The Inhibitory Effect Of Endothelial Progenitor Cells On The Proliferation And Phenotype Transformation Of Vascular Smooth Muscle Cells And The Underlying Mechanisms

Chapter 1 Effect of endothelial progenitor cell on the proliferation of co-cultured vascular smooth muscle cellsBackground Previous studies have demonstrated EPCs can delay the progress of vascular remodeling in blood vessel proliferating diseases such as hypertension, coronary artery disease, and vascular restenosis after stent deployment. It has been proposed that the proliferation and phenotype transformation of vascular smooth muscle cells(VSMCs) is the common basis of cell pathology for vessel proliferating diseases.Thus the present study aimed to explore the effect of EPCs on the proliferation of VSMCs in a VSMCs/EPCs co-culture system in vitro.Methods Mononuclear cells were isolated from fresh cord blood by 3%Gelatin and density gradient centrifugation. Isolated mononuclear cells were cultured in EBM-2 medium supplemented with 20%FBS, VEGF, bFGF and other growth factors. Flow cytometry and indirect immunofluorescence revealed that EPCs expressed hematopoietic progenitor cell-surface antigens CD34 and CD 133 as well as endothelial cell-surface antigens FLK-1, VWF, FLK-1, VE-cadherin, CD 146, CD31 and CD 105. Fluorescence microscopy showed that adherent EPCs are positive for Dil-ac-LDL uptaking and FITC-UEA-1 binding. The co-culture system of EPCs and VSMCs was established by Transwell permeable support,20%fetal bovine serum was used to stimulate the proliferation of VSMCs. In a VSMCs/EPCs co-culture system the DNA synthesis ability, total protein level and cell cycle of VSMCs was determined by BrdU marking method, protein quantitation and flow cytometry after co-culture for 6,12,24,48 and 72 h.Results After co-culture for 12,24,48 and 72 h, the DNA synthesis ability and total protein level of VSMCs significantly decreased compared with control group(P<0.05). Flow cytometry showed the percentage of S phase of VSMCs in VSMCs/EPCs co-culture group significantly decreased and the percentage of G1 phase increased markedly compared with control group(P< 0.05), The maximal inhibitory effect was obtained at the time of 48 h.Conclusion The early EPCs could inhibit the proliferation of VSMCs in vitro co-culture.Chapter 2 The effect of endothelial progenitor cells on angiotensin II induced proliferation and phenotype transformation of cultured rat vascular smooth muscle cellsBackground It has been proposed that phenotype transformation of VSMCs is pivotal for the proliferation for cardiovascular diseases such as hypertension, coronary artery disease, and vascular restenosis after stent deployment. Under pathological conditions, proliferation and phenotype transformation of VSMCs was regulated by a variety of vasoactive substances and cytokines. In order to further elucidate the inhibitory effect and its mechanisms of EPCs on the proliferation and phenotype transformation of VSMCs, the present study investigate the effect of pretreatment with EPC-CM on Ang II induced the proliferation, phenotype transformation and the MAPK and NF-κB signaling pathways in VSMCs.Methods After preparating early endothelial progenitor cell conditioned medium (E-EPC-CM), late endothelial progenitor cell conditioned medium (L-EPC-CM) and human umbilical vein endothelial cell conditioned medium (HUVEC-CM), the effect of E-EPC-CM, L-EPC-CM and HUVEC-CM on Ang II-induced DNA synthesis, total protein expression, cell survival and cycle progress was assessed by BrdU incorporation, total protein content, MTT assays and flow cytometry. RT-PCR and Western-blot were performed to analyze the effect of E-EPC-CM, L-EPC-CM and HUVEC-CM on AngⅡ-induced the expression of a-SM-actin and osteopontin in VSMCs. Moreover, the investigators further investigated the effect of E-EPC-CM on the AngⅡinduced phosphorylations of ERK, JNK, p38 MAPKs/NF-κB p65 (or nuclear translocation of p65) and the expressions of proto-oncogene c-myc and c-fos.Results The DNA synthesis, total protein contents and cell survival obviously increased in AngⅡ(10-6 mM) 48 h induced VSMCs compared with control group, while pretreatment with E-EPC-CM, L-EPC-CM and HUVEC-CM resulted in significant inhibitions in DNA synthesis, total protein content, and cell survival compared with the control group (P<0.05);The percentage of S phase cells significantly increased, while the percentage of cells in G1 phase decreased remarkably compared with control group, pretreatment with E-EPC-CM, L-EPC-CM, or HUVEC-CM led to sustained decrease in the percentage of cells in S phase and sustained increase in the percentage of cells in G1 phase after 48 h of stimulation with AngⅡ(P<0.05); The expression of theα-SM-actin mRNA and protein was significantly decreased and the osteopontin mRNA and protein was markerly increased compared with control group, suggesting that VSMCs was changed from contractile to synthesize type by angiotensinⅡ, pretreatment with E-EPC-CM, L-EPC-CM and HUVEC-CM could inhibit significantly the down-regulation of a-SM-actin expression and the up-regulation of osteopontin expression by AngⅡ(P<0.05). E-EPC-CM showed strongest inhibitory effect among all treatment groups(P<0.05). Western-blot showed that AngⅡtime dependently induced phosphorylation of p38, JNK, ERK/NF-κB p65 and significantly increased the expressions of c-myc and c-fos after AngⅡstimulation in VSMCs, while pretreatment with E-EPC-CM showed marked inhibitory effect on the AngⅡinduced phosphorylations of these MAPKs/NF-κB p65(nuclear translocation of p65) and the expressions of proto-oncogene c-myc and c-fos (P<0.05).Conclusion EPCs may inhibit AngⅡinduced proliferation and phenotype transformation of VSMCs through inactivating MAPKs and NF-κB signaling pathways as well as by reducing the expressions of proto-oncogene c-myc and c-fos. Chapter 3 The inhibitory effect of calcitonin gene-related peptide released from endothelial progenitor cells on proliferation and phenotype transformation of vascular smooth muscle cellsBackground Many biological functions of EPCs mainly attributed to cytokines by paracrine secretion, Previous studies showed that calcitonin gene-related peptide (CGRP) could significantly inhibit the proliferation and phenotype transformation of VSMCs induced by angiotensinⅡ. Moreover, studies demonstrated that early EPCs could express and secrete CGRP. In previous charpter, it was confirmed EPCs could inhibit the proliferation and phenotype transformation of VSMCs induced by angiotensinⅡ.According to CGRP released by EPCs by paracrine manner, we proposed that CGRP may be involved in this process.Methods The release and expression of CGRP was determined by ELISA and RT-PCR. E-EPC-CM was pre-incubated with functional blocking antibodies against CGRP for 1h or VSMCs was preteated with CGRP837(CGRP receptor antagonist) for 1h before VSMCs were pretreated with CM for 30 min. DNA synthesis ability, total protein levels, cell survival, MAPK/NF-κB signal transduction and expression of c-myc and c-fos of VSMCs induced by AngⅡ(10-6mol/L) were detected to assess the role of CGRP in AngⅡ-induced proliferation of VSMCs.Results The level of CGRP was significantly increased after 48 h and was higher than that observed in the L-EPC-CM and HUVEC-CM group.After pretreatment with anti-CGRP antibody and CGRP837 (CGRP receptor inhibitor), inhibitory effect of E-EPC-CM on DNA synthesis ability, total protein levels, cell survival, cycle progress and phenotype transformation of VSMCs induced by Ang II decreased obviously compared with the control group(P<0.05). Likewise, pretreatment with anti-CGRP antibody and CGRP837, in turn, partly attenuated the decreased activity of p38, JNK and ERK MAPKs and NF-κB p65(nuclear translocation of p65) induced by E-EPC-CM treatment in a time-dependent manner and partly cancelled the decreased expression of c-myc and c-fos induced by E-EPC-CM treatment (P<0.05).Conclusion The inhibitory effect of EPCs on proliferation and phenotype transformation of VSMCs may be achieved by the paracrine effect of CGRP.