Abstract 427: Transforming Growth Factor β/Smad3 Stimulates Smooth Muscle Cell Migration and Proliferation Through a Signaling Pathway that Involves C-X-C Chemokine Receptor Type 4 Expression and Activation of Mitogen-Activated Protein Kinase
Introduction:Intimal hyperplasia is a complex process that requires vascular smooth muscle cell (SMC) migration and proliferation. We have previously shown that levels of transforming growth factor beta (TGF-β) and its signaling protein Smad3 are elevated following arterial injury and contribute to intimal hyperplasia. In this study we exam whether these effects might be mediated through a mechanism that involves the chemokine stromal cell derived factor (SDF-1α) and its receptor C-X-C receptor type 4 (CXCR4).
Methods and Results: A microarray was used to better understand the signaling pathways through which TGF-β and Smad3 stimulate SMC proliferation and migration. Overexpression of Smad3 in SMCs followed by stimulation with TGF-β (5 ng/ml) for 6 hours resulted in significant alteration of ∼300 genes, the most dramatic effect being a >500 fold upregulation of CXCR4. Quantitative RT-PCR and Western blotting confirmed that Smad3/TGF- β increased CXCR4 transcription and protein expression by 155±9 and 24±5 -fold, respectively. Immunocytochemistry and flowcytometry analysis revealed a 6-fold increase in CXCR4 positive cells in TGF-β stimulated Smad3 overexpressing SMCs compared to control (P<0.05). TGF-β/Smad3 treated SMCs exhibited enhanced migration (4-fold VS control, P<0.01) in response to SDF-1α (100 ng/ml) which was eliminated after preincubation of SMCs with CXCR4 inhibitor AMD3100 (10 μM). Moreover, there was a 45±9% increase in proliferation (BrdU) in response to SDF-1α in TGF-β/Smad3 treated SMCs compared to GFP control (P<0.05). To explore the mechanism by which SDF-1α/CXCR4 produces proliferation and migration in SMCs, we studied the intracelluar signaling protein mitogen-activated protein kinase (MAPK), which is a known intermediate for both processes. Incubation of TGF-β/Smad3 SMCs with SDF-1α led to activation of MAPK (11-fold) as well as nuclear translocation of CXCR4 in a dose and time dependent manner.
Conclusions: Our data demonstrate that TGF-β through Smad3 stimulates expression of CXCR4 in medial vascular SMCs. These SMCs in response to SDF-1α then proliferate and migrate to the subintimal layer. These findings suggest a novel pathway through which TGF-β enhances intimal hyperplasia at sites of arterial injury.
- © 2012 by American Heart Association, Inc.