Bone morphogenetic protein 9 (BMP9) and BMP10 enhance tumor necrosis factor-α-induced monocyte recruitment to the vascular endothelium mainly via activin receptor-like kinase 2
2017; Elsevier BV; Volume: 292; Issue: 33 Linguagem: Inglês
10.1074/jbc.m117.778506
ISSN1083-351X
AutoresClaudia‐Gabriela Mitrofan, Sarah Appleby, Gerard B. Nash, Ziad Mallat, Edwin R. Chilvers, Paul D. Upton, Nicholas W. Morrell,
Tópico(s)Angiogenesis and VEGF in Cancer
ResumoBone morphogenetic proteins 9 and 10 (BMP9/BMP10) are circulating cytokines with important roles in endothelial homeostasis. The aim of this study was to investigate the roles of BMP9 and BMP10 in mediating monocyte–endothelial interactions using an in vitro flow adhesion assay. Herein, we report that whereas BMP9/BMP10 alone had no effect on monocyte recruitment, at higher concentrations both cytokines synergized with tumor necrosis factor-α (TNFα) to increase recruitment to the vascular endothelium. The BMP9/BMP10-mediated increase in monocyte recruitment in the presence of TNFα was associated with up-regulated expression levels of E-selectin, vascular cell adhesion molecule (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) on endothelial cells. Using siRNAs to type I and II BMP receptors and the signaling intermediaries (Smads), we demonstrated a key role for ALK2 in the BMP9/BMP10-induced surface expression of E-selectin, and both ALK1 and ALK2 in the up-regulation of VCAM-1 and ICAM-1. The type II receptors, BMPR-II and ACTR-IIA were both required for this response, as was Smad1/5. The up-regulation of cell surface adhesion molecules by BMP9/10 in the presence of TNFα was inhibited by LDN193189, which inhibits ALK2 but not ALK1. Furthermore, LDN193189 inhibited monocyte recruitment induced by TNFα and BMP9/10. BMP9/10 increased basal IκBα protein expression, but did not alter p65/RelA levels. Our findings suggest that higher concentrations of BMP9/BMP10 synergize with TNFα to induce the up-regulation of endothelial selectins and adhesion molecules, ultimately resulting in increased monocyte recruitment to the vascular endothelium. This process is mediated mainly via the ALK2 type I receptor, BMPR-II/ACTR-IIA type II receptors, and downstream Smad1/5 signaling. Bone morphogenetic proteins 9 and 10 (BMP9/BMP10) are circulating cytokines with important roles in endothelial homeostasis. The aim of this study was to investigate the roles of BMP9 and BMP10 in mediating monocyte–endothelial interactions using an in vitro flow adhesion assay. Herein, we report that whereas BMP9/BMP10 alone had no effect on monocyte recruitment, at higher concentrations both cytokines synergized with tumor necrosis factor-α (TNFα) to increase recruitment to the vascular endothelium. The BMP9/BMP10-mediated increase in monocyte recruitment in the presence of TNFα was associated with up-regulated expression levels of E-selectin, vascular cell adhesion molecule (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) on endothelial cells. Using siRNAs to type I and II BMP receptors and the signaling intermediaries (Smads), we demonstrated a key role for ALK2 in the BMP9/BMP10-induced surface expression of E-selectin, and both ALK1 and ALK2 in the up-regulation of VCAM-1 and ICAM-1. The type II receptors, BMPR-II and ACTR-IIA were both required for this response, as was Smad1/5. The up-regulation of cell surface adhesion molecules by BMP9/10 in the presence of TNFα was inhibited by LDN193189, which inhibits ALK2 but not ALK1. Furthermore, LDN193189 inhibited monocyte recruitment induced by TNFα and BMP9/10. BMP9/10 increased basal IκBα protein expression, but did not alter p65/RelA levels. Our findings suggest that higher concentrations of BMP9/BMP10 synergize with TNFα to induce the up-regulation of endothelial selectins and adhesion molecules, ultimately resulting in increased monocyte recruitment to the vascular endothelium. This process is mediated mainly via the ALK2 type I receptor, BMPR-II/ACTR-IIA type II receptors, and downstream Smad1/5 signaling. The vascular endothelium is a key regulator of vascular homeostasis with important roles in regulating blood pressure, coagulation, leukocyte trafficking, and angiogenesis (1.Rajendran P. Rengarajan T. Thangavel J. Nishigaki Y. Sakthisekaran D. Sethi G. Nishigaki I. The vascular endothelium and human diseases.Int. J. Biol. Sci. 2013; 9: 1057-1069Crossref PubMed Scopus (870) Google Scholar, 2.Cybulsky M.I. Gimbrone M.A. Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis.Science. 1991; 251: 788-791Crossref PubMed Scopus (1395) Google Scholar, 3.Poole J.C. Florey H.W. Changes in the endothelium of the aorta and the behaviour of macrophages in experimental atheroma of rabbits.J. Pathol. Bacteriol. 1958; 75: 245-251Crossref PubMed Scopus (142) Google Scholar). 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Although there is a growing body of evidence associating BMP9 signaling with neutrophil recruitment, the role of BMP9 and BMP10 in monocyte recruitment to the vascular endothelium has yet to be reported. In the current study we show, using an in vitro flow adhesion assay that both BMP9 and BMP10, in a concentration-dependent manner, synergistically enhance monocyte recruitment to TNFα-stimulated human aortic endothelial cells (HAECs). This occurs through the up-regulation of E-selectin, VCAM-1, and ICAM-1 on HAECs, and mainly via the type I receptor ALK2, the type II receptors BMPR-II/ACTR-IIA, and the downstream mediators Smad1/5. First, we investigated the role of BMP9 and BMP10 on monocyte recruitment to the vascular endothelium using an in vitro flow adhesion assay, which enables the quantification of real-time interactions between endothelial cells and leukocytes under conditions of physiological flow. As BMP9 has been reported to circulate at concentrations between 2 and 12 ng/ml in humans (18.David L. Mallet C. Keramidas M. Lamandé N. Gasc J.M. Dupuis-Girod S. Plauchu H. Feige J.J. Bailly S. Bone morphogenetic protein-9 is a circulating vascular quiescence factor.Circ. Res. 2008; 102: 914-922Crossref PubMed Scopus (312) Google Scholar, 43.Herrera B. Inman G.J. A rapid and sensitive bioassay for the simultaneous measurement of multiple bone morphogenetic proteins: identification and quantification of BMP4, BMP6 and BMP9 in bovine and human serum.BMC Cell Biol. 2009; 10: 20Crossref PubMed Scopus (112) Google Scholar), we exposed the endothelium to BMP9 or BMP10 at concentrations ranging from 0 to 5 ng/ml prior to the addition of TNFα, then assessed monocyte recruitment. Negligible monocyte recruitment was observed in HAECs treated with BMP9 (Fig. 1, A and B) or BMP10 (Fig. 1, A and C) alone. Although TNFα treatment, as previously reported (2.Cybulsky M.I. 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To examine whether this response was restricted to aortic cells, we also assessed the influence of BMP9 and BMP10 on TNFα-dependent recruitment of monocytes to blood outgrowth endothelial cell (BOEC) monolayers (46.Toshner M. Dunmore B.J. McKinney E.F. Southwood M. Caruso P. Upton P.D. Waters J.P. Ormiston M.L. Skepper J.N. Nash G. Rana A.A. Morrell N.W. Transcript analysis reveals a specific HOX signature associated with positional identity of human endothelial cells.PloS One. 2014; 9: e91334Crossref PubMed Scopus (41) Google Scholar). Similar to HAECs, BMP9 and BMP10 did not influence monocyte adhesion to BOECs, but enhanced the recruitment observed in response to TNFα (Fig. 1, F and G). Taken together, these data show that both BMP9 and BMP10 synergize with TNFα to enhance monocyte recruitment to the vascular endothelium in a concentration-dependent manner, at or above 1.5 ng/ml. Next, we used quantitative PCR (qPCR) and flow cytometry to identify whether pre-treatment with BMP9 or BMP10 increased expression of the endothelial selectins and adhesion molecules involved in monocyte recruitment in TNFα-stimulated HAECs. In accordance with previous studies (47.Wölle J. Hill R.R. Ferguson E. Devall L.J. Trivedi B.K. Newton R.S. Saxena U. Selective inhibition of tumor necrosis factor-induced vascular cell adhesion molecule-1 gene expression by a novel flavonoid: lack of effect on transcription factor NF-κB.Arterioscler. Thromb. Vasc. Biol. 1996; 16: 1501-1508Crossref PubMed Scopus (55) Google Scholar, 48.d'Alessio P. Moutet M. Coudrier E. Darquenne S. Chaudiere J. ICAM-1 and VCAM-1 expression induced by TNF-α are inhibited by a glutathione peroxidase mimic.Free Radic. Biol. Med. 1998; 24: 979-987Crossref PubMed Scopus (55) Google Scholar, 49.Jiang J. Fu W. Wang X. Lin P.H. Yao Q. Chen C. 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Sánchez-Duffhues G. Ten Dijke P. Yu P.B. Bone morphogenetic protein 6 and oxidized low-density lipoprotein synergistically recruit osteogenic differentiation in endothelial cells.Cardiovasc. Res. 2015; 108: 278-287Crossref PubMed Scopus (50) Google Scholar, 12.Csiszar A. Ahmad M. Smith K.E. Labinskyy N. Gao Q. Kaley G. Edwards J.G. Wolin M.S. Ungvari Z. Bone morphogenetic protein-2 induces proinflammatory endothelial phenotype.Am. J. Pathol. 2006; 168: 629-638Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 13.Csiszar A. Labinskyy N. Jo H. Ballabh P. Ungvari Z. Differential proinflammatory and prooxidant effects of bone morphogenetic protein-4 in coronary and pulmonary arterial endothelial cells.Am. J. Physiol. Heart Circ. Physiol. 2008; 295: H569-H577Crossref PubMed Scopus (59) Google Scholar), we next investigated whether treatment with BMP9 or BMP10 increased expression of these ligands in HAECs. BMP9 and BMP10 alone induced the expression of BMP2 by 3–4-fold in HAECs, whereas TNFα exerted a weak induction (Fig. 2G). However, pre-treatment
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