Abstract 16488: Overexpression of the Human Form of the Novel Extracellular Matrix Protein Podocan Inhibits Migration and Proliferation of Human Vascular Smooth Muscle Cells via Down-Regulation of the Wnt-Pathway
Background: Vascular smooth muscle cell (VSMC) biology modulates the clinical course of vascular disease. Podocan is a novel member of the small leucin rich repeat protein (SLRP) family. We have recently reported exuberant neointima formation in response to arterial injury in podocan knockout (KO) mice and increased migration and proliferation of VSMC derived from KO mice. Increased Wnt-pathway activation in KO VSMC has been shown. Now we report the effect of the human form of podocan on human VSMC migration and proliferation in vitro. In addition, we examined the role of the Wnt-pathway under these conditions
Methods and Results: Primary human aortic VSMC's were grown in DMEM under standard conditions. VSMC's were serially passaged before reaching confluence and all experiments were performed on VSMC's from passages 2 to 4. Podocan was highly expressed in a time dependent fashion in VSMC treated with podocan expression vector compared to vector control and untreated VSMC as assessed by Western Blot. Podocan overexpression resulted in a 29% reduction in VSMC migration compared to control as measured by the MTS assay (Promega) (0.395±0.075 vs. 0.557±0.097, P<0.05). Similarly, podocan overexpression significantly reduced VSMC proliferation in a time-dependent fashion. Using a BRDU incorporating assay (Roche) we found first a 9.7 % (at 12 h) and then a 32 % (at 24 h) inhibition of VSMC proliferation with podocan (0.152±0.003 vs. 0.223±0.007, P<0.05 at 24 hours). In addition, podocan overexpressing VSMC's showed a significant increase in phospho-beta catenin over non-phospho-beta catenin by Western Blot. Consistent with this inhibitory effect in podocan overexpressing VSMC we also found a significant reduction of the cell cycle activating Cyclin D1 protein as quantified by densitometry and expressed as % expression of non-transfected control cells (72% with podocan expression vector treated VSMC vs. 101% with vector alone, P<0.05).
Conclusions: The specific and time dependent inhibition of VSMC migration and proliferation by podocan strongly suggest podocan as a negative regulator of VSMC function via the Wnt-pathway. Our findings also point to podocan as a novel and potent therapeutic target for better treating conditions of accelerated arteriosclerosis.
- © 2011 by American Heart Association, Inc.