软骨寡聚基质蛋白在SD大鼠肺动脉平滑肌表型转化中的作用
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摘要
目的探讨软骨寡聚基质蛋白(COMP)对肺动脉平滑肌细胞(PASMCs)表型转化的影响。方法利用常氧和缺氧条件培养的SD大鼠(21%O_2或10%O_2饲养21d)与PASMCs(21%O_2或3%O_2培养48h),采用免疫组织化学染色、Western blot和q-PCR方法检测大鼠肺组织和PASMCs COMP表达;qPCR法检测特异性siRNA敲低COMP条件下,SM22α-、SMα-actin-和calponin mRNA水平的表达;Western blot法检测缺氧、siRNA-COMP或腺病毒过表达COMP后,SM22α、SMα-actin、calponin、osteopontin和proliferating cell nuclear antigen(PCNA)蛋白的表达。结果缺氧条件下,大鼠肺组织和PASMCs中COMP表达水平降低;COMP缺失引起PASMCs收缩表型蛋白SM22α和calponin的表达减少,osteopontin和PCNA表达增加;过表达COMP可以逆转缺氧引起的肺动脉平滑肌细胞SM22α和calponin表达的减少。结论 COMP有助于维持缺氧条件下肺动脉平滑肌处于收缩表型。
Objective To study effects of cartilage oligomeric matrix protein(COMP) on the phenotype switching of pulmonary arterial smooth muscle cells(PASMCs). Methods The expressions of COMP, SM22α, SMα-actin,calponin, osteopontin and proliferating cell nuclear antigen(PCNA) mRNAs or proteins were determined by Western blot,qPCR or immunohistochemistry in SD rats(21%O_2 or 10%O_2 for 21 days) and PASMCs(cultured under 21%O_2 or 3%O_2 for48 hours with/without siRNA or adenovirus treatment). Results The expression levels of COMP decreased in a chronic hypoxia rat PH model(P<0.05) and PASMCs. The expressions of SMα-actin、SM22α and calponin reduced in PASMCs under hypoxia or with the siRNA of COMP treatment under normoxia, but increased in PASMCs treated with adenovirus overexpressing COMP under hypoxia. The absorbance of cell counting kit-8 at 450 nm and the expressions of proliferating cell nuclear antigen(PCNA) and osteopontin increased in PASMCs with the siRNA of COMP under normoxia(P<0.05).Conclusion COMP could maintain the contratile phenotype in PASMC under hypoxia.
Objective To study effects of cartilage oligomeric matrix protein(COMP) on the phenotype switching of pulmonary arterial smooth muscle cells(PASMCs). Methods The expressions of COMP, SM22α, SMα-actin,calponin, osteopontin and proliferating cell nuclear antigen(PCNA) mRNAs or proteins were determined by Western blot,qPCR or immunohistochemistry in SD rats(21%O_2 or 10%O_2 for 21 days) and PASMCs(cultured under 21%O_2 or 3%O_2 for48 hours with/without siRNA or adenovirus treatment). Results The expression levels of COMP decreased in a chronic hypoxia rat PH model(P<0.05) and PASMCs. The expressions of SMα-actin、SM22α and calponin reduced in PASMCs under hypoxia or with the siRNA of COMP treatment under normoxia, but increased in PASMCs treated with adenovirus overexpressing COMP under hypoxia. The absorbance of cell counting kit-8 at 450 nm and the expressions of proliferating cell nuclear antigen(PCNA) and osteopontin increased in PASMCs with the siRNA of COMP under normoxia(P<0.05).Conclusion COMP could maintain the contratile phenotype in PASMC under hypoxia.
引文
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[12]Owens GK.Regulation of differentiation of vascular smooth muscle cells[J].Physiol Rev,1995,75(3):487~517.
[2]Kuhr FK,Smith KA,Song MY,et al.New mechanisms of pulmonary2arterial hypertension:role of Ca+signaling[J].Am J Physiol Heart Circ Physiol,2012,302(8):H1546~1562.
[3]Girgis RE,Mozammel S,Champion HC,et al.Regression of chronic hypoxic pulmonary hypertension by simvastatin[J].Am J Physiol Lung Cell Mol Physiol,2007,292(5):L1105~1110.
[4]Hedin U,Daum G,Clowes AW.Heparin inhibits thrombin-induced mitogen-activated protein kinase signaling in arterial smooth muscle cells[J].J Vasc Surg,1998,27(3):512~520.
[5]Pukac L,Huangpu J,Karnovsky MJ.Platelet-derived growth factorBB,insulin-like growth factor-I,and phorbol ester activate different signaling pathways for stimulation of vascular smooth muscle cell migration[J].Exp Cell Res,1998,242(2):548~560.
[6]Wagenseil JE,Mecham RP.Vascular extracellular matrix and arterial mechanics[J].Physiol Rev,2009,89(3):957~989.
[7]Oldberg A,Antonsson P,Lindblom K,et al.COMP(cartilage oligomeric matrix protein)is structurally related to the thrombospondins[J].J Biol Chem,1992,267(31):22346~22350.
[8]Nguyen BQ,Fife RS.Vitreous contains a cartilage-related protein[J].Exp Eye Res,1986,43(3):375~382.
[9]Riessen R,Fenchel M,Chen H,et al.Cartilage oligomeric matrix protein(thrombospondin-5)is expressed by human vascular smooth muscle cells[J].Arterioscler Thromb Vasc Biol,2001,21(1):47~54.
[10]Kipnes J,Carlberg AL,Loredo GA,et al.Effect of cartilage oligomeric matrix protein on mesenchymal chondrogenesis in vitro[J].Osteoarthritis Cartilage,2003,11(6):442~454.
[11]Chen FH,Thomas AO,Hecht JT,et al.Cartilage oligomeric matrix protein/thrombospondin 5 supports chondrocyte attachment through interaction with integrins[J].J Biol Chem,2005,280(38):32655~32661.
[12]Owens GK.Regulation of differentiation of vascular smooth muscle cells[J].Physiol Rev,1995,75(3):487~517.