α_vβ₃-Integrin antagonists inhibit thrombin-induced proliferation and focal adhesion formation in smooth muscle cells

Abstract

Sajid, M., R. Zhao, A. Pathak, S. S. Smyth, and G. A. Stouffer. ␣ v␤3-Integrin antagonists inhibit thrombin-induced proliferation and focal adhesion formation in smooth muscle cells. Am J Physiol Cell Physiol 285: C1330-C1338, 2003. First published July 23, 2003 10.1152 10. /ajpcell.00475.2002tegrin antagonists reduced neointimal formation following vascular injury in eight different animal models. Because ␣-thrombin contributes to neointimal formation, we examined the hypothesis that ␣ v␤3-integrins influence ␣-thrombin-induced signaling. Cultured rat aortic smooth muscle cells (RASMC) expressed ␣ v␤3-integrins as demonstrated by immunofluorescence microscopy and fluorescence-activated cell sorting analysis. Proliferative responses to ␣-thrombin were partially inhibited by anti-␤ 3-integrin monoclonal antibody F11 and by cyclic RGD peptides. Immunofluorescence microscopy showed that ␣-thrombin stimulated a rapid increase in the formation of focal adhesions as identified by vinculin staining and that this effect was partially inhibited by ␣ v␤3 antagonists. ␤3-Integrin staining was diffuse in quiescent RASMC and did not concentrate at sites of focal adhesions following thrombin treatment. ␣-Thrombin elicited a time-dependent increase in activation of c-Jun NH 2-terminal kinase-1 (JNK1) and in tyrosine phosphorylation of focal adhesion kinase (FAK). ␣ v␤3-Integrin antagonists partially inhibited increases in JNK1 activity but had no effect on FAK phosphorylation. In SMC isolated from ␤ 3-integrin-deficient mice, focal adhesion formation was impaired in response to thrombin but not sphingosine-1-phosphate, a potent activator of Rho. In summary, ␣ v␤3-integrins play an important role in ␣-thrombin-induced proliferation and focal adhesion formation in RASMC.receptors; vitronectin; integrins; focal adhesions PERCUTANEOUS CORONARY INTERVENTION (PCI) involves application of a controlled injury to a coronary artery to relieve focal obstruction. The injury stimulates a healing response, which, when overexuberant, leads to renarrowing of the artery in a process labeled "restenosis." Animal models have implicated ␣ v ␤ 3 -integrins in playing an important role in vascular healing responses. Studies in baboons, rats, rabbits, and pigs have shown that balloon angioplasty is a stimulus for ␤ 3 -integrin expression by vascular smooth muscle cells (SMC) (6,21,23) and that treatment with ␣ v ␤ 3 antagonists reduces (neo)intima formation after vascular injury (reviewed in Ref. 19).␣ v ␤ 3 -Integrins influence responses of cultured SMC to many extracellular agonists that may be important in healing responses in injured blood vessels. In addition to inhibiting responses to agents that directly bind ␣ v ␤ 3 , such as thrombospondin and osteopontin (23,24,27), ␣ v ␤ 3 antagonists also regulate SMC responses to platelet-derived growth factor (3, 4), epidermal growth factor (10), insulin-like growth factor-I (9), transforming growth factor-␤ (18), and ␣-thrombin (24). ␣-Thrombin is of particular interest because it is concentrated at ...

Thrombin Functions through Its RGD Sequence in a Non-canonical Conformation

“…These conditions may be mimicked by surface-coating or crystallographic packing and may reproduce the crowded environment of the extracellular matrix where thrombin migrates upon vascular injury (6 -8, 15, 16). However, recent studies have suggested that native thrombin in solution can interact with ␣ v ␤ 3 integrin in a way that can be inhibited specifically by RGD mimetics (17,18), which raises the possibility that exposure of the RGD sequence may also occur independent of matrix attachment. The conformation of molecule-2 reported here likely captures thrombin in the act of completely unraveling the RGD sequence and the 220-loop region, which may be revealed in future studies of the free form of the enzyme.…”

“…However, recent studies have shown that surface-absorbed thrombin, or its active site inhibited form, promote attachment, migration, and survival of endothelial cells via the ␣ ␤ 3 integrin (15,16). Other studies have provided evidence that thrombin in solution can interact with ␣ ␤ 3 integrin in a way that is inhibited by RGD mimetics (17,18). Hence, the enzyme may expose the RGD sequence independent of denaturation or proteolysis, as reported here.…”

Eptifibatide and abciximab inhibit insulin-induced focal adhesion formation and proliferative responses in human aortic smooth muscle cells

“…The cells were grown in media that was a 1:1 mixture of regular DMEM and smooth muscle proliferation medium with a glucose concentration of 15.27 mM. Cell proliferation, flow activated cell sorting (FACS) analysis, apoptosis assays, focal adhesion assays and cell adhesion assays were performed as previously described[ 4 , 9 ].…”

Effects of Thrombin on Interactions Between β 3 -Integrins and Extracellular Matrix in Platelets and Vascular Cells

“…We have also found that focal adhesion formation in response to thrombin treatment is impaired in SMCs isolated from ␤ 3 -integrin-deficient mice. 88 …”