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

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Enhanced external counterpulsation inhibits endothelial apoptosis via modulation of BIRC2 and Apaf-1 genes in porcine hypercholesterolemia Yan Xiong, Yu-feng Ren, Jia Xu, Da-ya Yang, Xiao-hong He, Jing-yun Luo, Jamal S. Rana, Yan Zhang, Zhen-sheng Zheng, Dong-hong Liu, Gui-fu Wu International Journal of Cardiology Volume 171, Issue 2, Pages 161-168 (February 2014) DOI: 10.1016/j.ijcard.2013.11.033 Copyright © 2013 The Authors Terms and Conditions

Fig. 1 Changes of blood lipid profiles in three groups. Levels of serum total cholesterone (Chol) and low-density lipoprotein (LDL) were recorded in three groups at different time points. Blue, red and green bars represent the Normal group, HC group and HC+EECP group respectively. Week 0 means baseline situation before they received a different diet feeding (week 0, baseline). Week 8, week 12 and week 15 represent the time points of initiation (pre-EECP), mid-stage (during-EECP) and termination of EECP (post-EECP) performed on the HC+EECP group respectively. Serum levels of total cholesterol and low-density lipoprotein among groups were similar at baselines, but significantly increased in animals of group HC and HC+EECP after 8weeks of cholesterol-rich diet feeding compared with the Normal group fed with normal diet, indicating a successful development of porcine model of hypercholesterolemia. Data were expressed as mean±SD; #P<0.05 compared to the Normal group; *P<0.05 compared with group HC. International Journal of Cardiology 2014 171, 161-168DOI: (10.1016/j.ijcard.2013.11.033) Copyright © 2013 The Authors Terms and Conditions

Fig. 2 Isolation and identification of vascular endothelial cells from the aortic endothelium with collagenase. Representative photomicrographs (×400) of isolated cells treated histocytochemically with CD31 antibody and incubated with DiI-Ac-LDL. Cells with cytomembrane and cytoplasmic staining of amber color indicate vascular endothelial cells (black arrow), and cells with red fluorescence were identified as viable vascular endothelial cells (white arrow), which consisted of the majority of total treated cells, indicating a successful endothelial cell isolation from aortic endothelium. International Journal of Cardiology 2014 171, 161-168DOI: (10.1016/j.ijcard.2013.11.033) Copyright © 2013 The Authors Terms and Conditions

Fig. 3 Effect of EECP on morphology of the endothelial cells in three groups observed under SEM and TEM. Representative micrographs of endothelium in three groups were directly observed by SEM. As presented by SEM (×1000), the orderly array and complete surface integrity of abdominal aortic endothelial cells appeared in the Normal group. (A) The endothelial cells had intact cytomembrane and were fusiform in shape, indicative of well consistency with blood flow direction. (B)However, endothelial cells were disorderly distributed and noticeably exfoliated, with significant shrinkage of cytomembrane and subsequently cell volume, characteristic of cells undergoing apoptosis in animals of the HC group. (C) EECP intervention brought a certain degree of alleviation from the above ultrastructural changes. (D) Normal ECs and (E) apoptotic ECs seen under SEM showed characteristics of micrographs (×3000) of ECs undergoing apoptosis manifested significant shrinkage of cytomembrane and subsequently cell volume. Correspondingly, under TEM (×5000), endothelial cells presented certain degrees of shrinking and blebbing of cytomembrane, marginalization, degeneration, and fragmentation of the nucleus, and even formation of apoptotic bodies in animals with hypercholesterolemia; compared with the integral cytomembrane and loosely puffed chromatin as seen in normal endothelial cells, EECP alleviated endothelial apoptosis by less degeneration and fragmentation of nucleus. (E, F, G and H) Representative micrographs of the normal and apoptotic ECs under TEM. (E) Normal ECs. (F) Apoptotic ECs at early stage. (G) Apoptotic ECs at mid-stage. (H) Apoptotic ECs at late stage. (I) Apoptotic bodies. Compared with the integral cytomembrane as well as loosely puffed chromatin as seen in normal endothelial cells, apoptotic endothelial cells showed shrinking and blebbing of cytomembrane; marginalization, degeneration, and fragmentation of the nucleus; and eventually formation of apoptotic bodies ( as shown in J). SEM: scanning electronic microscopy; TEM: transmission electronic microscopy. International Journal of Cardiology 2014 171, 161-168DOI: (10.1016/j.ijcard.2013.11.033) Copyright © 2013 The Authors Terms and Conditions

Fig. 4 Investigation of apoptotic isolated aortic endothelial cells by TUNEL technique. Representative immunocytochemical photomicrographs (×400) of apoptotic vascular endothelial cells (arrow indicates apoptotic endothelial cell with brown-stained nucleus documented by TUNEL technique) in three groups (as shown in A, B and C). Quantitative analysis of endothelial cell apoptosis was made by comparing apoptosis indices (AI) in endothelial cells. The AIs of group HC and HC+EECP were significantly higher than that of Normal (#P<0.05), whereas animals of the HC+EECP group had significantly lower AI than that of HC group (*P<0.05). Data were expressed as mean±SD; #P<0.05 compared to Normal; *P<0.05 compared with group HC. International Journal of Cardiology 2014 171, 161-168DOI: (10.1016/j.ijcard.2013.11.033) Copyright © 2013 The Authors Terms and Conditions

Fig. 5 Investigation of bioactivity of Caspase-3 with immunocytostaining of Cleaved Caspase-3 and transcriptional expression level of Caspase-3 in isolated aortic endothelial cells. Representative immunocytochemical photomicrographs (×400) of Cleaved Caspase-3 stained-positive aorta endothelial cells indicating apoptosis tendency in three groups (as indicated by arrow in A, B and C, top). Quantitative analysis of its bioactivity was made by comparing positive indices (PI) in endothelial cells. The PIs of group HC and HC+EECP were significantly higher than that of Normal (#P<0.05), whereas animals of the HC+EECP group had significantly lower PI than that of the HC group (*P<0.05). Representative electrophoresis bands of RT-PCR products of Caspase-3 genes were presented (bottom) and quantitatively analyzed by comparing with the internal standard β-actin. Relative mRNA expression of Caspase-3 gene was increased significantly in HC group, but EECP could significantly attenuate its expression. Data were expressed as mean±SD; #P<0.05 compared to Normal; *P<0.05 compared with group HC. International Journal of Cardiology 2014 171, 161-168DOI: (10.1016/j.ijcard.2013.11.033) Copyright © 2013 The Authors Terms and Conditions

Fig. 6 Positive immunostaining of Apaf-1 and BIRC2 in isolated aortic endothelial cells (as indicated by arrow). Representative immunocytochemical photomicrographs (×400) of Apaf-1 and BIRC2 in isolated aortic endothelial cells were shown above and quantitative analysis of Apaf-1 and BIRC2 expression was made. The protein expression of Apaf-1 gene was increased remarkably in the HC group but significantly declined after EECP treatment (bar graph, top). However, the BIRC2 expression was significantly restored by EECP (bar graph, bottom). Data were expressed as mean±SD; #P<0.05 compared to Normal; *P<0.05 compared with group HC. International Journal of Cardiology 2014 171, 161-168DOI: (10.1016/j.ijcard.2013.11.033) Copyright © 2013 The Authors Terms and Conditions

Fig. 7 Transcriptional expression of Apaf-1 and BIRC2 genes in isolated aortic endothelial cells. Representative electrophoresis bands of RT-PCR products of both genes Apaf-1 and BIRC2 were presented (top) and quantitatively analyzed by comparing with the internal standard β-actin (bottom). Relative mRNA expression of Apaf-1gene was increased significantly in the HC group but declined after EECP. However, mRNA expression of BIRC2 gene was decreased remarkably in the HC group; EECP could significantly restore its expression. Data were expressed as mean±SD; #P<0.05 compared to Normal; *P<0.05 compared with group HC. International Journal of Cardiology 2014 171, 161-168DOI: (10.1016/j.ijcard.2013.11.033) Copyright © 2013 The Authors Terms and Conditions