Blockade of CD47 function attenuates restenosis by promoting smooth muscle cell efferocytosis and inhibiting their migration and proliferation
2023; Elsevier BV; Volume: 299; Issue: 4 Linguagem: Inglês
10.1016/j.jbc.2023.104594
ISSN1083-351X
AutoresSuresh Govatati, Prahalathan Pichavaram, Raj Kumar, Gadiparthi N. Rao,
Tópico(s)Extracellular vesicles in disease
ResumoCluster of differentiation 47 (CD47) plays an important role in the pathophysiology of various diseases including atherosclerosis but its role in neointimal hyperplasia which contributes to restenosis has not been studied. Using molecular approaches in combination with a mouse vascular endothelial denudation model, we studied the role of CD47 in injury-induced neointimal hyperplasia. We determined that thrombin-induced CD47 expression both in human aortic smooth muscle cells (HASMCs) and mouse aortic smooth muscle cells. In exploring the mechanisms, we found that the protease-activated receptor 1-Gα protein q/11 (Gαq/11)–phospholipase Cβ3–nuclear factor of activated T cells c1 signaling axis regulates thrombin-induced CD47 expression in HASMCs. Depletion of CD47 levels using its siRNA or interference of its function by its blocking antibody (bAb) blunted thrombin-induced migration and proliferation of HASMCs and mouse aortic smooth muscle cells. In addition, we found that thrombin-induced HASMC migration requires CD47 interaction with integrin β3. On the other hand, thrombin-induced HASMC proliferation was dependent on CD47's role in nuclear export and degradation of cyclin-dependent kinase–interacting protein 1. In addition, suppression of CD47 function by its bAb rescued HASMC efferocytosis from inhibition by thrombin. We also found that vascular injury induces CD47 expression in intimal SMCs and that inhibition of CD47 function by its bAb, while alleviating injury-induced inhibition of SMC efferocytosis, attenuated SMC migration, and proliferation resulting in reduced neointima formation. Thus, these findings reveal a pathological role for CD47 in neointimal hyperplasia. Cluster of differentiation 47 (CD47) plays an important role in the pathophysiology of various diseases including atherosclerosis but its role in neointimal hyperplasia which contributes to restenosis has not been studied. Using molecular approaches in combination with a mouse vascular endothelial denudation model, we studied the role of CD47 in injury-induced neointimal hyperplasia. We determined that thrombin-induced CD47 expression both in human aortic smooth muscle cells (HASMCs) and mouse aortic smooth muscle cells. In exploring the mechanisms, we found that the protease-activated receptor 1-Gα protein q/11 (Gαq/11)–phospholipase Cβ3–nuclear factor of activated T cells c1 signaling axis regulates thrombin-induced CD47 expression in HASMCs. Depletion of CD47 levels using its siRNA or interference of its function by its blocking antibody (bAb) blunted thrombin-induced migration and proliferation of HASMCs and mouse aortic smooth muscle cells. In addition, we found that thrombin-induced HASMC migration requires CD47 interaction with integrin β3. On the other hand, thrombin-induced HASMC proliferation was dependent on CD47's role in nuclear export and degradation of cyclin-dependent kinase–interacting protein 1. In addition, suppression of CD47 function by its bAb rescued HASMC efferocytosis from inhibition by thrombin. We also found that vascular injury induces CD47 expression in intimal SMCs and that inhibition of CD47 function by its bAb, while alleviating injury-induced inhibition of SMC efferocytosis, attenuated SMC migration, and proliferation resulting in reduced neointima formation. Thus, these findings reveal a pathological role for CD47 in neointimal hyperplasia. Vascular smooth muscle cell (VSMC) migration and proliferation play a crucial role in the development of neointimal hyperplasia following percutaneous transluminal angioplasty (1Ebert M.L.A. Schmidt V.F. Pfaff L. von Thaden A. Kimm M.A. Wildgruber M. Animal models of neointimal hyperplasia and restenosis: species-specific differences and implications for translational research.JACC Basic Transl. Sci. 2021; 6: 900-917Crossref PubMed Scopus (12) Google Scholar). Cluster of differentiation 47 (CD47) also known as integrin associated protein is a ubiquitously expressed transmembrane glycoprotein that has been implicated in many normal and pathophysiological processes, including apoptosis, cell adhesion, cell migration & proliferation, and inflammation (2Sick E. Jeanne A. Schneider C. Dedieu S. Takeda K. Martiny L. CD47 update: a multifaceted actor in the tumour microenvironment of potential therapeutic interest.Br. J. Pharmacol. 2012; 167: 1415-1530Crossref PubMed Scopus (122) Google Scholar, 3Soto-Pantoja D.R. Kaur S. Roberts D.D. CD47 signaling pathways controlling cellular differentiation and responses to stress.Crit. Rev. Biochem. Mol. Biol. 2015; 50: 212-230Crossref PubMed Scopus (113) Google Scholar, 4Cham L.B. Torrez Dulgeroff L.B. Tal M.C. Adomati T. Li F. Bhat H. et al.Immunotherapeutic blockade of CD47 inhibitory signaling enhances innate and adaptive immune responses to viral infection.Cell Rep. 2020; 31: 107494Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 5Eladl E. Tremblay-LeMay R. Rastgoo N. Musani R. Chen W. Liu A. et al.Role of CD47 in hematological malignancies.J. Hematol. Oncol. 2020; 13: 96Crossref PubMed Scopus (63) Google Scholar, 6Hayat S.M.G. Bianconi V. Pirro M. Jaafari M.R. Hatamipour M. Sahebkar A. CD47: role in the immune system and application to cancer therapy.Cell Oncol. (Dordr.). 2020; 43: 19-30Crossref PubMed Scopus (96) Google Scholar). In recent years, it was demonstrated that CD47, as a bona fide "don't eat me" molecule, inhibits efferocytosis and thereby leads to the accumulation of cell debris in aortic plaques and as a result in the progression of atherosclerosis (7Kojima Y. Volkmer J.P. McKenna K. Civelek M. Lusis A.J. Miller C.L. et al.CD47-blocking antibodies restore phagocytosis and prevent atherosclerosis.Nature. 2016; 536: 86-90Crossref PubMed Scopus (359) Google Scholar, 8Kojima Y. Weissman I.L. Leeper N.J. The role of efferocytosis in atherosclerosis.Circulation. 2017; 135: 476-489Crossref PubMed Scopus (144) Google Scholar, 9Egaña-Gorroño L. Chinnasamy P. Casimiro I. Almonte V.M. Parikh D. Oliveira-Paula G.H. et al.Allograft inflammatory factor-1 supports macrophage survival and efferocytosis and limits necrosis in atherosclerotic plaques.Atherosclerosis. 2019; 289: 184-194Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 10Ye Z.M. Yang S. Xia Y.P. Hu R.T. Chen S. Li B.W. et al.LncRNA MIAT sponges miR-149-5p to inhibit efferocytosis in advanced atherosclerosis through CD47 upregulation.Cell Death Dis. 2019; 10: 138Crossref PubMed Scopus (133) Google Scholar, 11Mueller P.A. Kojima Y. Huynh K.T. Maldonado R.A. Ye J. Tavori H. et al.Macrophage LRP1 (Low-Density lipoprotein receptor-related protein 1) is required for the effect of CD47 blockade on efferocytosis and atherogenesis-brief report.Arterioscler. Thromb. Vasc. Biol. 2022; 42: e1-e9Crossref PubMed Scopus (8) Google Scholar). Although the role of CD47 in vascular diseases, particularly in atherosclerosis and stroke, has been studied (12Dou M. Chen Y. Hu J. Ma D. Xing Y. Recent advancements in CD47 signal transduction pathways involved in vascular diseases.Biomed. Res. Int. 2020; 2020: 4749135Crossref PubMed Scopus (8) Google Scholar, 13Isenberg J.S. Roberts D.D. Frazier W.A. CD47: a new target in cardiovascular therapy.Arterioscler. Thromb. Vasc. Biol. 2008; 28: 615-621Crossref PubMed Scopus (40) Google Scholar), very little is known on its role in neointimal hyperplasia. Previously, it was reported that activation of CD47 elicits proliferative response in cultured smooth muscle cells (SMCs) and that mechanical vascular injury increases its expression in the medial and neointimal region (14Sajid M. Hu Z. Guo H. Li H. Stouffer G.A. Vascular expression of integrin-associated protein and thrombospondin increase after mechanical injury.J. Investig. Med. 2001; 49: 398-406Crossref PubMed Scopus (28) Google Scholar). In addition, CD47 has been shown to inhibit inflammatory response triggered by the placement of stents and suppress in-stent restenosis (15Slee J.B. Alferiev I.S. Nagaswami C. Weisel J.W. Levy R.J. Fishbein I. et al.Enhanced biocompatibility of CD47-functionalized vascular stents.Biomaterials. 2016; 87: 82-92Crossref PubMed Scopus (31) Google Scholar, 16Inamdar V.V. Fitzpatrick E. Alferiev I. Nagaswami C. Spruce L.A. Fazelinia H. et al.Stability and bioactivity of pepCD47 attachment on stainless steel surfaces.Acta Biomater. 2020; 104: 231-240Crossref PubMed Scopus (5) Google Scholar). Neointimal hyperplasia that occurs after vascular interventions such as angioplasty, stent implantation, endarterectomy, and bypass grafting is a major contributing factor in restenosis and in-stent restenosis (17Goel S.A. Guo L.W. Liu B. Kent K.C. Mechanisms of post-intervention arterial remodelling.Cardiovasc. Res. 2012; 96: 363-371Crossref PubMed Scopus (74) Google Scholar). Phenotypic switching of SMCs from contractile to synthetic state and their subsequent migration from media to tunica intima leading to their enhanced proliferation in the intima is the primary cause of neointimal hyperplasia (18Schwartz S.M. deBlois D. O'Brien E.R. The intima. Soil for atherosclerosis and restenosis.Circ. Res. 1995; 77: 445-465Crossref PubMed Scopus (901) Google Scholar, 19Marx S.O. Totary-Jain H. Marks A.R. Vascular smooth muscle cell proliferation in restenosis.Circ. Cardiovasc. Interv. 2011; 4: 104-111Crossref PubMed Scopus (278) Google Scholar). Thus, exploring mechanisms underlying SMC migration and proliferation may lead to the development of new therapeutic targets against restenosis and in-stent restenosis. In response to injury, SMCs secrete an array of molecules that play a key role in vascular wall remodeling (20Lacolley P. Regnault V. Nicoletti A. Li Z. Michel J.B. The vascular smooth muscle cell in arterial pathology: a cell that can take on multiple roles.Cardiovasc. Res. 2012; 95: 194-204Crossref PubMed Scopus (533) Google Scholar). In addition, vascular injury induces the generation of thrombin, a potent mitogen, and chemotactic factor, in a time-dependent manner (21Gallo R. Padurean A. Toschi V. Bichler J. Fallon J.T. Chesebro J.H. et al.Prolonged thrombin inhibition reduces restenosis after balloon angioplasty in porcine coronary arteries.Circulation. 1998; 97: 581-588Crossref PubMed Scopus (87) Google Scholar). The recent work from our laboratory showed that thrombin via inducing the expression of interleukin-33 (IL-33) and cell division cycle 6 (CDC6) mediates VSMC migration and proliferation, respectively, leading to injury-induced vascular wall remodeling (22Janjanam J. Zhang B. Mani A.M. Singh N.K. Traylor Jr., J.G. Orr A.W. et al.LIM and cysteine-rich domains 1 is required for thrombin-induced smooth muscle cell proliferation and promotes atherogenesis.J. Biol. Chem. 2018; 293: 3088-3103Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 23Govatati S. Pichavaram P. Janjanam J. Zhang B. Singh N.K. Mani A.M. et al.NFATc1-E2F1-LMCD1-Mediated IL-33 expression by thrombin 1s required for injury-induced neointima formation.Arterioscler. Thromb. Vasc. Biol. 2019; 39: 1212-1226Crossref PubMed Scopus (19) Google Scholar, 24Govatati S. Pichavaram P. Janjanam J. Guo L. Virmani R. Rao G.N. Myristoylation of LMCD1 leads to its species-specific derepression of E2F1 and NFATc1 in the modulation of CDC6 and IL-33 expression during development of vascular lesions.Arterioscler. Thromb. Vasc. Biol. 2020; 40: 1256-1274Crossref PubMed Scopus (12) Google Scholar). Since vascular injury induces CD47 expression and its role in SMC proliferation and in-stent restenosis has been reported previously (14Sajid M. Hu Z. Guo H. Li H. Stouffer G.A. Vascular expression of integrin-associated protein and thrombospondin increase after mechanical injury.J. Investig. Med. 2001; 49: 398-406Crossref PubMed Scopus (28) Google Scholar, 15Slee J.B. Alferiev I.S. Nagaswami C. Weisel J.W. Levy R.J. Fishbein I. et al.Enhanced biocompatibility of CD47-functionalized vascular stents.Biomaterials. 2016; 87: 82-92Crossref PubMed Scopus (31) Google Scholar, 16Inamdar V.V. Fitzpatrick E. Alferiev I. Nagaswami C. Spruce L.A. Fazelinia H. et al.Stability and bioactivity of pepCD47 attachment on stainless steel surfaces.Acta Biomater. 2020; 104: 231-240Crossref PubMed Scopus (5) Google Scholar), we asked whether CD47 plays a role in thrombin-induced SMC migration and proliferation and injury-induced neointimal hyperplasia. In the present study, we show that CD47 while inhibiting SMC efferocytosis also mediates SMC migration and proliferation in vitro in response to thrombin and in vivo in response to injury, leading to neointimal hyperplasia and vascular wall remodeling. Previous studies have reported increased expression of CD47 in injured arteries as well as atherosclerotic plaques (7Kojima Y. Volkmer J.P. McKenna K. Civelek M. Lusis A.J. Miller C.L. et al.CD47-blocking antibodies restore phagocytosis and prevent atherosclerosis.Nature. 2016; 536: 86-90Crossref PubMed Scopus (359) Google Scholar, 14Sajid M. Hu Z. Guo H. Li H. Stouffer G.A. Vascular expression of integrin-associated protein and thrombospondin increase after mechanical injury.J. Investig. Med. 2001; 49: 398-406Crossref PubMed Scopus (28) Google Scholar). We have shown that thrombin– protease-activated receptor 1 (PAR1) signaling plays a role both in neointimal hyperplasia and atherosclerosis (22Janjanam J. Zhang B. Mani A.M. Singh N.K. Traylor Jr., J.G. Orr A.W. et al.LIM and cysteine-rich domains 1 is required for thrombin-induced smooth muscle cell proliferation and promotes atherogenesis.J. Biol. Chem. 2018; 293: 3088-3103Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 23Govatati S. Pichavaram P. Janjanam J. Zhang B. Singh N.K. Mani A.M. et al.NFATc1-E2F1-LMCD1-Mediated IL-33 expression by thrombin 1s required for injury-induced neointima formation.Arterioscler. Thromb. Vasc. Biol. 2019; 39: 1212-1226Crossref PubMed Scopus (19) Google Scholar, 24Govatati S. Pichavaram P. Janjanam J. Guo L. Virmani R. Rao G.N. Myristoylation of LMCD1 leads to its species-specific derepression of E2F1 and NFATc1 in the modulation of CDC6 and IL-33 expression during development of vascular lesions.Arterioscler. Thromb. Vasc. Biol. 2020; 40: 1256-1274Crossref PubMed Scopus (12) Google Scholar, 25Boro M. Govatati S. Kumar R. Singh N.K. Pichavaram P. Traylor Jr., J.G. et al.Thrombin-Par1 signaling axis disrupts COP9 signalosome subunit 3-mediated ABCA1 stabilization in inducing foam cell formation and atherogenesis.Cell Death Differ. 2021; 28: 780-798Crossref PubMed Scopus (11) Google Scholar). Based on these clues, we asked whether CD47 has any role in thrombin-induced vascular wall remodeling. To test this, first, we studied a time course effect of thrombin on CD47 expression in human aortic smooth muscle cells (HASMCs). We found that thrombin (0.5 U/ml) induces CD47 expression both at mRNA and protein levels in HASMCs in a time-dependent manner (Fig. 1, A and B). Immunofluorescence staining also showed a robust induction of CD47 in thrombin-treated HASMCs (Fig. 1C). Based on these observations, we next examined for the functional significance of CD47 in HASMCs. Depletion of CD47 levels by its siRNA (siCD47) substantially attenuated thrombin-induced HASMC migration and proliferation (Fig. 1, D and F). Cell viability by Trypan blue exclusion assay showed <5% of cell death both in siControl and siCD47-transfected cells. These results clearly infer that the decrease in thrombin-induced HASMC migration and proliferation by siCD47 was due to its effect on CD47 depletion rather than its cytotoxicity. Furthermore, interference with CD47 function by its blocking antibody (bAb, B6.H12; 10 μg/ml) also blunted thrombin-induced HASMC migration and proliferation (Fig. 1, E and G). Since our recent findings showed species-specific posttranslational modification of LMCD1 (LIM and cysteine-rich domains protein 1), a key transcriptional coregulator, in the modulation of VSMC migration and proliferation (24Govatati S. Pichavaram P. Janjanam J. Guo L. Virmani R. Rao G.N. Myristoylation of LMCD1 leads to its species-specific derepression of E2F1 and NFATc1 in the modulation of CDC6 and IL-33 expression during development of vascular lesions.Arterioscler. Thromb. Vasc. Biol. 2020; 40: 1256-1274Crossref PubMed Scopus (12) Google Scholar), we then tested for CD47 species specificity in thrombin-induced VSMC migration and proliferation. First, consistent with its expression in HASMCs, CD47 was also induced in mouse aortic smooth muscle cells (MASMCs) in response to thrombin (Fig. 1, H and I). Second, interference with CD47 function by its bAb (MIAP301; 10 μg/ml) attenuated thrombin-induced MASMC migration and proliferation as well (Fig. 1, J and K). Together, these results indicate that CD47 plays an important role in thrombin-induced VSMC migration and proliferation both in humans and mice. Previously, we have reported that activation of nuclear factor of activated T cells c1 (NFATc1), E2F1 (E2F transcription factor 1), and LMCD1 downstream to PAR1-Gαq/11 (Gα protein q/11)-phospholipase Cβ3 (PLCβ3) signaling was required for thrombin-induced IL-33 and CDC6 expression-mediating HASMC migration and proliferation, respectively (22Janjanam J. Zhang B. Mani A.M. Singh N.K. Traylor Jr., J.G. Orr A.W. et al.LIM and cysteine-rich domains 1 is required for thrombin-induced smooth muscle cell proliferation and promotes atherogenesis.J. Biol. Chem. 2018; 293: 3088-3103Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 23Govatati S. Pichavaram P. Janjanam J. Zhang B. Singh N.K. Mani A.M. et al.NFATc1-E2F1-LMCD1-Mediated IL-33 expression by thrombin 1s required for injury-induced neointima formation.Arterioscler. Thromb. Vasc. Biol. 2019; 39: 1212-1226Crossref PubMed Scopus (19) Google Scholar, 24Govatati S. Pichavaram P. Janjanam J. Guo L. Virmani R. Rao G.N. Myristoylation of LMCD1 leads to its species-specific derepression of E2F1 and NFATc1 in the modulation of CDC6 and IL-33 expression during development of vascular lesions.Arterioscler. Thromb. Vasc. Biol. 2020; 40: 1256-1274Crossref PubMed Scopus (12) Google Scholar). In view of these observations, we next studied the role of this signaling in thrombin-induced CD47 expression. Downregulation of PAR1, Gαq/11, PLCβ3 or NFATc1 but not E2F1 or LMCD1 levels using their respective siRNAs attenuated thrombin-induced CD47 expression both at mRNA and protein levels in HASMCs (Fig. 2, A–H). Previous studies have reported that endothelin-1 also stimulates NFATc1 via Gαq/11-PLCβ signaling in cardiac myocytes and neuronal crest cells (26Kawamura T. Ono K. Morimoto T. Akao M. Iwai-Kanai E. Wada H. et al.Endothelin-1-dependent nuclear factor of activated T lymphocyte signaling associates with transcriptional coactivator p300 in the activation of the B cell leukemia-2 promoter in cardiac myocytes.Circ. Res. 2004; 94: 1492-1499Crossref PubMed Scopus (45) Google Scholar, 27Ivey K. Tyson B. Ukidwe P. McFadden D.G. Levi G. Olson E.N. et al.Galphaq and Galpha11 proteins mediate endothelin-1 signaling in neural crest-derived pharyngeal arch mesenchyme.Dev. Biol. 2003; 255: 230-237Crossref PubMed Scopus (50) Google Scholar). Therefore, to demonstrate PAR1 specificity on NFATc1 activation, we examined for its involvement in endothelin-1-induced NFATc1 activation. Blockade of PAR1 by its antagonist SCH79797 (10 μM) while negating thrombin-induced NFATc1 activation, as measured by its nuclear translocation, had no effect on endothelin-1-induced NFATc1 activation in HASMCs (Fig. 2I). These observations clearly show the specificity of PAR1-Gαq/11-PLCβ3 signaling in thrombin-induced NFATc1 activation and its role in CD47 expression. CD47 is a multifunctional glycoprotein and exerts its biological effects by binding with various cell-surface receptors as well as extracellular matrix proteins (28Brown E.J. Frazier W.A. Integrin-associated protein (CD47) and its ligands.Trends Cell Biol. 2001; 11: 130-135Abstract Full Text Full Text PDF PubMed Scopus (684) Google Scholar). Thrombospondin 1 (TSP1), signal regulatory protein α (SIRPα), and integrins, particularly integrin αVβ3, are the high-affinity signaling partners of CD47 in mediating its biological effects in response to various cues (28Brown E.J. Frazier W.A. Integrin-associated protein (CD47) and its ligands.Trends Cell Biol. 2001; 11: 130-135Abstract Full Text Full Text PDF PubMed Scopus (684) Google Scholar, 29Barazi H.O. Li Z. Cashel J.A. Krutzsch H.C. Annis D.S. Mosher D.F. et al.Regulation of integrin function by CD47 ligands. Differential effects on alpha vbeta 3 and alpha 4beta1 integrin-mediated adhesion.J. Biol. Chem. 2002; 277: 42859-42866Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 30Matozaki T. Murata Y. Okazawa H. Ohnishi H. Functions and molecular mechanisms of the CD47-SIRPalpha signalling pathway.Trends Cell Biol. 2009; 19: 72-80Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 31Barclay A.N. Van den Berg T.K. The interaction between signal regulatory protein alpha (SIRPα) and CD47: structure, function, and therapeutic target.Annu. Rev. Immunol. 2014; 32: 25-50Crossref PubMed Scopus (502) Google Scholar, 32Israeli-Rosenberg S. Manso A.M. Okada H. Ross R.S. Integrins and integrin-associated proteins in the cardiac myocyte.Circ. Res. 2014; 114: 572-586Crossref PubMed Scopus (146) Google Scholar, 33Kale A. Rogers N.M. Ghimire K. Thrombospondin-1 CD47 signalling: from mechanisms to medicine.Int. J. Mol. Sci. 2021; 22: 4062Crossref PubMed Scopus (25) Google Scholar). Therefore, to understand the mechanisms by which CD47 mediates HASMC migration and proliferation, first, we studied a time course effect of thrombin on the expression of these molecules in HASMCs. We found that thrombin (0.5 U/ml) while having no effect on integrin αVβ3 levels, downregulated the expression levels of both TSP1 and SIRPα in HASMCs (Fig. 3A). Next, we tested the interaction of CD47 with TSP1, SIRPα, and integrin αVβ3 in response to thrombin. CD47 interacts only with integrin β3 but not TSP1, SIRPα, or integrin αV in response to thrombin (Fig. 3B). In addition, integrin β3 that was complexed with CD47 was found to be highly phosphorylated at Y773 and Y785 residues (Fig. 3B). A role for integrin β3 tyrosine phosphorylation in VSMC migration and proliferation in response to various agonists has been reported previously (34Slepian M.J. Massia S.P. Dehdashti B. Fritz A. Whitesell L. Beta3-integrins rather than beta1-integrins dominate integrin-matrix interactions involved in postinjury smooth muscle cell migration.Circulation. 1998; 97: 1818-1827Crossref PubMed Scopus (95) Google Scholar, 35Bendeck M.P. Nakada M.T. The beta3 integrin antagonist m7E3 reduces matrix metalloproteinase activity and smooth muscle cell migration.J. Vasc. Res. 2001; 38: 590-599Crossref PubMed Scopus (25) Google Scholar, 36Panchatcharam M. Miriyala S. Yang F. Leitges M. Chrzanowska-Wodnicka M. Quilliam L.A. et al.Enhanced proliferation and migration of vascular smooth muscle cells in response to vascular injury under hyperglycemic conditions is controlled by beta3 integrin signaling.Int. J. Biochem. Cell Biol. 2010; 42: 965-974Crossref PubMed Scopus (47) Google Scholar, 37Stouffer G.A. Hu Z. Sajid M. Li H. Jin G. Nakada M.T. et al.Beta3 integrins are upregulated after vascular injury and modulate thrombospondin- and thrombin-induced proliferation of cultured smooth muscle cells.Circulation. 1998; 97: 907-915Crossref PubMed Scopus (133) Google Scholar). To confirm the interactions between CD47 and integrin β3, we also performed coimmunofluorescence staining and proximity ligation assay (PLA). Our findings showed that CD47 interacts with integrin β3 in response to thrombin as demonstrated by coimmunofluorescence staining as well as PLA (Fig. 3, C and D). To explore the functional significance of these interactions, we next studied the role of these molecules in thrombin-induced HASMC migration and proliferation. In line with the lack of the effect of thrombin on CD47 interaction with TSP1 or SIRPα, blockade of their function by their neutralizing antibodies had no effect on thrombin-induced HASMC migration and proliferation (Fig. 3, E–G). However, consistent with enhanced interaction of integrin β3 with CD47 in response to thrombin, interference with integrin β3 function by its neutralizing antibody while having minimal effect on proliferation blocked thrombin-induced HASMC migration (Fig. 3, E and H). These findings imply that integrin β3 but not TSP1 or SIRPα is required for thrombin-induced CD47-mediated HASMCs migration. Since activation of integrin β3 is required only for thrombin-induced HASMC migration but not proliferation, we wanted to explore the mechanisms by which CD47 mediates thrombin-induced HASMC proliferation. We have previously reported that p21 cyclin-dependent kinase–interacting protein 1 (p21Cip1) translocated from the nucleus to the cytoplasm and undergoes proteasomal-mediated degradation in the modulation of monocyte chemoattractant protein 1-induced HASMC proliferation (38Janjanam J. Rao G.N. Novel role of cortactin in G protein-coupled receptor agonist-induced nuclear export and degradation of p21Cip1.Sci. Rep. 2016; 6: 28687Crossref PubMed Scopus (4) Google Scholar). Based on this information, we asked whether thrombin induced CD47-mediated HASMC proliferation requires p21Cip1 nuclear export and its degradation. To address this, first, we studied the effect of thrombin on CDK inhibitors in HASMCs. While having no major effect on the steady state levels of p27Kip1 or p57Kip2, thrombin downregulated p21Cip1 levels in a time-dependent manner (Fig. 4A). Thrombin had no apparent effect on p21Cip1 mRNA levels as compared to vehicle control (Fig. 4B). To understand the mechanism by which thrombin downregulates p21Cip1, we next examined its nuclear export, as mitogens trigger its translocation from the nucleus to the cytoplasm, where it undergoes degradation (39Hwang C.Y. Lee C. Kwon K.S. Extracellular signal-regulated kinase 2-dependent phosphorylation induces cytoplasmic localization and degradation of p21Cip1.Mol. Cell Biol. 2009; 29: 3379-3389Crossref PubMed Scopus (67) Google Scholar, 40Zhou B.P. Liao Y. Xia W. Spohn B. Lee M.H. Hung M.C. Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells.Nat. Cell Biol. 2001; 3: 245-252Crossref PubMed Scopus (932) Google Scholar). First, in control cells, most of p21Cip1 remained in the nucleus; however, in response to thrombin its levels both in the nucleus and cytoplasm appear to be decreased (Fig. 4, C–F). In addition, depletion of CD47 levels or blockade of its function led to the accumulation of p21Cip1 in the nucleus in response to thrombin, suggesting its role in p21Cip1 nuclear export (Fig. 4, C–F). To gain more evidence supporting this observation, we tested for CD47 and p21Cip1 interactions in the presence and absence of MG132, a proteasomal inhibitor (41Chen X. Barton L.F. Chi Y. Clurman B.E. Roberts J.M. Ubiquitin-independent degradation of cell-cycle inhibitors by the REGgamma proteasome.Mol. Cell. 2007; 26: 843-852Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). In response to thrombin, CD47 was found to be associated with p21Cip1 only in the cytoplasm but not in the nucleus and that in the presence of MG132 their interactions were further stabilized (Fig. 4G). In addition, coimmunofluorescence staining revealed the interaction of CD47 with p21Cip1 only in the cytoplasm in response to thrombin (Fig. 4H). Moreover, in the presence of MG132, p21Cip1 was stabilized and as such its association with CD47 particularly in the cytoplasm in response to thrombin was also increased (Fig. 4H). These observations reveal that CD47 while facilitating p21Cip1 nuclear export also binds with p21Cip1 in the cytoplasm and leads to its proteasomal-mediated degradation. In view of these observations, we next studied the effect of MG132 on thrombin-induced HASMC proliferation. MG132 substantially blocked thrombin-induced HASMC proliferation (Fig. 4I). Together, these observations reveal that CD47 binds with and mediates proteasomal degradation of p21Cip1 in the cytoplasm in response to thrombin in the modulation of HASMC proliferation. To understand the role of CD47 in injury-induced neointima formation in vivo, we used a mouse femoral artery guidewire injury (GI) model. In line with the effect of thrombin on CD47 expression in HASMCs and MASMCs in vitro, GI also induced CD47 expression in mouse femoral arteries in a time-dependent manner with maximum effect at 7 days post GI (Fig. 5A). In addition, double immunofluorescence staining for CD47 and smooth muscle myosin heavy chain (SMMHC) showed the expression of CD47 mostly in intimal SMCs in response to GI (Fig. 5B). Since thrombin-induced CD47 expression requires NFATc1 in HASMCs in vitro (Fig. 2F), we also wanted to test its potential role in injury-induced CD47 induction. To this end, application of NFATc1 siRNA in Pluronic gel perivascularly in vivo depleted GI-induced NFATc1 as well as CD47 expression levels (Fig. 5C). This observation suggests a role for NFATc1 in injury-induced CD47 expression. Since SMC migration and invasion from media to intima toward the injured luminal surface and their proliferation in the intimal region are critical factors for the development of neointimal hyperplasia (17Goel S.A. Guo L.W. Liu B. Kent K.C. Mechanisms of post-intervention arterial remodelling.Cardiovasc. Res. 2012; 96: 363-371Crossref PubMed Scopus (74) Google Scholar), we next tested the role of CD47 in these responses. Interference with CD47 function by its bAb (MIAP301) attenuated GI-induced SMC migration from media to luminal surface and their proliferation (Fig. 5, D and E). In line with these observations, CD47 bAb (MIAP301) also blunted GI-induced neointima formation substant
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