Abstract 281: Cytostatic Gene, l-Caldesmon, Suppressed Proliferation and Migration of Vascular Smooth Muscle Cells and Inhibits Neointimal Formation by Regulating the FAK-ERK Axis
Light type-caldesmon (l-CaD) is a cytostatic gene which is induced after mechanical stimuli to regulate cell growth and survival via modulation of cell shape and cytoskeleton. The aim of this study is to evaluate the inhibitory effect of l-CaD on neointimal formation after angioplasty like other cytostatic agents such as rapamycin and paclitaxel, which are predominantly used for drug eluting stents. Methods and Results: We tested cytostatic functions of l-CaD in human vascular smooth muscle cells (VSMCs) using assays for apoptosis, cell proliferation, and migration, and evaluated the expression pattern of each marker protein (focal adhesion kinase (FAK) and MAPKs) in VSMCs. Transfection of adenoviral vector encoding l-CaD (Ad-l-CaD) resulted in progressive loss of actin stress fibers and cell retraction. WST-1 proliferation assay demonstrated that l-CaD overexpression gradually decreased cell proliferation rate by 78% in VSMCs over 72 hours(p=0.011, n=6). ELISA demonstrated that Ad-l-CaD transfection increased the apoptosis rate by 75% in accordance with inhibition of bcl-2 expression and upregulation of caspase-3 activity, and reduced BrdU uptake by 49% (p=0.012, n=4 each vs. control of GFP alone). Furthermore, transfection of Ad-l-CaD inhibited migration of VSMCs induced by platelet-derived growth factor-BB (PDGF) by 36% (p=0.031, n=4). Immunoblotting analysis revealed that l-CaD overexpression reduced PDGF-induced phosphorylation of both FAK and extracellular signal regulated-kinase (ERK), but no significant reduction was seen in p38MAPK and SAPK/JNK. In balloon-injured rat carotid arteries, Ad-l-CaD transfection inhibited neointimal formation by 37% (Table⇓, p=0.042, n=6 each) without delaying reendothelializa-tion and thrombus occlusion at 14 days. Conclusions: Overexpression of l-CaD suppressed cell growth and survival in VSMCs and inhibited neointimal formation after experimental angio-plasty, partly by regulating the FAK-ERK axis.