A Key Role for Matrix Metalloproteinases and Neutral Sphingomyelinase-2 in Transplant Vasculopathy Triggered by Anti-HLA Antibody

Abstract

Background-Outcomes for organ transplantation are constantly improving because of advances in organ preservation, surgical techniques, immune clinical monitoring, and immunosuppressive treatment preventing acute transplant rejection. However, chronic rejection including transplant vasculopathy still limits long-term patient survival. Transplant vasculopathy is characterized by progressive neointimal hyperplasia leading to arterial stenosis and ischemic failure of the allograft. This work sought to decipher the manner in which the humoral immune response, mimicked by W6/32 anti-HLA antibody, contributes to transplant vasculopathy. Methods and Results-Studies were performed in vitro on cultured human smooth muscle cells, ex vivo on human arterial segments, and in vivo in a model consisting of human arterial segments grafted into severe combined immunodeficiency/beige mice injected weekly with anti-HLA antibodies. We report that anti-HLA antibodies are mitogenic for smooth muscle cells through a signaling mechanism implicating matrix metalloproteinases (MMPs) (membrane type 1 MMP and MMP2) and neutral sphingomyelinase-2. This mitogenic signaling and subsequent DNA synthesis are blocked in smooth muscle cells silenced for MMP2 or for neutral sphingomyelinase-2 by small interfering RNAs, in smooth muscle cells transfected with a vector coding for a dominant-negative form of membrane type 1 MMP, and after treatment by pharmacological inhibitors of MMPs (Ro28-2653) or neutral sphingomyelinase-2 (GW4869). In vivo, Ro28-2653 and GW4869 reduced the intimal thickening induced by anti-HLA antibodies in human mesenteric arteries grafted into severe combined immunodeficiency/beige mice. Key Words: anti HLA class I mAb Ⅲ metalloproteinases Ⅲ proliferation Ⅲ smooth muscle cells Ⅲ transplant vasculopathy P rogress in surgical techniques, organ preservation, and immunosuppressive drugs has improved the outcome of organ allografts. 1 However, the late outcome of organ transplants is impaired by transplant vasculopathy (also known as transplant arteriosclerosis or chronic vascular rejection), which occurs in up to 50% of heart transplant recipients within 5 years after transplantation. 2,3 Transplant vasculopathy is mediated by immune and nonimmune mechanisms (such as ischemia/reperfusion, inflammation, viral infection, and hypertension) that trigger diffuse and concentric intimal hyperplasia and lead finally to arterial stenosis, chronic ischemia, and functional loss of the grafted organ. 2,4,5 Conclusions-These Clinical Perspective on p 2734Both cell-mediated and humoral immunologic mechanisms are thought to participate in the pathogenesis of transplant vasculopathy. [5][6][7][8] Experimental and clinical studies suggest that B cells and donor-specific alloantibodies play a role in Received January 25, 2011; accepted October 11, 2011. From INSERM UMR-1048, Toulouse, France (S.G., M. Trayssac, N.A., J.T., D.C., M. Thomsen, A.N.-S., R.S.); Faculty of Medicine, Department of Biochemistry, University of Toulouse, Toulouse, Franc...

Mechanisms of human smooth muscle cell proliferation and transplant vasculopathy induced by HLA class I antibodies: In vitro and in vivo studies

“…Our data suggest that anti-HLA antibodies behave as stressinducing agents on SMC, activating the MMP/nSMase-2 pathway [31]. As reported for other stress-inducing agents (TNF-a, oxLDL, H 2 O 2 ), anti-HLA antibodies (1 lg/mL) rapidly activate nSMase-2 via an upstream activation of MT1-MMP and MMP-2.…”

“…We used the MMP inhibitor Ro28-2653 (oral gavage twice a week at 1 lg/10 g body weight) and the nSMase inhibitor GW4869 (intraperitoneal injection twice a week at 1 lg/1 g body weight). Both inhibitors efficiently reduced the development of TV induced by anti-HLA antibodies after 6 weeks of treatment [31]. GW4869 protected the human graft from intimal hyperplasia at about 50% and Ro28-2653 induced a decrease of more than 60% of the vascular lesions induced by anti-HLA antibodies.…”

“…In vitro studies show that anti-HLA antibodies directly activate signaling pathways involved in the migration and proliferation of SMC [17,18,30,31]. HLA class I antibodies increase the expression of the fibroblast growth factor receptor of SMC and a subsequent tyrosine phosphorylation of proteins involved in SMC proliferation [32].…”

Exosome reduction in vivo is associated with lower amyloid plaque load in the 5XFAD mouse model of Alzheimer's disease

“…Intraperitoneal administration of GW4869 for 21 days at 1.25 µg/g mouse body weight was able to cross the blood-brain barrier to inhibit nSMase2, alter brain and blood sphingolipid content, and affect spatial memory (Tabatadze et al, 2010). Another study using a lower dose (1 µg/g) twice weekly for 5 weeks was able to prevent the neointimal thickening of transplanted mesenteric arteries treated with an anti-human leukocyte antigen antibody (Galvani et al, 2011). With respect to inhibition of exosome secretion in vivo , GW4869 (1.25 µg/g) treatment for 5 days reduced lung multiplicities following Lewis lung carcinoma cell injection, which was reversed by a single tail-vein injection of Lewis lung carcinoma cell-derived exosomes expressing specific miRNA species (Fabbri et al, 2012).…”

Interacting Mechanisms in the Pathogenesis of Cardiac Allograft Vasculopathy

“…The binding of mouse anti-human monoclonal antibody to class I HLA molecules can activate certain effector pathways in EC that can promote inflammation independent of complement activation 52 . Repeated injection of one such mouse-anti-human antibody (W6/32) into immunodeficient mice bearing human artery interposition grafts can produce CAV-like lesions independent of T cells 53, 54 . It is not clear, however, how this antibody, which is much more strongly reactive with EC than SMC, causes SMC proliferation and accumulation in an expanded intima.…”