Expression of Smooth Muscle-specific Molecules and Phenotypic Changes in HITASY Cells by Serum Withdrawal

Human internal thoracic artery cells (HITASY) were used to explore the vascular smooth muscle cells (SMCs) converted from a synthetic into a contractile phenotype characterized by serum withdrawal, which may contribute to neointimal formation and restenosis after vascular injury. Confluent monolayer of HITASY cells cultured in M199 serum-free medium exhibited morphological and functional characteristics consistent with a differentiated phenotype. Smooth muscle cell contractile phenotype was determined by observing the expression of smooth muscle myosin heavy chain (SMMHC, a marker of fully differentiated smooth muscle ) and smooth muscle alpha-actin ( a marker for all smooth muscle, including immature smooth muscle). Serum withdrawal induced a prominent decrease in extracellular matrix protein synthesis resulting in a 58% lower cell number. Nonsignificant proliferation HITASY cells treated with platelet derived growth factor (PDGF) was detected. Western blot analysis revealed a reversible upregulation of smooth muscle α-actin, calponin, caldesmon and SMMHC. Further, RT-PCR also indicated the expression of smooth muscle alpha 22 gene in HITASY cells after serum withdrawal. Vasoactive agonist stimulated robust calcium oscillations that coupled cell contraction in HITASY cell area or length on average. Differential display PCR was used to screen differential expression genes in HITASY cells, and the data showed that HITASY cells have the potential to express cellular repressor of E1A-stimulated genes, only after cultured in medium serum-free. Following readdition 10% serum, SMCs underwent a reversible dedifferentiation, followed by proliferation. These findings support the pivotal role of SMCs phenotype modulation in vitro.