| Hypoxia pulmonary hypertension (HPH) is characterized by a process ofpulmonary vasoconstriction, pulmonary hypertrophy and pulmonary vascularremodeling. An important pathological feature of pulmonary hypertension isincreased medial thickening of the pulmonary artery attributable to hypertrophyand hyperplasia of pulmonary arterial smooth muscle cell (PASMC). Ourprevious studies have shown that anti-proliferative Sodium tanshinone IIAsulphonate (STS) significantly inhibited both the pulmonary vascularremodeling induced by hypoxia in rats and the PASMC proliferation in culture.Other investigators also have reported that STS inhibits PASMC proliferation invitro and in vivo. To date, however, the mechanism by which STS inhibitsPASMC proliferation has not been fully elucidated. Cell proliferation is tightly controlled by cyclin-dependent kinases (CDK)and cyclin-dependent kinases inhibitor (CDKI) during the cell cycle. As one ofthe important CDKIs, p27kip1 (p27) was originally identified as a protein capableof inhibiting G1 cyclin/CDK complexes. It has been shown that p27 gene is theonly CDKI which was decreased in hypoxic exposure of mice. Otherinvestigators have found that overexpression of p27 was associated withattenuated PASMC proliferation. The S phase kinase associated protein 2 (Skp-2)was found as a member of the large eukaryotic family of F-box proteins whichfunctions specifically for the degradation of p27. After activated by thephosphorylatedAkt (p-Akt), Skp-2 mediates the proteolysis of p27.In the present study, we employed STS to investigate the roles of p27 andits closely-related kinase Skp2 in the progression of pulmonary vascularremodeling and HPH. In addition, we constructed the HS-p27-pAcGFP1-1expression plasmid (HRE enhancer-sm22 promoter driven p27 expressionplasmids), to test its role of inhibiting the hypoxia-induced PASMCproliferation.Part I: Cyclin-dependent kinase inhibitor p27 is required for inhibition ofhypoxia-induced pulmonary hypertension and remodeling by STS1. ObjectiveTo investigate therole of the CDK inhibitors p27 in the inhibitory effect ofSTS on PASMC proliferation in vitro and on the development ofhypoxia-induced pulmonaryhypertension and remodeling in vivo.2 MethodsSprague-Dawley rats treated with or without STS were challenged byintermittent chronic hypoxia exposure for 4 weeks to establish hypoxic pulmonary hypertension models. Subsequently, hemodynamic and pulmonarypathomorphology data were gathered. Additionally, PASMC were cultured todetermine the anti-proliferation effect of STS under hypoxia exposure. Westernblotting or reverse transcriptional polymerase chain reaction (RT-PCR) wereadopted to test p27, Skp-2 and p-Akt changes in rat lung tissue and culturedPASMC. Cell proliferation was detected by MTT assay. Cell cycle percentagewas detected byflow cytometry analysis.3 ResultsChronic hypoxia significantly increased mean pulmonary arterial pressure(mPAP), right ventricular hypertrophy index (RVHI) and medial width ofpulmonary arterioles, accompanied with decreased expression of p27 in rats. Asexpected, STS treatment repressed the elevation of mPAP, RVHI, and attenuatedthe remodeling of pulmonary arterioles induced by chronic hypoxia.Interestingly, STS treatment also induced the expression of p27 both in vivo andin vitro. This study also showed that STS treatment suppressed the proliferationof PASMC and elevated the expression of p27 under hypoxia exposure. Inaddition, experiments both in vivo and in vitro consistently demonstrated anincreased expression of Skp-2 and p-Akt kinases under hypoxia. Besides, allthese changes were alleviated in the presence of STS.4 ConclusionsSTS can effectively attenuate the remodeling of pulmonary arteriolesinduced by chronic hypoxia. The underlying mechanism may partially bethrough the increased p27 by inhibiting Skp-2 through Akt signal pathway.Therefore, targeting up-regulation of p27 might provide new strategies fortreatment of HPH. Partâ…¡The role of HS-p27-pAcGFP1-1 in inhibiting the proliferation ofPASMC with specificity and effectiveness1. ObjectiveMake a HRE enhancer-sm22 promoter driven p27 expression plasmidwhich can be efficently expressed in hypoxia and capable of inhibiting theproliferation of PASMC.2 Methods(1) To construct five plasmid expression vectors pcDNA3.1-p27,sm22-pAcGFP1-1,HRE-sm22-pAcGFP1-1,sm22-p27-pAcGFP1-1,HS-p27-pAcGFP1-1 by PCR,enzyme restriction and ligation techniques.(2) To transfect the vector pcDNA3.1-p27 into PASMC; total protein wereextracted and p27 expression were detected by Western blot, cell proliferationwere detected byMTTassay.(3) To transfect the vector sm22-pAcGFP1-1 into PASMC, A10, 3T3, A549,and 293 cells, green fluorescence protein were examined with fluorescencemicroscopy.(4) To transfect the vector HRE-sm22-pAcGFP1-1 into PASMC innormoxic (control) or hypoxia circumstance, green fluorescence protein wereexamined with fluorescence microscopy.(5) To transfect the vector HS-p27-pAcGFP1-1 into PASMC in hypoxiacircumstance, total protein were extracted, p27 and Skp2 expression weredetected by Western blot.Cell proliferation was detected by MTT assay. Cellcycle percentage was detected byflow cytometry analysis.3 Results(1) p27-pcDNA3.1,sm22-pAcGFP1-1,HRE-sm22-pAcGFP1-1, sm22-p27-pAcGFP1-1,HS-p27-pAcGFP1-1 were constructed successfully andidentified bysequencing.(2) Overexpression of p27 in transfected p27-pcDNA3.1 was confirmed inthe protein level. The proliferation of PASMC was suppressed in tansfectingp27.(3) Green fluorescence protein was expressed in PASMC and A10 cell withtransfecting sm22-pAcGFP1-1.(4) The expression of Green fluorescence protein in transfectedHRE-sm22-pAcGFP1-1 markedlyincreased in PASMC hypoxia.(5) Overexpression of p27 in transfected HS-p27-pAcGFP1-1 wasconfirmed from the protein level. The proliferation and cell cycle progression ofPASMC was suppressed in tansfecting HS-p27-pAcGFP1-1.4 Conclusions:Recombinant vectors HS-p27-pAcGFP1-1 can obviously, specificlyinhibited the hypoxia-induced PASMC proliferation.
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