Elevated Soluble Flt 1 Inhibits Endothelial Repair in PR 3-ANCA – Associated Vasculitis

Antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis exhibits endothelial damage, but the capacity for vessel repair in this disorder is not well understood. Here, we observed a marked increase in serum levels of soluble Flt1 (sFlt1), a potent inhibitor of vascular endothelial growth factor, in patients with active ANCA-associated vasculitis compared with patients during remission and other controls. Serum levels of sFlt1 correlated with C5a, an anaphylatoxin released after complement activation. Serum from patients with acute ANCA-associated vasculitis disrupted blood flow in the chicken chorioallantoic membrane assay, suggesting an antiangiogenic effect. Preincubation with excess human vascular endothelial growth factor prevented this effect. Anti–proteinase-3 (PR3) mAb and serum containing PR3-ANCA from patients with active vasculitis both induced a significant and sustained release of sFlt1 from monocytes, whereas anti-myeloperoxidase (MPO) mAb or polyclonal antibodies did not. However, the serum containing polyclonal PR3-ANCA did not induce release of sFlt1 from cultured human umbilical vein endothelial cells. In summary, these data suggest that anti-PR3 antibodies, and to a much lesser extent anti-MPO antibodies, increase sFlt1 during acute ANCA-associated vasculitis, leading to an antiangiogenic state that hinders endothelial repair. J Am Soc Nephrol 23: 155–164, 2012. doi: 10.1681/ASN.2010080858 Antineutrophil cytoplasm antibody (ANCA)– associated vasculitidies (AAVs) are a group of necrotizing small vessel vasculitidies that includeWegener’s granulomatosis, microscopic polyangiitis, and Churg-Strauss syndrome. Frequent manifestations of ANCA-associated vasculitis include crescentic GN and alveolar hemorrhage, both of which are due to necrotizing capillaritis. ANCA-associated vasculitis is characterized by the presence of autoantibodies directed against neutrophil antigens, specifically myeloperoxidase (MPO) andproteinase-3 (PR3) (termed MPO-ANCA and PR3-ANCA, respectively). In addition, a potential novel ANCA autoantigen, lysosomal membrane associated protein-2, was recently described.1,2 Accumulating data demonstrate the pathogenic roles of ANCA in vivo and in vitro in priming neutrophils and initiating endothelial damage. ANCA activate neutrophils3 and monocytes Received August 19, 2010. Accepted August 28, 2011. S.L. R. and R.J.P. contributed equally to this work. A.D.S. and F.F. are senior co-authors. Published online ahead of print. Publication date available at www.jasn. org. Correspondence: Dr. Fadi Fakhouri, ITUN, U643, Department of Nephrology and Immunology, CHU de Nantes, 44000 Nantes, France. Email: fadi.fakhouri@univ-nantes.fr Copyright © 2012 by the American Society of Nephrology J Am Soc Nephrol 23: 155–164, 2012 ISSN : 1046-6673/2301-155 155 in vitro,4 promoting their degranulation and respiratory burst5 as well as stimulating upregulation of adhesion molecules that lead to their augmented endothelial rolling, adhesion, and cell transmigration.6,7 Immunization of MPO-deficient mice with murine MPO in vivo leads to the development of MPO-ANCA, which are sufficient to passively transfer disease to naïve recipients, with the development of necrotizing renal and pulmonary vasculitis.8 In a distinct autoimmune animal model, susceptible rats immunized with human MPO develop vasculitis, pulmonary capillaritis, and focal necrotizing GN in association with MPO-ANCA.9 Similarly, a new model of disease induced by anti-PR3 antibodies was recently reported.10 Finally, the results of a single case report demonstrated the pathogenicity of MPO-ANCA in human patients after the transplacental transfer of anti-MPO antibodies to a newborn.11 ANCA ultimately cause endothelial damage by activating circulating leukocytes and releasing various proteases, and the number of detached apoptotic endothelial cells in the circulation seems to correlate with disease activity in patients with AAV.12 Moreover, the number of circulating CD34 hematopoietic progenitor cells and, to a lesser extent, endothelial progenitor cells, which may contribute to the repair of damaged microvasculature, tend to increase during the remission phases of AAV.13,14 Despite increased understanding of angiogenesis and the critical role of vascular endothelial growth factor (VEGF) in mediating vessel repair, little is known regarding the regulation of endothelial cell survival and vessel regeneration in patients with AAV. VEGF is the most potent growth factor for endothelial cells, and it promotes endothelial cell proliferation and neovasculogenesis and prevents endothelial cell apoptosis. There are various VEGF isoforms that result from alternative splicing, with VEGF 121 and 167 being the most important isoforms in humans.15,16 Similarly, several types of VEGF receptors were identified, with VEGF-R1 and VEGF-R2 being the two main receptors. Soluble Flt1 (sFlt1), a circulating form of VEGF-R1 that lacks the transmembrane domain of the intact protein, results from an alternative splicing of VEGF-R1 and also likely results from the cleavage of membrane-bound Flt1 ectodomain by the disintegrin and metalloproteinase/TNF-a–converting enzyme (ADAM/TACE) family of metalloproteases.17 sFlt1 is mainly secreted by monocytes,18,19 endothelial cells, and placental cytotrophoblasts20 and displays potent inhibitory effects on VEGF. Elevated levels of sFlt1 lead to functional VEGF deficiency and promote an effective antiangiogenic state, which may be of significant clinical importance, as demonstrated by the association of elevated serum sFlt1 levels with placental vascular dysfunction and preeclampsia.21,22 Moreover, there is a close association between sFlt1 production and inflammation, which was demonstrated in a murine model of repeated fetal loss in which sFlt1 release was promoted through complement activation, specifically the anaphylatoxin C5a.23 A study using a murine model of retinopathy of prematurity underscored the link between C5a and angiogenesis through the augmented secretion of sFlt1 by monocytes.24 In this study, we assessed the role of sFlt1 in patients with AAV, diseases characterized by monocyte activation and in which alternative complement components have been implicated in their pathophysiology.Wemeasured sFlt1serum levels in patients with AAV at different disease points and investigated whether increased sFlt1 leads to an effective antiangiogenic state in these individuals.