Influence Of PDGF-BB And PKGIα On Morphology And Synthetic Phenotype Marker Genes Of Vascular Smooth Muscle Cells

Objective:To investigate the influence of PDGF-BB and cGMP dependent protein kinase I a (PKGIa) on morphology and expression of synthetic phenotypic marker genes of rat vascular smooth muscle cell.Methods:(1) Recombinant adenovirus encoding gene PKGIα(pAd-PKGIα) was generated by site-specific recombination. Viral tittering was proceeded to determine the titer of the adenoviral stock; (2) Explant culture and growth of rat primary rat aortic smooth muscle cells (RASMCs);(3)RT-PCR was applied to detect the expression of PKGIa in RASMCs and CS-54 cells infected by pAd-PKGIα. Western-blotting assay was applied for detecting the activation of PKGIa by an isozyme-selective activator 8-APT-cGMP; (4) Live cell imaging was applied to observe the influence of PDGF-BB and PKGIa on morphology of RASMCs and CS-54 cells;(5) Immunofluorescence assay was applied to observe the1 influence of PDGF-BB and PKGIa on cytoskeleton of RASMCs and CS-54 cells; (6) Real-time PCR was applied to detect the influence of PDGF-BB and PKGIa on expression of synthetic phenotypic marker genes in CS-54 cells; (7) Western-blotting was applied to detect the influence of PDGF-BB and PKGIαon protein expression of synthetic phenotypic marker genes in CS-54 cells;Results:(1) Plaque titration on 293 cells showed titers of 5×10¹⁰ pfu/ml and 7.5×10¹⁰ pfu/ml for pAd-PKGI a and pAd-LacZ respectively. The multiplicity of infection were both 100; (2) Cells cultured by explant-attachment method were proved to be vascular smooth muscle cells (VSMC) via morphology and immunofluorescence assay. (3) Expression of PKGIαmRNA in both CS-54 cells and RASMCs infected with recombinant adenoviral construct pAd-PKGIa were significantly increased. And 8-APT-cGMP treatment leaded to activation of PKGIa; (4) PDGF-BB has an influence on morphology and cytoskeleton organization of CS-54 cells and RASMCs, and increase of the expression and the activity of PKGI a can reverse the effect; (5) PDGF-BB was proved to increase the expression of a synthetic phenotypic marker gene Epiregulin. PKGIαand 8-APT-cGMP can reduce the expression of Epiregulin to control level. Similar results were not found in other synthetic phenotypic marker genes like OPN and MMP2.Conclusion:PDGF-BB could cause obvious changes in morphology and cytoskeleton of VSMCs, leading to a phenotypic modulation from contractile phenotype to synthetic one. Treatment of PKGI and 8-APT-cGMP could reverse this procedure. PDGF-BB was proved to increase the expression of a synthetic phenotypic marker gene Epiregulin. Meanwhile, PKGIa and 8-APT-cGMP can reduce the expression of Epiregulin to control level. There were no similar results in other two synthetic phenotypic marker genes OPN or MMP2.