Endothelial nitric oxide synthase Asp298Glu (894G/T) gene polymorphism as a possible risk factor for the coronary slow flow phenomenon among Iranians.

BACKGROUND: Mounting evidence indicates an association between endothelial dysfunction and the coronary slow flow phenomenon (CSFP). In the present study, we aimed to evaluate the possible role of endothelial nitric oxide synthase (eNOS) 894G/T and interleukin-1ß (IL-1ß) 315C/T polymorphisms as possible risk factors for CSFP. METHODS: This prospective study enrolled patients with CSFP and individuals with normal coronary arteries. Genotypes were assessed using regular polymerase chain reaction and direct Sanger-sequencing techniques. RESULTS: The study population consisted of 267 individuals: 180 patients with CSFP (49 women [27.2%]) at a median age of 55 (48-62) years and 87 controls with normal coronary arteries (56 women [64.4%]) at a median age of 47 (41-58) years. The allelic distribution of eNOS 894G/T was significantly associated with CSFP (odds ratio [OR], 1.58; 95% confidence interval (CI), 1.04-2.42; P = 0.03). This polymorphism increased the risk of CSFP under the dominant model (OR 1.73; 95% CI I.02-2.95; P = 0.04). However, the allelic frequencies (1.05; 95% CI 0.68-1.59; P = 0.83) and genotypic frequencies (0.88; 95% CI 0.52-1.49; P = 0.63) of the IL-1ß 315C/T polymorphism were not associated with the incidence of CSFP in the Iranian population. CONCLUSIONS: The CSFP and control groups were statistically different regarding the eNOS 894G/T polymorphism. Our findings also demonstrated that the IL-1ß 315C/T polymorphism was not a risk factor for CSFP.

Tongmai Yangxin pill reduces myocardial No-reflow via endothelium-dependent NO-cGMP signaling by activation of the cAMP/PKA pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: The Tongmai Yangxin pill (TMYX) is derived from the Zhigancao decoction recorded in Shang han lun by Zhang Zhongjing during the Han dynasty. TMYX is used for the clinical treatment of chest pain, heartache, and qi-yin-deficiency coronary heart disease. Previous studies have confirmed that TMYX can improve vascular endothelial function in patients with coronary heart disease by upregulating nitric oxide activity and then regulating vascular tension. Whether TMYX can further improve myocardial NR by upregulating NO activity and then dilating blood vessels remains unclear. AIM OF THE STUDY: This study aimed to reveal whether TMYX can further improve myocardial NR by upregulating NO activity and then dilating blood vessels. The underlying cAMP/PKA and NO-cGMP signaling pathway-dependent mechanism is also explored. MATERIALS AND METHODS: The left anterior descending coronary arteries of healthy adult male SD rats were ligated to establish the NR model. TMYX (4.0 g/kg) was orally administered throughout the experiment. Cardiac function was measured through echocardiography. Thioflavin S, Evans Blue, and TTC staining were used to evaluate the NR and ischemic areas. Pathological changes in the myocardium were assessed by hematoxylin-eosin staining. An automated biochemical analyzer and kit were used to detect the activities of myocardial enzymes and myocardial oxidants, including CK, CK-MB, LDH, reactive oxygen species, superoxide dismutase, malonaldehyde, and NO. The expression levels of genes and proteins related to the cAMP/PKA and NO/cGMP signaling pathways were detected via real-time fluorescence quantitative PCR and Western blot analysis, respectively. A microvascular tension sensor was used to detect coronary artery diastolic function in vitro. RESULTS: TMYX elevated the EF, FS, LVOT peak, LVPWd and LVPWs values, decreased the LVIDd, LVIDs, LV-mass, IVSd, and LV Vols values, demonstrating cardio-protective effects, and reduced the NR and ischemic areas. Pathological staining showed that TMYX could significantly reduce inflammatory cell number and interstitial edema. The activities of CK, LDH, and MDA were reduced, NO activity was increased, and oxidative stress was suppressed after treatment with TMYX. TMYX not only enhanced the expression of Gs-α, AC, PKA, and eNOS but also increased the expression of sGC and PKG. Furthermore, TMYX treatment significantly decreased ROCK expression. We further showed that TMYX (25-200 mg/mL) relaxed isolated coronary microvessels. CONCLUSIONS: TMYX attenuates myocardial NR after ischemia and reperfusion by activating the cAMP/PKA and NO/cGMP signaling pathways, further upregulating NO activity and relaxing coronary microvessels.

Tongmai Yangxin pill reduces myocardial no-reflow by regulating apoptosis and activating PI3K/Akt/eNOS pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Tongmai Yangxin pill (TMYX) is derived from the Zhigancao decoction recorded in Shang han lun by Zhang Zhongjing during the Han dynasty and was further improved by Professor Ruan Shiyi, a cardiovascular expert at Tianjin University of Traditional Chinese Medicine. TMYX is used for the clinical treatment of chest pain, heartache, and qi-yin-deficiency coronary heart disease and can improve vascular endothelial function in patients with angina pectoris or coronary heart disease by up-regulating nitric oxide activity and then regulating vascular tension. Whether TMYX can further improve myocardial no-reflow by up-regulating NO activity and then dilating blood vessels remains unclear. AIM OF THE STUDY: This study aimed to reveal whether TMYX can further improve myocardial NR by up-regulating NO activity and then dilating blood vessels. The mechanism underlying PI3K/Akt/eNOS pathway activation and apoptosis regulation is also explored. MATERIALS AND METHODS: The left anterior descending coronary arteries of healthy adult male SD rats were ligated to establish a NR model. The rats were assigned to 14 groups: control, sham, NR, TMYX (4.0 g/kg), sodium nitroprusside (SNP), Tongxinluo capsule (TXL), PI3K blocker (LY), TMYX + LY, SNP + LY, TXL + LY, eNOS blocker (L-NAME), TMYX + L-NAME, SNP + L-NAME, and TXL + L-NAME groups. Cardiac function was measured through echocardiography. Thioflavin S, Evans Blue, and TTC staining were adopted to evaluate NR and ischemic areas. Cell inflammation degree and edema were assessed by hematoxylin-eosin staining. Automated biochemical analyzer and kit were used to detect the activities of myocardial oxidants, including reactive oxygen species, super oxide dismutase, malonaldehyde, and NO. The expression levels of genes and proteins in the PI3K/Akt/eNOS signaling pathway and apoptosis were detected via real-time fluorescence quantitative PCR and Western blot analysis, respectively. A microvascular tension sensor was adopted to detect coronary artery diastolic function in vitro. RESULTS: TMYX reduced NR and ischemic areas; suppressed LV-mass; enhanced EF, FS, LVOT peak, and LVSV; and improved cardiac structure and function. Moreover, it decreased creatine kinase (CK), CK-MB, and lactic dehydrogenase activities. TMYX increased NO and super oxide dismutase activities; inhibited malonaldehyde activity; reduced muscle fiber swelling and inflammatory cell infiltration; and improved vasodilation in vitro. In the NR myocardium, TMYX stimulated myocardial PI3K activities and PI3K (Tyr458) phosphorylation and enhanced Akt activities and Akt phosphorylation at Tyr315. TMYX increased the activities of eNOS and the phosphorylation of eNOS at Ser1177 in the NR myocardium and attenuated cardiomyocyte apoptosis by increasing the expression of Bcl-2 and decreasing that of caspase-3 and Bax. All these effects of TMYX were abolished by the specific inhibitors of PI3K (LY) and eNOS (L-NAME). CONCLUSIONS: TMYX attenuates myocardial NR after ischemia and reperfusion by activating the PI3K/Akt/eNOS pathway and regulating apoptosis, further up-regulating NO activity and relaxing coronary microvessels.

Platelet-Specific p38α Deficiency Improved Cardiac Function After Myocardial Infarction in Mice.

OBJECTIVE: MAPKs (mitogen-activated protein kinases), especially p38, play detrimental roles in cardiac diseases and cardiac remodeling post-myocardial infarction. However, the activation and function of MAPKs in coronary thrombosis in vivo and its relationship with clinical outcomes remain poorly understood. APPROACH AND RESULTS: Here, we showed that p38α was the major isoform expressed in human and mouse platelets. Platelet-specific p38α-deficient mice presented impaired thrombosis and hemostasis but had improved cardiac function, reduced infarct size, decreased inflammatory response, and microthrombus in a left anterior descending artery ligation model. Signaling analysis revealed that p38 activation was one of the earliest events in platelets after treatment with receptor agonists or reactive oxygen species. p38α/MAPK-activated protein kinase 2/heat shock protein 27 and p38α/cytosolic phospholipases A2 were the major pathways regulating receptor-mediated or hydrogen peroxide-induced platelet activation in an ischemic environment. Moreover, the distinct roles of ERK1/2 (extracellular signal-regulated kinase) in receptor- or reactive oxygen species-induced p38-mediated platelet activation reflected the complicated synergistic relationships among MAPKs. Analysis of clinical samples revealed that MAPKs were highly phosphorylated in platelets from preoperative patients with ST-segment-elevation myocardial infarction, and increased phosphorylation of p38 was associated with no-reflow outcomes. CONCLUSIONS: We conclude that p38α serves as a critical regulator of platelet activation and potential indicator of highly thrombotic lesions and no-reflow, and inhibition of platelet p38α may improve clinical outcomes in subjects with ST-segment-elevation myocardial infarction.

Relationship between methylenetetrahydrofolate reductase gene polymorphism and the coronary slow flow phenomenon.

OBJECTIVE: To investigate the correlation between methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism and the coronary slow flow phenomenon (CSFP), and to discover the involvement of genetic factors in CSFP. PARTICIPANTS AND METHODS: Seventy-five patients with normal angiographic coronary arteries were recruited between June 2012 and June 2013. MTHFR C677T genotypes were sequenced by pyrosequencing, whereas the concentration of homocysteine (Hcy) was determined using the enzymatic cycling assay. RESULTS: Compared with the controls, the CSFP patients had higher Hcy concentrations and higher male morbidity. The CSFP patients showed higher frequencies of MTHFR 677(TT+TC) genotypes and the 677T allele compared with the controls. Plasma Hcy levels and male morbidity were correlated positively with the average corrected TIMI frame count. Multiple linear regression and logistic regression analysis indicated that both Hcy and male sex were risk factors for CSFP. MTHFR C677T genotypes and the frequency distribution of 677T allele complied with Hardy-Weinberg equilibrium. CONCLUSION: CSFP was associated with a high level of plasma Hcy, and men were more vulnerable to CSFP. By regulating the plasma Hcy level, the MTHFR C677T gene polymorphism and folic acid level might be involved in the occurrence of CSFP.

Paraoxonase-1 activity and oxidative stress in patients with anterior ST elevation myocardial infarction undergoing primary percutaneous coronary intervention with and without no-reflow.

BACKGROUND: Reperfusion and ischemic injuries are pathogenetic mechanisms of no-reflow. Oxidative stress plays a critical role during ischemia as well as during the reperfusion phase following ST elevation myocardial infarction (STEMI). We sought to investigate the relationship between no-reflow with paraoxonase-1 (PON-1) activity and oxidative stress markers (total antioxidant capacity (TAC), total oxidant status (TOS), oxidative stress index (OSI), lipid hydro-peroxide (LOOH)) in patients with anterior STEMI undergoing primary percutaneous coronary intervention (PCI). METHODS: In this study, 319 consecutive anterior STEMI patients undergoing primary PCI were prospectively included (mean age 56.5 ± 12.5 years). The patients were divided into two groups as normal flow (n = 231) and no-reflow (n = 88) groups. Serum PON-1 activity was measured spectrophotometrically. TAC and TOS levels were determined by using an automated measurement method. LOOH levels were measured by ferrous oxidation with xylenol orange assay. RESULTS: PON-1 activity and TAC levels were significantly lower and TOS, OSI and LOOH levels were significantly higher in patients with no-reflow compared to normal flow group (p < 0.05, for all). On multivariate logistic regression analysis, PON-1 activity (ß = 0.976, 95% CI = 0.962-0.990, p = 0.001) and OSI (ß = 1.094, 95% CI = 1.042-1.148, p < 0.001) as well as diabetes, infarction time, thrombus score and initial SYNTAX score were independently associated with no-reflow. CONCLUSION: In patients with no-reflow compared with normal flow, oxidants are increased, while serum PON-1 activity and antioxidants are decreased. This result shows that increased oxidative stress has a role in the pathogenesis of no-reflow.

Cytochrome c release in acute myocardial infarction predicts poor prognosis and myocardial reperfusion on contrast-enhanced magnetic resonance imaging.

BACKGROUND: Myocardial ischemia and reperfusion injury in ST-segment elevation myocardial infarction (STEMI) can trigger no-flow, resulting in myocardial necrosis and apoptosis, even a poor prognosis. Cytochrome c can induce an apoptotic process. The aim of our study was to assess the relationship between systemic cytochrome c levels and the occurrence of no-reflow in STEMI. METHODS: One hundred and sixty patients with STEMI undergoing a primary percutaneous coronary intervention (PPCI) were randomly chosen. Patients were divided into two groups defined by the mean cytochrome c peak level after PPCI. No-reflow was assessed using three different methods after PPCI: myocardial blush grade, electrocardiographic ST-resolution, and microvascular obstruction (MO) assessed by cardiovascular magnetic resonance imaging. The primary clinical end points were major adverse cardiovascular events (defined as cardiac death, reinfarction, or new congestive heart failure). Clinical follow-up was carried out for 1 year. RESULTS: Patients with a cytochrome c level of at least the mean peak level had a greater creatine kinase-MB isoenzyme peak level (P=0.044), a lower left ventricular ejection fraction (P=0.029), a significantly higher occurrence of early MO (P=0.008), and a significantly larger extent of early MO (P=0.020). The cytochrome c peak level was elevated in patients with early MO (P=0.025), myocardial blush grade 0-1 (P=0.002), and ST-resolution less than 30% (P=0.003) after PPCI. A higher incidence of cardiac death at the 1-year follow-up was found in the patients with cytochrome c levels of at least the mean peak level (log rank, P=0.029). CONCLUSION: Cytochrome c levels above the mean peak level were related to no-reflow and mortality in patients with STEMI.

Endothelial nitric oxide synthase levels and their response to exercise in patients with slow coronary flow.

BACKGROUND: Endothelial dysfunction plays a key role in the aetiopathogenesis of slow coronary flow (SCF) even if there is no obstructive epicardial lesion. Reduced plasma levels of endothelial nitric oxide synthase (eNOS) are an important indicator of endothelial dysfunction. We aimed to determine plasma levels of eNOS and their relationship with exercise in patients with SCF. METHODS: Twenty-two patients with SCF in at least one coronary artery and 17 healthy individuals were included in this study. The TIMI frame count method was used to determine SCF. Plasma levels of eNOS before and after effort were determined in the patient and control groups. RESULTS: Basal eNOS levels in the patient group were lower than in the control group (p = 0.040), and plasma eNOS levels after exercise decreased more significantly in the patient group compared to the control group (p = 0.002). Median decreases of eNOS in response to exercise were higher in the SCF group than in the control group (p < 0.001), and the decrease observed in the control group was not statistically significant (p = 0.35). There were significantly negative correlations between TIMI frame count and plasma levels of eNOS at baseline and after exercise (r = -0.51, p = 0.015, r = -0.58, p = 0.005, respectively). Moreover, there was also a positive correlation between the rate-pressure product and plasma levels of eNOS after exercise in patients with SCF (r = 0.494, p = 0.019). CONCLUSION: Our findings indicate an important pathophysiological relationship between the severity of SCF in which endothelial dysfunction plays a role in its pathogenesis and the level of circulating plasma levels of eNOS.

Evaluation of coronary microvascular function and nitric oxide synthase intron 4a/b polymorphism in patients with coronary slow flow.

OBJECTIVE: Slow coronary flow (SCF) is reported to be associated with increased risk of cardiovascular disease. We have used coronary flow reserve measurement by transthoracic Doppler echocardiography to determine coronary microvascular function in patients with SCF and to determine whether the intron 4a/b polymorphism of the eNOS gene influences coronary endothelial function. METHODS: Overall, 96 patients with SCF and 79 controls were enrolled in the study. Coronary flow was quantified according to the thrombolysis in myocardial infarction (TIMI) frame count (TFC) on angiogram. Coronary diastolic peak flow velocities (DPFV) were measured with color Doppler flow mapping at baseline and after dipyridamole infusion. Coronary flow reserve was calculated as the ratio of hyperemic to baseline DPFV. The eNOS 4a/b polymorphism was detected by PCR. Patients with diabetes were excluded from the study. RESULTS: The SCF group was comparable to the control group in terms of demographic and clinical characteristics, except for hemoglobin and HDL-cholesterol levels, TFC of the left anterior descending artery, the circumflex artery, and the right coronary artery; the mean TFC was higher in the SCF group. Hyperemic DPFV and the hyperemic/baseline DPFV ratio were significantly lower in the SCF group when compared with the control group. However, baseline DPFV were similar in both groups. The number of patients with eNOS4 a/a and eNOS4 a/b phenotypes was statistically higher in SCF groups. The frequency of allele 'a' of the eNOS4 gene was also statistically higher in the SCF group. When patients were grouped according to the presence or absence of allele 'a' of the eNOS4 gene, statistically significant differences were found in the TFC of the left anterior descending artery, the circumflex artery; mean TFC; baseline DPFV; and hyperemic/baseline DPFV. Univariate analysis in which eNOS4 b/b was used as the referent group showed that the presence of allele 'a' of the eNOS4 gene significantly predicted SCF (odds ratio: 2.79, 95% confidence interval: 1.32-5.89; P=0.007). In multivariate analysis using a model adjusted for variables with a P value lower than 0.10 in univariate analyses, the presence of allele 'a' of the eNOS4 gene was found to be an independent predictor of SCF (odds ratio: 3.22, 95% confidence interval: 1.28-8.82; P=0.013). CONCLUSION: The presence of allele 'a' may be a risk factor for microvascular endothelial dysfunction and higher TFCs in SCF patients.

Intravenous nicorandil preserves endothelial junctions by decreasing endothelin-1 via activation of ATP-sensitive K+ channel.

BACKGROUND: It has been verified that nicorandil can attenuate myocardial no-reflow. However; the effects of nicorandil on endothelial junctions and Endothelin-1 (ET-1) are unknown. METHODS: 40 mini-swines randomized into 5 study groups: 8 in control, 8 nicorandil pretreatment, 8 in glibenclamide (KATP channel blocker)-treated, 8 in nicorandil and glibenclamide-pretreated and 8 in sham-operated. Acute myocardial infarction and reperfusion model was created with three-hour occlusion of the left anterior descending coronary artery followed by one-hour reperfusion. RESULTS: In control group, plasma ET-1 significantly increased, ET-1 or VE-cadherin level in the reflow and no-reflow myocardium was significantly higher or lower than that in normal myocardium. Compared with the control group, nicorandil significantly decreased plasma ET-1 and myocardial tissue ET-1, maintained VE-cadherin level. However, glibenclamide abrogated the protective effect of nicorandil. CONCLUSIONS: The beneficial effect of nicorandil on endothelial junctions could be due to its effect on ET-1 via the activation of KATp channel.