Contribution Of Circulating Smooth Muscle Progenitor Cells In Hypoxia-induced Muscularization Of Non-muscular Pulmonary Arterioles

Chronic hypoxia is one of the key elements which lead to pulmonary artrial remodeling(PAR) and pulmonary hypension (PH), and the muscularization of non-muscular pulmonaryarteriole is one of the most important characteristics of hypoxia-induced PAR. It is reportedthat the source of new formation of vascular smooth muscle cells (VSMC) in muscularizednon-muscular pulmonary arterioles is due to the migration and proliferation from theadjecent pulmonary arteries (PA), however, the effects of clinical treatments targeting onthe VSMCs migration and proliferation are not encouraging, suggesting that there underliesother mechanisms. Myocardin is a co-activitor of serum response factor (SRF) involved insmooth muscle cells (SMCs) and cardiac myocyte differentiation. Transfection thefibroblast and stem cells with myocardin over-expression vectors enabled them to expressthe molecular markers of SMCs, and transplantation of bone marrow-derived mesenchymalstem cells (MSCs) with forced-expression of myocardin to the infarcted area improved thecardiac function, and further results revealed that transplantated MSC effectivelydifferentiated into cardiac myocytes under the regulation of myocardin. It is reported thatthere exits smooth muscle progenitor cells (SPCs) in the circulation, and the SPCs mayhome to atherosclerosis (AS) plaques and ischemic lesions in the limbs under hypoxicconditions. Moreover, chronic hypoxia mobilized the stem cells to the peripheralcirculation and induced the circulating c-kit⁺ progenitor cells to the pulmonary arteryadventitial remodeling and formation of vasa vasorum in the PA. Based on the observationmentioned above, we speculate that the circulating SPCs home to the wall of PA under hypoxia, and sequently differentiate into VSMCs under the control of myocardin, whichlead to the muscularization of non-muscular pulmonary arterioles. This study was dividedinto three parts.Partâ… :Isolation, Identification and Differentiation of Rat Circulating Smooth MuscleProgenitor CellsObjective: To isolate the rat circulating smooth muscle progenitors cells (SPCs) and toidentify its phenotypes, by which to make preparation for the following studies. Methods:Wistar rats were intraperitoneally injected with G-CSF for consecutive five days, and theMNCs were isolated from circulating blood by density gradient centrifugation overFicoll-paque (1.077g/ml) on the 6th day, then the magnet-activated cell sorting (MACS)was used to separate the CXCR4⁺/PDGFRβ⁺ progenitor cells from the MNCs. After thedifferentiation of the CXCR4⁺/PDGFRβ⁺ progenitor cells were induced byPDGF-BB-enriched medium or hypoxia(1%O₂), immunofluorescence, RT-PCR andwestern blot were used to detect the SMCs markers, and TEM was performed to examinethe ultrastructures of differentiated cells. Results: the CXCR4⁺/PDGFRβ⁺ progenitor cellswere separated successfully from the circulating MNCs of G-CSF mobilized rats, and itspurity reached to 93.7% by FCM. Results of immunofluroscence revealed that theprogenitor cells expressed CXCR4 and PDGFRβ. After induced by PDGF-BB, theprogenitor cells underwent the morphological changes which turned from small round cellsinto polygon and spindle-like cells and showed a "hilly valley" appearance that of VSMCswhen it was confluent. The results of immunofluorescence, RT-PCR and western blotshowed that theα-SMA, SM22α, calponin and SM-MHC were potive forPDGF-BB-induced differentiated cells, and the myofilaments, dense bodies, dense patcheswere observed in the differentiated cells by TEM. When the progenitor cells cultured under hypoxia condition, the cells underwent the morphological changes similar with thePDGF-BB-induced differentiated cells. Furthermore, the markers of VSMCs includingα-SMA, SM22α, calponin and SM-MHC were occurred in hypoxia-induced differentiatedcells as well as the ultrastructural structures of VSMCs were showed by TEM. Conclusion:the rat circulating CXCR4⁺/PDGFRβ⁺ progenitor cells are a real subpopulation of SPCs,and hypoxia contributes to its differentiation into SMCs in vitro.Partâ…¡:Expression and Contribution of Myocardin in the Differentiation ofCXCR4⁺/PDGFRβ⁺ Progenitor Cell into SMCs in vitroObjective: To detect the expression of myocardin in the differentiated CXCR4⁺/PDGFRβ⁺progenitor cells and to investigate the contribution of myocardin in the differentiation of theCXCR4⁺/PDGFRβ⁺ progenitor cells into SMCs in vitro. Methods: The lentivirusinterfering vectors targeting rat myocardin mRNA (lentivirus-GFP-shMyocd) and thenegative control lentivirus interfering vector (lentivirus-GFP-NC) were constructed withthe lentiviral plasmid pGCSIL-GFP. After the CXCR4⁺/PDGFRβ⁺ progenitor cells wereinduced to differentiate into SMCs by PDGF-BB and hypoxia respectively,immunofluscence, RT-PCR and western blot were performed to detect the myocardinexpression; finally, the CXCR4⁺/PDGFRβ⁺ progenitor cells were pre-transducted with thelentivirus interfering vectors by diffenent timepoints before hypoxia, and then RT-PCR andwestern blot were adopted to examine myocardin and SMCs markers expression. Results:the lentivirus-GFP-shMyocd and lentivirus-GFP-NC RNAi plasmids were constructedsuccessfully, and its tite reached to 1×10⁹ TU/ml and 5×10⁹ TU/ml, respectively. The resultsof RT-PCR and western blot revealed that the freshly isolated CXCR4⁺/PDGFRβ⁺progenitor cells did not express myocardin, while induced by PDGF-BB or hypoxia, themyocardin began to express on day 7, and reached to the peak level on day 14 while decreased on day 21. Pre-treatment the CXCR4⁺/PDGFRβ⁺ progenitor cells withlentivirus-GFP-shMyocd suppressed the hypoxia-induced expression of myocardin, andfollowing the down-regulation of SMCs markers,α-SMA, SM22α, calponin and SM-MHCat the mRNA and protein levels. Conclusion" hypoxia-induced expression of myocardin inthe CXCR4⁺/PDGFRβ⁺ progenitor cells gorvers the SMC differentiation of progenitor cellsin vitro.Partâ…¢:Contribution of Myocardin-Regulated Differentiation of SPCs in Hypoxia-InducedMuscularization of Non-muscular Pulmonary ArteriolesObjective: To investigate whether chronic hypoxia induce the circulating SPCs to home tothe pulmonary artery as well as the role of homed SPCs in the hypoxia-inducedmuscularization of non-muscular pulmonary arterioles. Methods: the bone marrow cells(BMC) chimeric mice were created by transplantating the BMCs from GFP transgenicC57BL6/J mice to the sub-lethal ⁶⁰Co irradiated WT C57BL6/J mice, and the chimericmice were subjected to hypoxia (10%O₂) for 4 wk, the distribution of GFP signal andexpression of GFP were examined by laser scanning confocal microscopy and western blotanalysis respectively. After transducted the CXCR4⁺/PDGFRβ⁺ SPCs with or without thelentivirus, the SPCs were infused to wistar rats via jugular veins. The mPAP was detectedafter 6 wk of hypoxia and then the lungs were exteriorized, the muscularization degree wasassessed by immunohistrochemistrical staining ofα-SMA and elastic fiber staining; themean wall thickness of muscularized pulmonary arterioles was measured. The myocardinmRNA expression in muscularized pulmonary arterioles was analysized by Q-PCR on RNAisolated from laser capture microdissection-separated arterioles. At the same time, thedistribution and differentiation of infused SPCs were examined by immunoflurescentstaining ofα-SMA on froze sections. Results" There occured obvious GFP signals in the PA of the BMCs chemiric mice after 4 wk of hypoxia, and the result of western blot furtherconfirmed that the GFP protein was significantly detected in hypoxic BMCs chemiric micethan normoxic chemiric mice. In anothor 6wk of hypoxia wistar rat model, the meanpalmonary arties pressue (mPAP), muscularization degree of alveolus arterioles and themean wall thickness of muscularized arterioles were greater than that of normoxic rats. TheGFP⁺ cells were located in the muscularized arterioles of the rats infused with theprogenitor cells, and parts of them wereα-SMA positive. Morover, the muscularizationdegree of alveolus arterioles, the mean wall thickness of muscularized arterioles and eventhe mPAP of the rats infused with lentivirus-GFP-shMyocd-transducted-progenitor cellswere decreased than the rats infused with lentivirus-GFP-NC-transducted progenitor cells.Finally, the myocardin mRNA in muscularized arterioles of the rats infused withlentivirus-GFP-shMyocd-transducted-progenitor cells was down-regulated than the ratsinfused with lentivirus-GFP-NC-transducted progenitor cells or the hypoxic rats infusedwith progenitor cell, which were parallelled with the changes of mPAP. Conclusion:Hypoxia induces SPCs to home to the pulmonary artery and to express myocardin. Thehomed SPCs differentiate into VSMC under the control of myocardin, and thusparticipating in the hypoxia-induced muscularization of non-muscular pulmonary arterioles.Conclusion:Taken together, the present study was the first report to show that hypoxia can induce theCXCR4⁺/PDGFRβ⁺ SPCs to locate in the alveolar arterioles and differentiate into SMCs, inwhich myocardin up-regulation was involved, contributing to the muscularization ofalveolar arterioles, eventually leading to the PVR and PAH.