Enhanced external counterpulsation inhibits endothelial apoptosis via modulation of BIRC2 and Apaf-1 genes in porcine hypercholesterolemia.

OBJECTIVES: Enhanced external counterpulsation (EECP) could improve endothelium-dependent vasodilatation of carotid artery and restore imbalance of nitric oxide and endothein-1 in patients with coronary artery disease. Our study was designed to test the hypothesis that long-term EECP may protect vascular endothelial cells from apoptosis by modifying apoptosis-related gene expression. METHODS: Eighteen male Yorkshire pigs were randomly assigned to three groups: usual diet (Normal), high cholesterol diet (HC) and high cholesterol diet plus EECP (HC+EECP). Vascular endothelial cells were isolated from the aortic endothelium and identified by CD31 staining and DiI-Ac-LDL reaction. Morphological changes were observed by both scanning and transmission electronic microscopes. TUNEL technique was applied to detect the apoptotic index of vascular endothelial cells. Two genes, Apaf-1 and BIRC2, were chosen for exploring the potential mechanisms of action at the molecular level. RESULTS: EECP brought a certain degree of alleviation from ultrastructural changes such as shrinking and blebbing of cytomembrane, marginalization, degeneration, and fragmentation of the nucleus. EECP also significantly reduced apoptotic indices while compared with that of control (177±12‰ vs. 237±23‰, P<0.05). The Apaf-1 expression at both protein and mRNA level in pigs of HC+EECP group was significantly decreased than those of the HC group (P<0.05), whereas the BIRC2 expression was significantly enhanced after EECP treatment, documented by immunostaining and semi-quantitative RT-PCR analysis, respectively (P<0.05). CONCLUSIONS: EECP could protect vascular endothelial cells from apoptosis, thereby delaying the progression of early atherosclerotic lesions possibly through transcriptional down-regulation of pro-apoptotic gene Apaf-1, and up-regulation of anti-apoptotic gene BIRC2.

Enhanced external counterpulsation promotes growth cytokines-mediated myocardial angiogenesis in a porcine model of hypercholesterolemia.

BACKGROUND: Enhanced external counterpulsation (EECP) improves ischemia in patients with refractory angina pectoris, but the mechanism remains unclear. To explore the mechanisms of EECP action, we detected progenitor cells presenting any of the following markers CD34(+), CD29(+), and CD106(+). METHODS: Growth cytokines-mediated progenitor cell mobilization and associated angiogenesis potential were assessed in a porcine model of hypercholesterolemia. Twenty-four male domestic swines were randomly assigned to 4 groups: normal diet (control, n = 6), hypercholesterolemic diet (CHOL, n = 6), hypercholesterolemic diet with administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) (rhG-CSF, n = 6), and hypercholesterolemic diet with EECP treatment (EECP, n = 6). EECP was applied 2 hours every other day for a total of 36 hours. Serum levels of vascular endothelial growth factor (VEGF) and granulocyte colony-stimulating factor (G-CSF), peripheral blood progenitor cell counts, level of regional angiogenesis, and expression of VEGF and stromal cell derived factor 1alpha (SDF-1alpha) in porcine myocardium were assessed, respectively. RESULTS: A porcine model of hypercholesterolemia-induced arteriosclerosis was successfully established. There was no significant difference in serum levels of VEGF among the four groups. The serum levels of G-CSF in the EECP group increased significantly at week 15 and week 18 ((38.3 +/- 5.6) pg/ml at week 15 vs (26.2 +/- 3.7) pg/ml at week 12, P < 0.05, and (46.9 +/- 6.1) pg/ml at week 18 vs (26.2 +/- 3.7) pg/ml at week 12, P < 0.01). The serum levels of G-CSF in group 3 increased also significantly after receiving rhG-CSF injection for five days ((150 +/- 13.9) pg/ml at week 18 vs (24.8 +/- 5.4) pg/ml at week 12, P < 0.01). Compared to other groups and other time points, progenitor cell counts increased significantly after 2-hour EECP treatment (108 +/- 13 vs 26 +/- 6 per 10(5) leukocytes, P < 0.01), but not at week 18. The progenitor cell counts also increased significantly after subcutaneous injection of rhG-CSF for five days compared to the week 12 (baseline) (180 +/- 21 vs 25 +/- 7 per 10(5) leukocytes, P < 0.01). There was no significant difference among the four groups at other time points. Moreover, the expression of VEGF and SDF-1alpha and the level of regional angiogenesis in myocardium increased significantly in both EECP and rhG-CSF groups. CONCLUSIONS: The results demonstrated that EECP could facilitate angiogenesis in the myocardium of atherosclerotic swines by increasing endogenous G-CSF, inducing an enhanced mobilization of progenitor cells and augmenting myocardial expression of VEGF and SDF-1alpha.

[Effect of external counterpulsation on morphological damage of endomembrane and endothelium-dependent vasodilatation in the carotid arteries in hypercholesterolemic pigs].

OBJECTIVE: To explore the effect of long-term enhanced external counterpulsation (EECP) on morphological damage of endomembrane and endothelium-dependent vasodilatation of the carotid arteries of hypercholesterolemic pigs. METHODS: Eighteen male infant pigs were randomly divided into three groups according to the contents of their diet: the normal control group (n=6), the high-cholesterol feeding control group (n=6) and EECP group (n=6). Porcine model of hypercholesterolemia was reproduced by feeding animals with high-cholesterol diet. After completion of EECP treatment for 36 hours in EECP group, carotid arterial rings were harvested from all animals. Both scanning and transmission electron microscopy was employed to observe the changes in morphology of their endomembrane. At the same time, their vasodilatation response to variant dose of acetylcholine (Ach) was detected. RESULTS: The surface of endothelium in the normal control group was smooth, and endothelial cells were in regular streamline array, and they were almost in same size, attaching closely to matrix without smooth muscle cell proliferation and lipid infiltration in intima. In contrast, the endothelial cells of hypercholesterolemic pigs were in irregular array, with marked desquamation, resulting in loose linkage. Smooth muscle cells were found to invade into intimal layer and proliferated, and foam cells could also be found in intimal layer. In hypercholesterolemic pigs treated with EECP, slight intimal damage was found. In addition, with Ach dose of 10(-8) mol/L to 10(-5)mol/L, endothelium-dependent vasodilatation ratio in hypercholesterolemic pigs with or without EECP treatment, was significantly lower than that of the normal control group (all P<0.05). However, endothelium-dependent vasodilatation ratio in pigs with EECP treatment was obviously higher compared with hypercholesterolemic pigs without EECP treatment with the dosage of Ach concentration ranged from 10(-7) mol/L to 10(-5) mol/L (all P<0.05). CONCLUSION: Long-term EECP ameliorates both the morphological damage and the impaired endothelium-dependent vasodilatation function resulting from hypercholesterolemia, contributing to prevention of atherosclerosis.

[Effects of external counterpulsation on shear stress and production of nitric oxide and cGMP in canines with myocardial infarction].

OBJECTIVE: To investigate the effects of external counterpulsation (ECP) on shear stress and signal transduction in canines with myocardial infarction. METHODS: Nineteen healthy dogs were randomly divided into control, ischemia, and ischemia plus ECP groups. Myocardial infarction was induced in the latter two groups by ligation of the left anterior descending artery (LAD). Serum and aorta NO levels of the dogs were determined by modified nitrate reductase method, and serum and aorta cyclic guanosine monophosphate (cGMP) levels by radioimmunoassay. RESULTS: The shear stress in the truncus brachiocephalicus decreased after LAD ligation, but increased significantly after 2 h of ECP treatment. Serum and aorta NO levels in ECP and control groups were significantly higher than those in the ischemic group (P<0.05). Serum and aorta cGMP levels in control group and ECP group after LAD ligation were also significantly higher than those in the ischemic group (P<0.05). CONCLUSION: ECP can increase the shear stress and increase NO and cGMP levels in dogs with myocardial ischemia, which might be an important mechanism of ECP for protection of the ischemic myocardium.

Effect of external counterpulsation on plasma nitric oxide and endothelin-1 levels.

Enhanced external counterpulsation (EECP) significantly augments diastolic blood flow and has been postulated to improve endothelial function by increased shear stress. We examined the effects of EECP on plasma nitric oxide and endothelin-1 (ET-1) levels. Plasma nitrate and nitrite (NOx) and ET-1 levels were measured serially in 13 patients with coronary artery disease who received 1-hour daily treatments of EECP over 6 weeks. During the course of EECP therapy, plasma NOx progressively increased and plasma ET-1 progressively decreased. After 36 hours of EECP, there was a 62 +/- 17% increase in plasma NOx compared with baseline (43.6 +/- 4.3 vs 27.1 +/- 2.6 micromol/L, p <0.0001) and a 36 +/- 8% decrease in plasma ET-1 (76.7 +/- 9.5 vs 119.5 +/- 8.5 pg/L, p <0.0001). At 3 months after completion of EECP, NOx remained 12 +/- 11% above baseline (p = 0.002), and ET-1 remained 11 +/- 10% below baseline (p = 0.0068). Our data provides neurohormonal evidence to support the hypothesis that EECP improves endothelial function.

[Establishment of a pig model of chronic ally enhanced external counterpulsation].

OBJECTIVE: To establish a pig model of chronic external counterpulsation. METHODS: Twelve pigs were anaesthetized with sodium pentobarbital (< or =30 mg/kg.b.w.) and 846 mixture (< or =0.1 ml/kg.b.w.) and counterpulsed in a lateral position for 2 h every two days (totally 36 h) with 0.025 to 0.04 MPa/cm(2) pressure. RESULTS: External counterpulsation was successfully completed in all the animals. Combined administration of sodium pentobarbital and 846 mixture resulted in good anesthetic effect with reduced anesthetic dosage and minimal side effect on the viscera (the liver, kidney and heart, etc). CONCLUSION: The pig model of chronic external counterpulsation has been successfully established. Combined use of sodium pentobarbital and 846 mixture is recommended for chronic external counterpulsation.

[Effects of enhanced external counterpulsation in atherosclerosis and NF-kappaB expression: a pig model with hypercholesterolemia].

OBJECTIVE: To study the effects of enhanced external counterpulsation (EECP) on the vascular morphology, and endothelial function using experimentally induced hypercholesterolemic pigs. METHODS: Thirty five male pigs were randomly divided into three groups: 7 normal control animals, 11 hypercholesterolemic animals, and 17 hypercholesterolemic animals receiving EECP. Serum cholesterol was measured. The coronary arteries and aortas were sampled for histopathologic and ultrastructural examination. The NF-kappaB protein expression of porcine coronary arteries was investigated by immunofluorescence. RESULTS: Compared with the normal controls, serum cholesterol levels were significantly higher in the hypercholesterolemic animals with or without EECP. The plaque/intimal area ratio of the aorta decreased significantly in animals receiving EECP [(3.33 +/- 2.40)%, versus (12.03 +/- 7.12)% in those without EECP, P < 0.05]. Lipid deposition, endothelial damage and proliferation of smooth muscle cells were less severe in animals receiving EECP than those not. Moreover, activation and expression of NF-kappaB also decreased significantly (P < 0.05) in animals receiving EECP. CONCLUSIONS: EECP improves the morphology and function of vascular endothelium, and retards the development and progression of atherosclerosis, likely through the inhibition of NF-kappaB signaling pathway.

[Effect of chronic enhanced external counterpulsation on arterial endothelial cells of porcine with hypercholesteremia].

OBJECTIVE: To verify whether chronic enhanced external counterpulsation (EECP) may repair artery endothelial cells (ECs) damage resulted from hypercholesteremia in pigs. METHODS: EECP was performed for 36 hours in pigs with hypercholesteremia and the left descending artery (LDA) was isolated for scanning electron microscopy (SEM). The ECs were collected from the thoracic aorta and analyzed by proteomic technique. RESULTS: The ECs of hypercholesteremia pigs showed irregular arrangement with obvious desquamation of coronary ECs and formation of atherosclerotic plaque, and these lesions were milder in EECP-treated pigs. Six over-expressed proteins were detected in the endothelial cells in EECP-treated pigs in comparison with those of the hypercholesteremia pigs. CONCLUSION: Chronic EECP helps restore cell morphology and repair functional damage of ECs resulted from hypercholesteremia by regulating endothelial protein expressions, and consequently improves cell adhesion and intracellular metabolism and reduces EC apoptosis.

Microvessel angiogenesis: a possible cardioprotective mechanism of external counterpulsation for canine myocardial infarction.

BACKGROUND: Enhanced external counterpulsation (EECP) has been demonstrated to be effective in the treatment of patients with coronary artery disease (CAD). It has been proposed that the beneficial effects of EECP observed in clinical studies may be due to the formation of new blood vessels (angiogenesis) and collateral development. However, there is a relative paucity of basic studies to support the proposed mechanisms. METHODS: Twelve Beagle dogs were anesthetized with 3% sodium pentobarbital, 1 mg/kg intraperitoneal injection and mechanically ventilated for the development of myocardial infarction. After coronary occlusion, all animals were randomly assigned to either EECP or control. EECP was given one hour per day, 5 days a week, for a total of 28 to 30 hours treatment over a 6-week course. Immunohistochemical studies of alpha-actin and von Willebrand factor (vWF) were used to detect newly developed microvessels. Systemic and local vascular endothelial growth factor (VEGF) were identified by enzyme linked immunosorbent assay (ELISA) and reverse-transcriptional polymerase chain reaction (RT-PCR) analysis. RESULTS: There was a significant increase in the density of microvessels per mm(2) in the infarcted regions of EECP group compared to control group (vWF, 15.2 +/- 6.3 versus 4.9 +/- 2.1, P < 0.05; alpha-actin, 11.8 +/- 5.3 versus 3.4 +/- 1.2, P < 0.05), along with significant increase of positive vWF and alpha-actin stained area. Both immunohistochemical staining and RT-PCR analysis documented a significant increase in VEGF expression. These factors associated with angiogenesis corresponded to improved myocardial perfusion by 99mTc-sestamibi single-photon emission computed tomography. CONCLUSION: Microvessel angiogenesis may be a mechanism of action for the improved myocardial perfusion after EECP therapy.

Effects of enhanced external counterpulsation on stress radionuclide coronary perfusion and exercise capacity in chronic stable angina pectoris.

Enhanced external counterpulsation (EECP) is an effective noninvasive treatment for patients with coronary artery disease (CAD). EECP has been demonstrated to improve anginal class and time to ST-segment depression during exercise stress testing. This study assesses the efficacy of EECP in improving stress-induced myocardial ischemia using radionuclide perfusion treadmill stress tests (RPSTs). The international study group enrolled patients from 7 centers with chronic stable angina pectoris and a baseline ischemic pre-EECP RPST. Patients' demographic and clinical characteristics were recorded. A baseline pre-EECP maximal RPST was performed within 1 month before EECP treatment. The results were compared with a follow-up RPST performed within 6 months of completion of a 35-hour course of EECP. Four centers performed post-EECP RPST to the same level of exercise as pre-EECP, whereas 3 centers performed maximal RPST post-EECP. The study enrolled 175 patients (155 men and 20 women). Improvement in angina, defined by > or =1 Canadian Cardiovascular Society angina class change, was reported in 85% of patients. In the centers performing the same level of exercise, 81 of 97 patients (83%) had significant improvement in RPST perfusion images. Patients who underwent maximal RPST revealed improvement in exercise duration (6.61 +/- 1.88 pre-EECP vs 7.41 +/- 2.03 minutes post-EECP, p <0.0001); 42 of the 78 patients (54%) in this group showed significant improvement in RPST perfusion images. Thus, EECP was effective in improving stress myocardial perfusion in patients with chronic stable angina at both comparable (baseline) and at maximal exercise levels.