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.

[Effect of chronic enhanced external counterpulastion on gene expression profiles of arterial endothelial cells of pigs fed with high-cholesterol diet].

OBJECTIVE: To investigate the effect of chronic enhanced external counterpulastion (EECP) on gene expression profiles of arterial endothelial cells (ECs) of pigs fed with high-cholesterol diet. METHODS: Eight male pigs were fed with high-cholesterol diet for 12 weeks to induce arteriosclerosis and subjected to EECP for accumulative 36 h (2 h every other day for 18 sessions). Another 8 pigs on cholesterol-enriched diet and 6 normally fed pigs served as the arteriosclerosis model group and normal control group, respectively, and the high-cholesterol diet was maintained until the end of EECP treatment. The coronary artery was then isolated for transmission electro microscopy, and the abdominal aorta was observed using Sudan III staining. The gene expression profiles in ECs from the thoracic aorta using cDNA microarrays. RESULTS: Macrophages and foam cells were detected beneath the ECs in the coronary artery of pigs in the model group, but not in the other two groups. The ratios of Sudan III-positive area in the celiac aorta were significantly lower in normal control and EECP groups than in the model control group (P<0.05). Compared with the normal control group, the gene expressions of integrins-beta1 and CTGF were up-regulated in the model group. Compared with the model group, the expressions of integrins-beta1, CTGF and VCAM-1 were down-regulated and eNOS up-regulated in EECP group. CONCLUSION: Chronic EECP may reduce endothelial injury, down-regulate the gene expression level of integrin-beta1, CTGF and VCAM-1, lower cholesterol uptake and attenuate arterial endothelial inflammation to protect the pigs fed with high-cholesterol diet from arteriosclerosis.