循环平滑肌祖细胞在低氧性肺无肌细动脉肌化中的作用
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摘要
长期慢性低氧是引起肺动脉重塑(Pulmonary artery remodeling，PAR)进而导致肺动脉高压(Pulmonary hypertension，PH)的重要因素。肺无肌细动脉肌化是低氧引起的PAR的重要特征之一。既往认为，引起肺无肌细动脉肌化的机制主要是由于临近动脉的血管平滑肌细胞(Vascular smooth muscle cells，VSMC)迁移及增殖而来，但临床针对VSMC的迁移和增殖治疗效果不明显，提示尚存在其他的机制。Myocardin是血清反应因子(Serum response factor，SRF)的一种共辅助因子，参与平滑肌细胞(Smooth muscle cells，SMCs)及心肌细胞的分化，是SMCs和心肌细胞分化所必须的。用myocardin过表达载体转染纤维母细胞和干细胞均可使其表达SMCs的分子标志，且移植到心急梗死灶中的骨髓间充质干细胞(Bone marrow-derived mesenchymal stemceils，MSCs)强制表达myocardin后心功能得到明显改善，结果证实移植的MSCs在myocardin的调控下有效分化为心肌细胞。已经证实循环血液存在平滑肌祖细胞(Smooth muscle progenitor cells，SPCs)。在低氧条件下SPCs可以归巢到动脉粥样硬化斑块及缺血的肢体等病灶中并发挥生物学作用。慢性低氧不仅动员骨髓干细胞而且可诱导循环c-kit~+祖细胞参与肺动脉外膜重塑及肺动脉滋养血管的形成。鉴于以上研究基础，我们设想：在慢性低氧条件下，循环SPCs归巢至肺无肌细动脉，在myocardin的调控下分化为VSMCs从而参与了低氧性肺无肌细动脉的肌化。本研究分三个部分。
第一部分：大鼠循环平滑肌祖细胞的分离、鉴定及分化
目的：分离大鼠循环血液中的平滑肌祖细胞(SPCs)并鉴定其表型，为后续研究打下基础。方法：采用单核细胞集落刺激因子(M-CSF)注射法动员大鼠骨髓干细胞，第六天收集循环血液经密度梯度离心法获得单个核细胞(MNCs)，用磁激活的细胞分选法(MACS)分离具有CXCR4~+／PDGFRβ~+表型的祖细胞。采用富含PDGF-BB的培养液常规条件下诱导祖细胞分化，用细胞免疫荧光，RT-PCR，Western blot检测分化细胞的SMCs表型标记物，透射电镜检测分化细胞的超微结构从而判断分离的祖细胞的SMCs分化潜能；低氧(1％O_2)条件下培养上述祖细胞，观察祖细胞的SMCs分化情况，用细胞免疫荧光，RT-PCR，Western blot检测分化细胞的SMCs表型标记物，透射电镜检测分化细胞的超微结构从而判断低氧诱导的分化细胞是否为典型的SMCs。结果：从G-CSF动员的大鼠外周血液MNCs中成功分离出具有CXCR4~+／PDGFRβ~+表型的祖细胞，FCM证实纯度达93．7％，细胞免疫荧光证实祖细胞表达CXCR4及PDGFRβ。经富含PDGF-BB的无血清培养液诱导后，CXCR4~+／PDGFRβ~+祖细胞发生了典型的形态学变化，从小、圆形细胞转变为多角形、梭型细胞，细胞长满后具有SMCs的“峰谷”特征；免疫荧光、RT-PCR及western blot检测到分化细胞表达α-SMA，SM22α，calponin及SM-MHC，透射电镜证实分化细胞具有肌丝、密体，密斑等SMCs超微结构；低氧条件下，CXCR4~+／PDGFRβ~+祖细胞具有和PDGF-BB诱导下相似的形态学变化；免疫荧光、RT-PCR及Western blot检测到分化细胞表达α-SMA，SM22α，calponin及SM-MHC，透射电镜亦证实分化细胞具有肌丝、密体，密斑等SMCs超微结构。结论：大鼠循环CXCR4+~／PDGFRβ~+祖细胞为平滑肌祖细胞，体外低氧可诱导其分化为成熟的SMCs。
第二部分：Myocardin在CXCR4~+／PDGFRβ~+祖细胞体外SMC分化中的表达及作用
目的：检测Myocardin在CXCR4~+／PDGFRβ~+祖细胞分化过程中的表达，探讨干扰myocardin后对CXCR4~+／PDGFRβ~+祖细胞分化为SMCs的影响。方法：以慢病毒质粒pGCSIL-GFP为基础构建针对myocardin mRNA靶点的慢病毒干扰质粒(lentivirus-GFP-shMyocd)及阴性对照质粒(lentivirus-GFP-NC)；用有效靶点的myocardin慢病毒干扰质粒转染CXCR4+~／PDGFRβ~+祖细胞获得稳定表达，将稳定转染干扰病毒的祖细胞置于低氧条件下诱导分化，用RT-PCR及western blot检测不同时间点分化细胞内的myocardin及SMCs分化标志基因的表达。结果：成功构建滴度为1×10~9TU／ml的lentivirus-GFP-shMyocd和5×10~9TU／ml的lentivirus-GFP-NC干扰病毒。RT-PCR及western blot证实刚分离的CXCR4~+／PDGFRβ~+祖细胞不表达myocardin，PDGF-BB和低氧诱导后7天后表达myocardin，14天达到峰值，21天分化细胞成熟时其表达下降。用lentivirus-GFP-shMyocd预转染CXCR4~+／PDGFRβ~+祖细胞后再将细胞置于低氧条件下分化，RT-PCR及western blot结果显示lentivirus-GFP-shMyocd质粒有效减低了低氧所诱导的myocardin表达，敲减后的祖细胞中SMCs分化标记基因表达趋势与myocardin表达下降趋势相一致。结论：体外低氧诱导CXCR4~+／PDGFRβ~+祖细胞中myocardin的表达介导了祖细胞向SMCs分化。
第三部分：Myocardin调控CXCR4~+/PDGFRβ~+ SPCs参与低氧性肺无肌细动脉肌化
目的：探讨慢性低氧是否诱导循环平滑肌祖细胞(SPCs)归巢至肺无肌细动脉以及该归巢的SPCs在低氧诱导的肺无肌细动脉肌化中的作用。方法：GFP转基因C57BL6／J小鼠骨髓细胞移植到经过亚致死量~(60)Co照射的Wt C57BL6／J小鼠以形成骨髓细胞嵌合小鼠，嵌合成功的小鼠低氧(10％O_2)饲养4wk，采用激光共聚焦显微镜观察肺动脉上GFP荧光的分布，western blot分析肺组织内GFP蛋白的表达；用慢病毒干扰质粒转染CXCR4~+／PDGFRβ~+ SPC形成稳定表达细胞，将该稳定转染的SPCs经颈静脉输入Wistar大鼠。经6wk低氧后检测大鼠平均肺动脉压(mPAP)，处死大鼠取出肺组织，免组织化学染色检测肺血管α-SMA表达，结合弹力纤维染色判断肺动脉肌化程度，测定大鼠肌化血管壁的平均厚度；显微切割肌化血管并经定量PCR检测肌化肺动脉中myocardin mRNA的表达；同时冰冻切片经免疫荧光标记VEVSMC的α-SMA，激光共聚焦显微镜下观察α-SMA的表达及定位，结合GFP的表达情况判断输入的SPCs在肌化动脉上的定位及分化情况。结果：骨髓细胞嵌合小鼠经4wk低氧后观察到肺血管中具有较多的GFP信号，western blot亦证实嵌合小鼠低氧下较常氧条件下肺组织内GFP蛋白显著增加。Wistar大鼠经6wk低氧后，mPAP、肺动脉肌化程度及肌化血管平均厚度明较常氧组升高。接受SPCs输注低氧大鼠较未接受SPCs输注大鼠其mPAP、肺动脉肌化程度、肌化血管平均厚度均高于单纯低氧组。显微镜下见SPCs输注低氧大鼠肌化动脉壁见GFP阳性细胞，部分细胞同时α-SMA阳性。接受lentivirus-GFP-shMyocd转染SPCs低氧大鼠其mPAP、肺动脉肌化程度、肌化动脉平均厚度较lentivirus-GFP-NC转染SPCs低氧大鼠明显减低。最后，对显微切割分离的肌化肺动脉组织myocardin mRNA定量分析表明，输注转染lentivirus-GFP-shMyocd的SPCs低氧大鼠肌化肺动脉myocardinmRNA较转染对照质粒祖细胞低氧大鼠及单纯输注祖细胞低氧大鼠均显著下降，并与其mPAP及肌化程度变化相一致。结论：低氧诱导归巢到肺动脉的CXCR4~+/PDGFRβ~+SPCs表达myocardin并调控其分化为VSMCs从而参与低氧性肺无肌细动脉肌化。
结论：
结合前面三部分的结果，我们认为：低氧能诱导循环CXCR4~+／PDGFRβ~+的SPCs归巢至肺无肌细动脉并促进归巢的SPCs表达myocardin；在myocardin的介导下归巢的SPCs进而分化为VSMC。该过程可能在低氧性肺无肌细动脉的肌化过程中起重要作用。
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.
PartⅠ:Isolation, Identification and Differentiation of Rat Circulating Smooth MuscleProgenitor Cells
Objective: 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%O_2), 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.
PartⅡ:
Expression and Contribution of Myocardin in the Differentiation ofCXCR4~+/PDGFRβ~+ Progenitor Cell into SMCs in vitro
Objective: 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 1×10~9 TU/ml and 5×10~9 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.
PartⅢ:
Contribution of Myocardin-Regulated Differentiation of SPCs in Hypoxia-InducedMuscularization of Non-muscular Pulmonary Arterioles
Objective: 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 ~(60)Co irradiated WT C57BL6/J mice, and the chimericmice were subjected to hypoxia (10%O_2) 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.
第一部分：大鼠循环平滑肌祖细胞的分离、鉴定及分化
目的：分离大鼠循环血液中的平滑肌祖细胞(SPCs)并鉴定其表型，为后续研究打下基础。方法：采用单核细胞集落刺激因子(M-CSF)注射法动员大鼠骨髓干细胞，第六天收集循环血液经密度梯度离心法获得单个核细胞(MNCs)，用磁激活的细胞分选法(MACS)分离具有CXCR4~+／PDGFRβ~+表型的祖细胞。采用富含PDGF-BB的培养液常规条件下诱导祖细胞分化，用细胞免疫荧光，RT-PCR，Western blot检测分化细胞的SMCs表型标记物，透射电镜检测分化细胞的超微结构从而判断分离的祖细胞的SMCs分化潜能；低氧(1％O_2)条件下培养上述祖细胞，观察祖细胞的SMCs分化情况，用细胞免疫荧光，RT-PCR，Western blot检测分化细胞的SMCs表型标记物，透射电镜检测分化细胞的超微结构从而判断低氧诱导的分化细胞是否为典型的SMCs。结果：从G-CSF动员的大鼠外周血液MNCs中成功分离出具有CXCR4~+／PDGFRβ~+表型的祖细胞，FCM证实纯度达93．7％，细胞免疫荧光证实祖细胞表达CXCR4及PDGFRβ。经富含PDGF-BB的无血清培养液诱导后，CXCR4~+／PDGFRβ~+祖细胞发生了典型的形态学变化，从小、圆形细胞转变为多角形、梭型细胞，细胞长满后具有SMCs的“峰谷”特征；免疫荧光、RT-PCR及western blot检测到分化细胞表达α-SMA，SM22α，calponin及SM-MHC，透射电镜证实分化细胞具有肌丝、密体，密斑等SMCs超微结构；低氧条件下，CXCR4~+／PDGFRβ~+祖细胞具有和PDGF-BB诱导下相似的形态学变化；免疫荧光、RT-PCR及Western blot检测到分化细胞表达α-SMA，SM22α，calponin及SM-MHC，透射电镜亦证实分化细胞具有肌丝、密体，密斑等SMCs超微结构。结论：大鼠循环CXCR4+~／PDGFRβ~+祖细胞为平滑肌祖细胞，体外低氧可诱导其分化为成熟的SMCs。
第二部分：Myocardin在CXCR4~+／PDGFRβ~+祖细胞体外SMC分化中的表达及作用
目的：检测Myocardin在CXCR4~+／PDGFRβ~+祖细胞分化过程中的表达，探讨干扰myocardin后对CXCR4~+／PDGFRβ~+祖细胞分化为SMCs的影响。方法：以慢病毒质粒pGCSIL-GFP为基础构建针对myocardin mRNA靶点的慢病毒干扰质粒(lentivirus-GFP-shMyocd)及阴性对照质粒(lentivirus-GFP-NC)；用有效靶点的myocardin慢病毒干扰质粒转染CXCR4+~／PDGFRβ~+祖细胞获得稳定表达，将稳定转染干扰病毒的祖细胞置于低氧条件下诱导分化，用RT-PCR及western blot检测不同时间点分化细胞内的myocardin及SMCs分化标志基因的表达。结果：成功构建滴度为1×10~9TU／ml的lentivirus-GFP-shMyocd和5×10~9TU／ml的lentivirus-GFP-NC干扰病毒。RT-PCR及western blot证实刚分离的CXCR4~+／PDGFRβ~+祖细胞不表达myocardin，PDGF-BB和低氧诱导后7天后表达myocardin，14天达到峰值，21天分化细胞成熟时其表达下降。用lentivirus-GFP-shMyocd预转染CXCR4~+／PDGFRβ~+祖细胞后再将细胞置于低氧条件下分化，RT-PCR及western blot结果显示lentivirus-GFP-shMyocd质粒有效减低了低氧所诱导的myocardin表达，敲减后的祖细胞中SMCs分化标记基因表达趋势与myocardin表达下降趋势相一致。结论：体外低氧诱导CXCR4~+／PDGFRβ~+祖细胞中myocardin的表达介导了祖细胞向SMCs分化。
第三部分：Myocardin调控CXCR4~+/PDGFRβ~+ SPCs参与低氧性肺无肌细动脉肌化
目的：探讨慢性低氧是否诱导循环平滑肌祖细胞(SPCs)归巢至肺无肌细动脉以及该归巢的SPCs在低氧诱导的肺无肌细动脉肌化中的作用。方法：GFP转基因C57BL6／J小鼠骨髓细胞移植到经过亚致死量~(60)Co照射的Wt C57BL6／J小鼠以形成骨髓细胞嵌合小鼠，嵌合成功的小鼠低氧(10％O_2)饲养4wk，采用激光共聚焦显微镜观察肺动脉上GFP荧光的分布，western blot分析肺组织内GFP蛋白的表达；用慢病毒干扰质粒转染CXCR4~+／PDGFRβ~+ SPC形成稳定表达细胞，将该稳定转染的SPCs经颈静脉输入Wistar大鼠。经6wk低氧后检测大鼠平均肺动脉压(mPAP)，处死大鼠取出肺组织，免组织化学染色检测肺血管α-SMA表达，结合弹力纤维染色判断肺动脉肌化程度，测定大鼠肌化血管壁的平均厚度；显微切割肌化血管并经定量PCR检测肌化肺动脉中myocardin mRNA的表达；同时冰冻切片经免疫荧光标记VEVSMC的α-SMA，激光共聚焦显微镜下观察α-SMA的表达及定位，结合GFP的表达情况判断输入的SPCs在肌化动脉上的定位及分化情况。结果：骨髓细胞嵌合小鼠经4wk低氧后观察到肺血管中具有较多的GFP信号，western blot亦证实嵌合小鼠低氧下较常氧条件下肺组织内GFP蛋白显著增加。Wistar大鼠经6wk低氧后，mPAP、肺动脉肌化程度及肌化血管平均厚度明较常氧组升高。接受SPCs输注低氧大鼠较未接受SPCs输注大鼠其mPAP、肺动脉肌化程度、肌化血管平均厚度均高于单纯低氧组。显微镜下见SPCs输注低氧大鼠肌化动脉壁见GFP阳性细胞，部分细胞同时α-SMA阳性。接受lentivirus-GFP-shMyocd转染SPCs低氧大鼠其mPAP、肺动脉肌化程度、肌化动脉平均厚度较lentivirus-GFP-NC转染SPCs低氧大鼠明显减低。最后，对显微切割分离的肌化肺动脉组织myocardin mRNA定量分析表明，输注转染lentivirus-GFP-shMyocd的SPCs低氧大鼠肌化肺动脉myocardinmRNA较转染对照质粒祖细胞低氧大鼠及单纯输注祖细胞低氧大鼠均显著下降，并与其mPAP及肌化程度变化相一致。结论：低氧诱导归巢到肺动脉的CXCR4~+/PDGFRβ~+SPCs表达myocardin并调控其分化为VSMCs从而参与低氧性肺无肌细动脉肌化。
结论：
结合前面三部分的结果，我们认为：低氧能诱导循环CXCR4~+／PDGFRβ~+的SPCs归巢至肺无肌细动脉并促进归巢的SPCs表达myocardin；在myocardin的介导下归巢的SPCs进而分化为VSMC。该过程可能在低氧性肺无肌细动脉的肌化过程中起重要作用。
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.
PartⅠ:Isolation, Identification and Differentiation of Rat Circulating Smooth MuscleProgenitor Cells
Objective: 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%O_2), 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.
PartⅡ:
Expression and Contribution of Myocardin in the Differentiation ofCXCR4~+/PDGFRβ~+ Progenitor Cell into SMCs in vitro
Objective: 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 1×10~9 TU/ml and 5×10~9 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.
PartⅢ:
Contribution of Myocardin-Regulated Differentiation of SPCs in Hypoxia-InducedMuscularization of Non-muscular Pulmonary Arterioles
Objective: 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 ~(60)Co irradiated WT C57BL6/J mice, and the chimericmice were subjected to hypoxia (10%O_2) 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.
引文
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