Abstract 11759: Poldip 2 Regulates Adhesion and Spreading of Vascular Smooth Muscle Cells Through Its Interaction With Kindlin-2 and Talin
Vascular smooth muscle cell (VSMC) migration is a key step in atherosclerosis. Cell migration depends on adhesion to a substrate, extension of lamellipodia, and formation of focal adhesions (FA). Polymerase Delta-Interacting Protein 2 (Poldip2) overexpression impairs VSMC migration. We hypothesize that Poldip2 interaction with FA proteins Kindlin-2 (KIN) and Talin (TL) increases VSMC adhesion and spreading through integrin activation.
Methods: VSMCs were transduced with control adenovirus (AdGFP) or adenovirus overexpressing Poldip2 (AdPol). Transduced VSMCs were plated on different matrices (Poly-L-Lysine (PLL), Collagen I (Col), Fibronectin (FN), and Vitronectin (VN)). Adherence of VSMCs was measured using fluorescence, and area was calculated using Image Pro. Strength of adhesion was measured using a centrifugation assay. Interaction of Poldip2 with KIN and TL was assessed using the Duolink assay and immunocytochemistry (ICC). Quantification of integrin expression was measured by flow cytometry.
Results: AdPol-transduced VSMCs adhered significantly more to PLL, Col, VN (p<0.05), and potentially to FN (p=0.07). AdPol VSMCs spread more compared to AdGFP VSMCs on Col, FN and VN at 2 hours after seeding (Medians(μ m2) 440.7 vs. 291.7, 280.3 vs. 244, 232.2 vs. 201.6 respectively p=0.0001 for all comparisons, n=750 cells in each group from three separate experiments); spreading on PLL was similar (p=0.391). AdPol cells adhered more strongly to Col, FN and PLL compared to AdGFP cells. Poldip2 interacted with both TL and KIN, and colocalized with each of them in focal adhesions. AdPol cells had higher levels of activated β1 integrin compared to AdGFP cells (32.8 % vs. 22.9%, p=0.055, n=2), but there was no difference in β1, β3 or αvβ3 integrin expression on the cell surface.
Conclusion: Poldip2 regulates cell adhesion and spreading through interaction with the focal adhesion proteins Kindlin-2 and Talin and through increased activation of β1 integrins.
- © 2012 by American Heart Association, Inc.