Establishment Of Human Cell Model That Adult Vascular Smooth Muscle Cell Phenotypes Can Convert Reversibly And Changes Of Plasminogen Activator Inhibitor-1 During This Conversion

Objective Vascular smooth muscle cells (VSMCs) perform diverse functions that can be categorized as contractile and synthetic. A classic view was that adult VSMCs have the capacity to modulate their phenotypes in a bi-directional manner in order to perform these diverse functions. However, this view has been challenged, in part because there is no evidence that an adult synthetic VSMCs can acquire the ability to contract. With the study of heterogeneity among VSMCs there is an idea of the selective VSMCs expansion that there is no a prior need to invoke VSMC phenotype shift to explain the emergence of a noncontractile phenotype. Instead, noncontractile or synthetic functions may arise by selective expansion of cell subpopulations. Then whether VSMCs need phenotype shift is controversial. In human there is little support forthe functions of different subpopulation, because isolation of pure human VSMC subsets has been proven difficult. The present study aimed to establish human VSMCs clones model that can stably passage and maintain its physiological characters. (2)We sought characters for VSMCs clones that can convert reversibly between two phenotypes and induced VSMCs to perform contractile function in vitro. (3)The changes of PAI-1 in cloned VSMC stimulated with PDGF-BB and then observed that there is relationship between PDGF-BB and PAI-1 by different dose and time of PDGF-BB.Method (DEstablish human cell model that adult VSMC can convert reversibly between noncontractile and contractile phenotypes by means of cloning; (2)stable VSMC clones were divided into 3 groups. Ml99 with serum group^ Ml 99 with serum withdrawal and M199 with restoration of serum. Study VSMCs characters and the relative of phenotype and contractile state: ?Evaluation of the morphology of cloned VSMCs ; ?Assessment of cell proliferation and DNA synthesis , proliferative and migrative response to PDGF-BB, then the response to PDGF-BB with different dose and different stimuli time by counting with a hemacytomeler, measuring incorporation of [3H] thymidine and wounding assay; ㏄rotein synthesis assay by measuring in corporation of [3H] leucine.; ㏕he contraction of cloned VSMC in response to histamine or angiotensin II by two approaches-measuring planar area and cell length and then Receptor antagonist Pyrilamine and losartan were added 10 minutes before adding of agonist so that we can observe whether contraction of cloned VSMC is inhibited. ?Expression of contractile apparatus proteins incloned VSMC by western blot analysis. (3) The changes of PAI-1 in cloned VSMC stimulated with PDGF-BB and then observed that there is relationship between PDGF-BB and PAI-1 by different dose and time of PDGF-BB.Result We have successfully applied a cloning strategy to obtain VSMC clones-HITB5 that can stably passage and maintain its physiological characters. HITB5 can convert reversibly between non-contractile and contractile phenotypes. The synthetic VSMC can acquire the ability to contract in vitro. (2)HITB5 that can contract in serum and exist in synthetic phenotype is its inherent physiological character. It can convert completely into contractile phenotype 72h after serum withdrawal. (3)Serum withdrawal induced synthetic HITB5 state into contractile state: ?Morphological changes: after serum withdrawal, HITB5 cell underwent a striking change to a highly elongated cell and was more refractive to phase-contrast light. The population of cell organized to form dense, multilayered cell bundles that appeared as well-aligned ridges reminiscent of the organization of VSMCs in blood vessels. This morphology can be maintained for 2w. Two days after serum restoration, however, the cell bundle disassembled and the cells lost the organized pattern that is indistinguishable from to that before serum withdrawal, which suggested serum withdrawal induced reversible morphological changes in HITB5. (2)On serum withdrawal, HITB5 proliferation and [3H] thymidine in corporation fell to near 0, which suggested DNA synthesis stopped. Production of solu...