mg dose of telcagepant adequately blocks the peripheral CGRP receptor in healthy men based on a previous study conducted by our group in which we used topical capsaicin applications to elicit CGRP release in human skin and assessed the increase in dermal blood flow (DBF) using laser Doppler. Telcagepant inhibited the increase in DBF following capsaicin application and the subsequent analysis of the pharmacokinetic/pharmacodynamic relationship suggested that telcagepant engages the CGRP receptor with an EC90 of approximately 900 n m 42 . The concentration–response curve above 900 nM was relatively flat indicating that at or above this plasma concentration, telcagepant is maximally blocking the peripheral CGRP receptor in healthy men. In the capsaicin study, an early formulation of telcagepant was used that had lower bioavailability than the formulation used in this trial. The plasma concentrations achieved in this study are estimated to be approximately two- to four-fold higher than 900 n m and we are therefore confident that adequate blockade of the peripheral CGRP receptor was achieved 10, 43 .

    Despite the use of a high clinical dose in the present study (i.e. 500 mg), telcagepant alone (prior to NTG treatment) did not exert any unwanted cardiovascular effects either on the peripheral or on the central circulation of healthy men. The radial AIx was marginally lower when telcagepant was administered compared with placebo (P= 0.040), which results mostly from an unexplained difference in the pre-dose values and therefore merely reflects normal variability of the measurement. Our observations are in agreement with the findings of Petersen et al. who reported the absence of effect of olcegepant (a distinct CGRP receptor antagonist) on cerebral blood flow, temporal and radial artery diameter and oscillometric blood pressure measurements 15 . These data suggest that although CGRP circulates under measurable concentrations in the blood in basal conditions, it does not exert a homeostatic vasodilator activity in humans under resting conditions. As a consequence, a CGRP receptor antagonist, in contrast to the triptans, seems devoid of vasoconstrictor effects under resting conditions. In addition, in all recent studies telcagepant has been generally well tolerated and appears to have a profile consistent with safe use in patients with cardiovascular disease 10, 43-46 .

    A number of limitations of the present study need to be taken into account. First, the vascular effects of telcagepant were investigated in healthy men. Though CGRP has been demonstrated to play a role in ischaemic preconditioning in rats and is believed to mediate the cardioprotective effects of NTG-induced preconditioning in rabbits, there was no evidence of a major role of CGRP in regulating ischaemic blood flow in dogs 47-49 . Because of these differences between species and the lack of human studies, additional studies may be useful in patients with known cardiovascular disease 23 . In a first study in patients with stable cardiovascular disease, telcagepant did not appear to exacerbate spontaneous ischaemia 50 . Nevertheless, this study was performed under standardized resting conditions, whereas the effect of CGRP antagonism in cardiovascular patients might be rather different during normal daily activities or exercise. Second, central pressure effects were not measured invasively but were estimated by use of a generalized transfer function. This transfer function has received some criticism and has not been convincingly validated in young healthy subjects 39 . In addition calibrating the peripheral pressure wave form by use of brachial SBP and DBP might further flaw the prediction of aortic blood pressures 32 . However, in this study the use of radial AIx and aortic AIx yield the same conclusions, confirming that the untransformed radial pressure wave form provides similar information on pressure wave augmentation as the aortic PWF does. Third, the present study was not performed in migraine patients, the future patient population of telcagepant. This might be relevant as migraine patients are known to have alterations in arterial function when compared with healthy volunteers 51 .

    In summary, this study shows that telcagepant, at a high clinical dose for the treatment of migraine headache, had no clinically relevant effect on NTG-induced haemodynamic changes in healthy male volunteers. There was also no measurable vasoconstrictor effect of telcagepant as such in both the central and peripheral vascular bed. These results support the favourable cardiovascular safety profile of CGRP-receptor antagonists and indicate that CGRP is not involved in NTG-induced vasodilation in humans.


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