Von Willebrand factor regulates complement on endothelial cells
2016; Elsevier BV; Volume: 90; Issue: 1 Linguagem: Inglês
10.1016/j.kint.2016.03.023
ISSN1523-1755
AutoresDamien Noone, Magdalena Riedl, Fred G. Pluthero, Mackenzie Bowman, M. Kathryn Liszewski, Lily Lu, Yi Quan, Steve Balgobin, Reinhard Schneppenheim, Sonja Schneppenheim, Ulrich Budde, Paula James, John P. Atkinson, Nades Palaniyar, Walter H.A. Kahr, Christoph Licht,
Tópico(s)Renal Diseases and Glomerulopathies
ResumoAtypical hemolytic uremic syndrome and thrombotic thrombocytopenic purpura have traditionally been considered separate entities. Defects in the regulation of the complement alternative pathway occur in atypical hemolytic uremic syndrome, and defects in the cleavage of von Willebrand factor (VWF)-multimers arise in thrombotic thrombocytopenic purpura. However, recent studies suggest that both entities are related as defects in the disease-causing pathways overlap or show functional interactions. Here we investigate the possible functional link of VWF-multimers and the complement system on endothelial cells. Blood outgrowth endothelial cells (BOECs) were obtained from 3 healthy individuals and 2 patients with Type 3 von Willebrand disease lacking VWF. Cells were exposed to a standardized complement challenge via the combination of classical and alternative pathway activation and 50% normal human serum resulting in complement fixation to the endothelial surface. Under these conditions we found the expected release of VWF-multimers causing platelet adhesion onto BOECs from healthy individuals. Importantly, in BOECs derived from patients with von Willebrand disease complement C3c deposition and cytotoxicity were more pronounced than on BOECs derived from normal individuals. This is of particular importance as primary glomerular endothelial cells display a heterogeneous expression pattern of VWF with overall reduced VWF abundance. Thus, our results support a mechanistic link between VWF-multimers and the complement system. However, our findings also identify VWF as a new complement regulator on vascular endothelial cells and suggest that VWF has a protective effect on endothelial cells and complement-mediated injury. Atypical hemolytic uremic syndrome and thrombotic thrombocytopenic purpura have traditionally been considered separate entities. Defects in the regulation of the complement alternative pathway occur in atypical hemolytic uremic syndrome, and defects in the cleavage of von Willebrand factor (VWF)-multimers arise in thrombotic thrombocytopenic purpura. However, recent studies suggest that both entities are related as defects in the disease-causing pathways overlap or show functional interactions. Here we investigate the possible functional link of VWF-multimers and the complement system on endothelial cells. Blood outgrowth endothelial cells (BOECs) were obtained from 3 healthy individuals and 2 patients with Type 3 von Willebrand disease lacking VWF. Cells were exposed to a standardized complement challenge via the combination of classical and alternative pathway activation and 50% normal human serum resulting in complement fixation to the endothelial surface. Under these conditions we found the expected release of VWF-multimers causing platelet adhesion onto BOECs from healthy individuals. Importantly, in BOECs derived from patients with von Willebrand disease complement C3c deposition and cytotoxicity were more pronounced than on BOECs derived from normal individuals. This is of particular importance as primary glomerular endothelial cells display a heterogeneous expression pattern of VWF with overall reduced VWF abundance. Thus, our results support a mechanistic link between VWF-multimers and the complement system. However, our findings also identify VWF as a new complement regulator on vascular endothelial cells and suggest that VWF has a protective effect on endothelial cells and complement-mediated injury. Atypical hemolytic uremic syndrome (aHUS) and thrombotic thrombocytopenic purpura (TTP) define variants of the extending spectrum of thrombotic microangiopathies (TMAs). aHUS occurs due to the dysfunctional regulation of the complement alternative pathway (AP) caused by mutations or inhibiting autoantibodies affecting the function of complement activators or regulators with subsequent microvascular endothelial cell (EC) injury and the formation of platelet microthrombi.1Noris M. Remuzzi G. Atypical hemolytic-uremic syndrome.N Engl J Med. 2009; 361: 1676-1687Crossref PubMed Scopus (928) Google Scholar TTP, by contrast, is caused by either a deficiency of a disintegrin and metalloproteinase with a thrombospondin type 1 motif member 13 (ADAMTS13) or autoantibodies inhibiting its function, allowing ultra-large VWF-multimers to be released from ECs into the bloodstream, bind platelets, and produce occlusive thrombi.2Tsai H.M. Pathophysiology of thrombotic thrombocytopenic purpura.Int J Hematol. 2010; 91: 1-19Crossref PubMed Scopus (171) Google Scholar, 3George J.N. Measuring ADAMTS13 activity in patients with suspected thrombotic thrombocytopenic purpura: when, how, and why?.Transfusion. 2015; 55: 11-13Crossref PubMed Scopus (21) Google Scholar Traditionally aHUS and TTP were considered 2 distinct pathologies, although both ultimately lead to a microangiopathic hemolytic anemia.4Tsai H.M. Untying the knot of thrombotic thrombocytopenic purpura and atypical hemolytic uremic syndrome.Am J Med. 2013; 126: 200-209Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar However, recognizing their significant clinical overlap, the concept of a disease spectrum with shared underlying pathogenetic mechanisms, for instance, complement AP defects, was also proposed.5Chapin J. Eyler S. Smith R. et al.Complement factor H mutations are present in ADAMTS13-deficient, ticlopidine-associated thrombotic microangiopathies.Blood. 2013; 121: 4012-4013Crossref PubMed Scopus (19) Google Scholar, 6Noris M. Mescia F. Remuzzi G. STEC-HUS, atypical HUS and TTP are all diseases of complement activation.Nat Rev Nephrol. 2012; 8: 622-633Crossref PubMed Scopus (284) Google Scholar The finding of decreased C3 levels7Noris M. Ruggenenti P. Perna A. et al.Hypocomplementemia discloses genetic predisposition to hemolytic uremic syndrome and thrombotic thrombocytopenic purpura: role of factor H abnormalities. Italian Registry of Familial and Recurrent Hemolytic Uremic Syndrome/Thrombotic Thrombocytopenic Purpura.J Am Soc Nephrol. 1999; 10: 281-293PubMed Google Scholar and increased complement AP activity in TTP patients,8Feng S. Kroll M.H. Nolasco L. et al.Complement activation in thrombotic microangiopathies.Br J Haematol. 2013; 160: 404-406Crossref PubMed Scopus (16) Google Scholar along with the experimental observation that serum from TTP patients can result in complement deposition on ECs,9Ruiz-Torres M.P. Casiraghi F. Galbusera M. et al.Complement activation: the missing link between ADAMTS-13 deficiency and microvascular thrombosis of thrombotic microangiopathies.Thromb Haemost. 2005; 93: 443-452Crossref PubMed Scopus (81) Google Scholar further supports such an overlap. A recent study found that 80% of an aHUS patient cohort carried at least 1 non-synonymous change in ADAMTS13.10Feng S. Eyler S.J. Zhang Y. et al.Partial ADAMTS13 deficiency in atypical hemolytic uremic syndrome.Blood. 2013; 122: 1487-1493Crossref PubMed Scopus (61) Google Scholar Recent studies further linked the complement AP and its principal regulator, complement factor H (CFH), with VWF and its cleaving protease, ADAMTS13.11Turner N.A. Moake J. Assembly and activation of alternative complement components on endothelial cell-anchored ultra-large von Willebrand factor links complement and hemostasis-thrombosis.PLoS One. 2013; 8: e59372Crossref PubMed Scopus (88) Google Scholar, 12Nolasco L. Nolasco J. Feng S. et al.Human complement factor H is a reductase for large soluble von Willebrand factor multimers—brief report.Arterioscler Thromb Vasc Biol. 2013; 33: 2524-2528Crossref PubMed Scopus (33) Google Scholar, 13Feng S. Liang X. Cruz M.A. et al.The interaction between factor H and Von Willebrand factor.PLoS One. 2013; 8: e73715Crossref PubMed Scopus (38) Google Scholar, 14Rayes J. Roumenina L.T. Dimitrov J.D. et al.The interaction between factor H and VWF increases factor H cofactor activity and regulates VWF prothrombotic status.Blood. 2014; 123: 121-125Crossref PubMed Scopus (57) Google Scholar Most recently, the assembly and activation of complement AP components was shown on ultra-large VWF-multimer strings secreted from and anchored to ECs (human umbilical vein ECs [HUVECs]).11Turner N.A. Moake J. Assembly and activation of alternative complement components on endothelial cell-anchored ultra-large von Willebrand factor links complement and hemostasis-thrombosis.PLoS One. 2013; 8: e59372Crossref PubMed Scopus (88) Google Scholar We aimed to explore the possible functional link between VWF-multimers and the complement system using BOECs from patients with Type 3 von Willebrand disease (VWD) lacking functional VWF.15Bowman M. Tuttle A. Notley C. et al.The genetics of Canadian type 3 von Willebrand disease: further evidence for co-dominant inheritance of mutant alleles.J Thromb Haemost. 2013; 11: 512-520Crossref PubMed Scopus (58) Google Scholar We utilized a standardized strategy of exposing BOECs to a complement challenge,16Liszewski M.K. Atkinson J.P. Membrane cofactor protein (MCP; CD46). Isoforms differ in protection against the classical pathway of complement.J Immunol. 1996; 156: 4415-4421PubMed Google Scholar, 17Barilla-LaBarca M.L. Liszewski M.K. Lambris J.D. et al.Role of membrane cofactor protein (CD46) in regulation of C4b and C3b deposited on cells.J Immunol. 2002; 168: 6298-6304Crossref PubMed Scopus (111) Google Scholar, 18Triantafilou K. Hughes T.R. Triantafilou M. et al.The complement membrane attack complex triggers intracellular Ca2+ fluxes leading to NLRP3 inflammasome activation.J Cell Sci. 2013; 126: 2903-2913Crossref PubMed Scopus (256) Google Scholar which allowed us to study EC responses ultimately leading to a TMA phenotype (i.e., complement deposition, VWF release, platelet adhesion, and EC injury). Unexpectedly, we found that endothelial complement fixation was increased on VWD BOECs, and those pathologic consequences of complement fixation such as EC injury and cytotoxicity as well as platelet adhesion were enhanced, findings in keeping with VWF acting to downregulate complement-mediated EC injury. This has physiological implications since (primary) glomerular ECs (GECs) show heterogeneous VWF expression both in vitro and in vivo. BOECs are late outgrowth endothelial colony-forming cells derived from circulating endothelial progenitor cells isolated from peripheral blood.19Timmermans F. Van Hauwermeiren F. De Smedt M. et al.Endothelial outgrowth cells are not derived from CD133+ cells or CD45+ hematopoietic precursors.Arterioscler Thromb Vasc Biol. 2007; 27: 1572-1579Crossref PubMed Scopus (306) Google Scholar, 20Timmermans F. Plum J. Yoder M.C. et al.Endothelial progenitor cells: identity defined?.J Cell Mol Med. 2009; 13: 87-102Crossref PubMed Scopus (424) Google Scholar For this study we generated BOECs from 3 healthy adult donors and 2 patients with VWD and confirmed their EC phenotype and stability in an early and late passage (Supplementary Figure S1A–D). Using flow cytometry, we determined that the expression level of the membrane-anchored complement regulators CD46, CD55, and CD59 was similar in control and VWD BOECs (Figure 1). Using reverse transcription quantitative real-time PCR (RT-qPCR), we also confirmed that the baseline gene expression of these complement regulators was similar between controls and HUVECs (Supplementary Figure S1E). Of note, in both BOECs and HUVECs the expression level of CD59 was about 10-fold higher as compared to CD46 and CD55. VWF was lacking from plasma of Type 3 VWD patients (Figure 2a, lanes 3 and 4). In addition, VWF was found in cell lysates of control BOECs but lacking from VWF BOECs (Figure 2b), and VWF mRNA expression in Type 3 VWD BOECs was only 5.6% of that in controls (Figure 2c). We established a method of complement fixation on ECs using (blocking) antibodies specific to the membrane-anchored complement regulators CD46, CD55, and CD59 followed by exposure to 50% normal human serum (NHS) in serum-free media (SFM). Of note, the use of anti-CD46 or -CD55 alone was not sufficient to enhance C3c deposition. The use of anti-CD59 antibody, however, was alone able to significantly increase C3c deposition, and the use of all 3 antibodies was additive (Supplementary Figure S2A, B). Using 50% NHS was more effective than 25% or 10% NHS (Supplementary Figure S2E). This method combines the effects of complement induction via sensitization (via the classical pathway) and complement amplification (via the alternative pathway [AP])—a strategy that has previously been utilized.16Liszewski M.K. Atkinson J.P. Membrane cofactor protein (MCP; CD46). Isoforms differ in protection against the classical pathway of complement.J Immunol. 1996; 156: 4415-4421PubMed Google Scholar, 17Barilla-LaBarca M.L. Liszewski M.K. Lambris J.D. et al.Role of membrane cofactor protein (CD46) in regulation of C4b and C3b deposited on cells.J Immunol. 2002; 168: 6298-6304Crossref PubMed Scopus (111) Google Scholar, 18Triantafilou K. Hughes T.R. Triantafilou M. et al.The complement membrane attack complex triggers intracellular Ca2+ fluxes leading to NLRP3 inflammasome activation.J Cell Sci. 2013; 126: 2903-2913Crossref PubMed Scopus (256) Google Scholar The contribution of the classical pathway was confirmed using C1q-deficient NHS (CompTech, Tyler, TX), which reduced EC complement (C3c) fixation by ∼80% (Supplementary Figure S2C, D). C5b-9 deposition on ECs was determined by immunofluorescence (Supplementary Figure S2F), which confirmed full activation of the complement cascade. Given the ∼10-fold higher surface expression of CD59 on BOECs (confirmed by both RT-qPCR and flow cytometry) (Supplementary Figure S1E; data not shown for flow cytometry), the fact that the monoclonal anti-CD59 antibody is an IgG2b—an isotype known to activate complement via the classical pathway—and the fact that the mouse monoclonal antibodies to CD46 and CD55 are IgG1 and typically non–complement-activating, complement fixation to BOECs in our model is likely due to anti-CD59 antibody-initiated classical pathway activation.21Seino J. Eveleigh P. Warnaar S. et al.Activation of human complement by mouse and mouse/human chimeric monoclonal antibodies.Clin Exp Immunol. 1993; 94: 291-296Crossref PubMed Scopus (46) Google Scholar Of note, complement fixation was also achieved by incubating BOECs with a non–complement-specific, rabbit polyclonal anti–β2-microglobulin antibody (Supplementary Figure S3). In keeping with their lower expression level, antibodies to PECAM/CD31 (mouse monoclonal IgG1) and endoglin/CD107a (mouse monoclonal IgG2a) were not able to induce complement fixation (data not shown). Taken together, we have established a reliable and reproducible method of complement fixation on ECs. VWF, stored in Weibel–Palade bodies, was released upon complement fixation. By using 2 different antibodies against VWF we were able to identify stored VWF (cells permeabilized) and released VWF (cells non-permeabilized). We observed that over time VWF was released, and after 60 minutes control BOECs were almost completely depleted of (intracellular) VWF (Figure 3). In aHUS dysregulation of the complement AP is hypothesized to ultimately lead to EC activation, a procoagulant endothelium, and platelet aggregation. To investigate the functional role of VWF in this context, we quantified platelet adhesion to complement-challenged BOECs grown in a microfluidic flow chamber comparing control to VWD BOECs. Calcein-labeled platelets (15 × 107 per ml) were perfused at 2 dynes/cm2 for 10 to 20 minutes over control and VWD BOECs. Platelet adhesion was observed on VWF strings (Figure 4a) when BOECs were treated with 50% NHS, a phenomenon that was enhanced after EC preincubation with CD46, CD55, and CD59 antibodies (20 platelets per high-power field vs. 84 platelets per high-power field at X4 magnification; P < 0.05). Of note, platelets adhered only to control BOECs and not to VWD BOECs devoid of VWF (P < 0.001) (Figure 4b–d). We conclude from these results that platelet adhesion to BOECs in response to complement fixation is VWF dependent. To investigate the functional relevance of VWF for complement fixation on ECs, we performed flow cytometry detecting C3c deposition on control and VWD BOECs as follows: (i) 50% NHS, (ii) functional blockade of 1 membrane-anchored complement regulator (CD46) with 50% NHS, and (iii) combined sensitization and functional blockade using monoclonal antibodies to the 3 main complement regulators (CD46, CD55, and CD59) with 50% NHS. We observed an increase of C3c deposition on VWD BOECs after preincubation with CD46, CD55, and CD59 antibodies as compared to control BOECs (i.e., increase in median fluorescence intensity from 3450 ± 1599 to 5861 ± 3332, P < 0.05, N = 5; Figure 5). Thus, under complement stress, the absence of VWF resulted in increased complement fixation. Cytotoxicity was quantified by the measurement of lactate dehydrogenase (LDH) released from BOECs into the supernatant. LDH release, reflective of cell damage, correlated with incremental complement fixation. LDH release of VWD BOECs was 46% ± 14% when treated with 10% NHS, 49% ± 16% when treated with 10% NHS after treatment with the anti-CD46 antibody, and 81% ± 20% when treated with 10% NHS after preincubation with the CD46, CD55, and CD59 antibodies, as compared to 31% ± 15%, 30% ± 12%, and 48% ± 28%, respectively, in control BOECs (P < 0.01, P < 0.05, P < 0.05, N = 5; Figure 6). As previously described, VWF expression is not homogeneous but demonstrates a heterogeneous expression pattern in ECs.22Aird W.C. Phenotypic heterogeneity of the endothelium: II. Representative vascular beds.Circ Res. 2007; 100: 174-190Crossref PubMed Scopus (778) Google Scholar, 23Pusztaszeri M.P. Seelentag W. Bosman F.T. Immunohistochemical expression of endothelial markers CD31, CD34, von Willebrand factor, and Fli-1 in normal human tissues.J Histochem Cytochem. 2006; 54: 385-395Crossref PubMed Scopus (585) Google Scholar Studying primary GECs we found decreased VWF gene expression and decreased protein levels (Figure 7a and b ). By immunofluorescence, we confirmed that these findings resulted from VWF heterogeneity. VWF was positive in 61.5% of the fixed and stained GECs, only minimally present in 7.7%, but completely absent in 31%. GECs were costained for CD144, CD31, or both, in order to ensure retention of their EC phenotype (Figure 7c). Thrombotic microangiopathy (TMA) defines a growing spectrum of diseases sharing the pathologic consequences of the disruption of the integrity of the vascular endothelium. A variety of insults can be responsible for the EC injury, which subsequently recruits the inflammatory and coagulatory systems and leads to transient or permanent impairment or loss of organ function.1Noris M. Remuzzi G. Atypical hemolytic-uremic syndrome.N Engl J Med. 2009; 361: 1676-1687Crossref PubMed Scopus (928) Google Scholar, 24Ruggenenti P. Noris M. Remuzzi G. Thrombotic microangiopathy, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura.Kidney Int. 2001; 60: 831-846Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar Complement-mediated TMA has been defined as atypical hemolytic uremic syndrome (aHUS) and is caused by loss- or gain-of-function mutations in complement factors participating in maintaining the balance between complement activation and regulation, or the presence of autoantibodies with similar functional consequences.1Noris M. Remuzzi G. Atypical hemolytic-uremic syndrome.N Engl J Med. 2009; 361: 1676-1687Crossref PubMed Scopus (928) Google Scholar, 25George J.N. Nester C.M. Syndromes of thrombotic microangiopathy.N Engl J Med. 2014; 371: 1847-1848Crossref PubMed Scopus (522) Google Scholar We developed an ex vivo model utilizing blood outgrowth endothelial cells (BOECs), which allows us to study the functional consequences of complement activation on ECs under static and microfluidic conditions. The synchronous blockade of the 3 surface regulators CD46, CD55, and CD59 via specific inhibiting antibodies allowed us to achieve complement fixation on BOECs recruiting the classical and alternative pathways. EC complement fixation resulted in release of VWF as previously also reported by others,26Hattori R. Hamilton K.K. McEver R.P. et al.Complement proteins C5b-9 induce secretion of high molecular weight multimers of endothelial von Willebrand factor and translocation of granule membrane protein GMP-140 to the cell surface.J Biol Chem. 1989; 264: 9053-9060Abstract Full Text PDF PubMed Google Scholar, 27Nakashima S. Qian Z. Rahimi S. et al.Membrane attack complex contributes to destruction of vascular integrity in acute lung allograft rejection.J Immunol. 2002; 169: 4620-4627Crossref PubMed Scopus (58) Google Scholar, 28Ota H. Fox-Talbot K. Hu W. et al.Terminal complement components mediate release of von Willebrand factor and adhesion of platelets in arteries of allografts.Transplantation. 2005; 79: 276-281Crossref PubMed Scopus (33) Google Scholar and enhanced platelet adhesion and cell injury. Under fluidic conditions we found platelets adhering to VWF-multimers.29Bryckaert M. Rosa J.P. Denis C.V. et al.Of von Willebrand factor and platelets.Cell Mol Life Sci. 2015; 72: 307-326Crossref PubMed Scopus (130) Google Scholar, 30Chauhan A.K. Motto D.G. Lamb C.B. et al.Systemic antithrombotic effects of ADAMTS13.J Exp Med. 2006; 203: 767-776Crossref PubMed Scopus (180) Google Scholar, 31De Ceunynck K. De Meyer S.F. Vanhoorelbeke K. Unwinding the von Willebrand factor strings puzzle.Blood. 2013; 121: 270-277Crossref PubMed Scopus (111) Google Scholar By contrast, until recently, complement involvement in the pathogenesis of TTP was categorically excluded, and genetic or autoimmune defects in the cleavage of VWF-multimers via ADAMTS13 were claimed to be the sole cause.2Tsai H.M. Pathophysiology of thrombotic thrombocytopenic purpura.Int J Hematol. 2010; 91: 1-19Crossref PubMed Scopus (171) Google Scholar, 4Tsai H.M. Untying the knot of thrombotic thrombocytopenic purpura and atypical hemolytic uremic syndrome.Am J Med. 2013; 126: 200-209Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar Evidence for the functional interactions of the 2 systems as recently collected in aHUS and TTP patients10Feng S. Eyler S.J. Zhang Y. et al.Partial ADAMTS13 deficiency in atypical hemolytic uremic syndrome.Blood. 2013; 122: 1487-1493Crossref PubMed Scopus (61) Google Scholar, 32Reti M. Farkas P. Csuka D. et al.Complement activation in thrombotic thrombocytopenic purpura.J Thromb Haemost. 2012; 10: 791-798Crossref PubMed Scopus (113) Google Scholar, 33Choi H.S. Cheong H.I. Kim N.K. et al.ADAMTS13 gene mutations in children with hemolytic uremic syndrome.Yonsei Med J. 2011; 52: 530-534Crossref PubMed Scopus (4) Google Scholar and in vitro12Nolasco L. Nolasco J. Feng S. et al.Human complement factor H is a reductase for large soluble von Willebrand factor multimers—brief report.Arterioscler Thromb Vasc Biol. 2013; 33: 2524-2528Crossref PubMed Scopus (33) Google Scholar, 13Feng S. Liang X. Cruz M.A. et al.The interaction between factor H and Von Willebrand factor.PLoS One. 2013; 8: e73715Crossref PubMed Scopus (38) Google Scholar, 14Rayes J. Roumenina L.T. Dimitrov J.D. et al.The interaction between factor H and VWF increases factor H cofactor activity and regulates VWF prothrombotic status.Blood. 2014; 123: 121-125Crossref PubMed Scopus (57) Google Scholar, 34Wu T.C. Yang S. Haven S. et al.Complement activation and mortality during an acute episode of thrombotic thrombocytopenic purpura.J Thromb Haemost. 2013; 11: 1925-1927Crossref PubMed Scopus (41) Google Scholar fundamentally challenges the proposed dichotomy. It was first reported in static conditions that components of the complement cascade, including C3 and C5, were present on EC-secreted VWF-multimers.11Turner N.A. Moake J. Assembly and activation of alternative complement components on endothelial cell-anchored ultra-large von Willebrand factor links complement and hemostasis-thrombosis.PLoS One. 2013; 8: e59372Crossref PubMed Scopus (88) Google Scholar Tati et al. subsequently showed that, under shear conditions, and in the absence of ADAMTS13, C3 bound to histamine-induced VWF-multimers, to VWF adherent platelets, and to ECs secreting VWF.35Tati R. Kristoffersson A.C. Stahl A.L. et al.Complement activation associated with ADAMTS13 deficiency in human and murine thrombotic microangiopathy.J Immunol. 2013; 191: 2184-2193Crossref PubMed Scopus (56) Google Scholar While the functional implications remained unclear,35Tati R. Kristoffersson A.C. Stahl A.L. et al.Complement activation associated with ADAMTS13 deficiency in human and murine thrombotic microangiopathy.J Immunol. 2013; 191: 2184-2193Crossref PubMed Scopus (56) Google Scholar a complement amplifying effect of the association of the complement AP and VWF was recently proposed.36Turner N. Nolasco L. Nolasco J. et al.Thrombotic microangiopathies and the linkage between von Willebrand factor and the alternative complement pathway.Semin Thromb Hemost. 2014; 40: 544-550Crossref PubMed Scopus (39) Google Scholar We aimed to study the functional interactions between complement and VWF in TMA pathogenesis utilizing BOECs from Type 3 VWD patients. This approach allowed us to take advantage of a naturally occurring EC line expressing essentially no VWF, equivalent to a VWF-null EC, to study the functional interactions of VWF and the complement system. It was expected that VWF-multimers act as a complement amplifier on ECs, thus less complement activation would be observed on VWD BOECs (cells devoid of VWF). Contrary to our expectation, we found that VWD BOECs demonstrated increased C3c deposition when compared to control BOECs even though the expression level of membrane-anchored complement regulators was not different from that in BOECs. Importantly, VWD BOECs exhibited decreased survival of complement-mediated cytotoxicity. Taken together, our data suggest that VWF is secreted upon complement fixation and serves as a complement regulator on EC surfaces, protecting ECs from complement deposition. This adds to the recent finding that secreted VWF might act as a cofactor for complement factor I–mediated cleavage and inactivation of C3b.37Feng S. Liang X. Kroll M.H. et al.von Willebrand factor is a cofactor in complement regulation.Blood. 2015; 125: 1034-1037Crossref PubMed Scopus (53) Google Scholar The vascular endothelium is diverse, including VWF expression.22Aird W.C. Phenotypic heterogeneity of the endothelium: II. Representative vascular beds.Circ Res. 2007; 100: 174-190Crossref PubMed Scopus (778) Google Scholar In the pulmonary vascular endothelium, VWF expression is strongest in veins and very weak in capillaries.38Kawanami O. Jin E. Ghazizadeh M. et al.Heterogeneous distribution of thrombomodulin and von Willebrand factor in endothelial cells in the human pulmonary microvessels.J Nippon Med Sch. 2000; 67: 118-125Crossref PubMed Scopus (30) Google Scholar, 39Muller A.M. Skrzynski C. Skipka G. et al.Expression of von Willebrand factor by human pulmonary endothelial cells in vivo.Respiration. 2002; 69: 526-533Crossref PubMed Scopus (29) Google Scholar By contrast, immunohistochemical studies of normal human kidney tissue obtained from biopsies and autopsy specimens showed that the fenestrated glomerular endothelium has patchy positivity for VWF.23Pusztaszeri M.P. Seelentag W. Bosman F.T. Immunohistochemical expression of endothelial markers CD31, CD34, von Willebrand factor, and Fli-1 in normal human tissues.J Histochem Cytochem. 2006; 54: 385-395Crossref PubMed Scopus (585) Google Scholar Testing primary GECs, we confirmed less VWF by both RT-qPCR and Western blot. In addition, immunofluorescence revealed that the overall reduced VWF expression level was caused by the complete absence of VWF from about one-third of GECs rather than an overall reduced expression level. As the capacity of VWF to act as endothelial complement regulator will depend on its abundance, heterogeneous VWF expression may explain the susceptibility of the glomerular endothelium to the complement-mediated injury in aHUS. The elevated VWF expression level of the brain microvasculature,40Dorovini-Zis K. Prameya R. Bowman P.D. Culture and characterization of microvascular endothelial cells derived from human brain.Lab Invest. 1991; 64: 425-436PubMed Google Scholar, 41Bernas M.J. Cardoso F.L. Daley S.K. et al.Establishment of primary cultures of human brain microvascular endothelial cells to provide an in vitro cellular model of the blood-brain barrier.Nat Protoc. 2010; 5: 1265-1272Crossref PubMed Scopus (161) Google Scholar on the other hand, could explain minimal central nervous system involvement during aHUS and predominant central nervous system manifestation during TTP. In this study we determined that VWF-deficient ECs (Type 3 VWD BOECs) resulted in the upregulation of complement-mediated EC injury, contrary to previous suggestions and recent publications. We were able to demonstrate that VWF-multimers released by ECs contribute to EC protection by acting as complement regulator. The lack of VWF resulted in increased complement deposition and cytotoxicity. Our results provide evidence for a functional link between complement and VWF. Further work is needed to elucidate the exact mechanism of TMA pathogenesis. Ethics approval was obtained from the Research Ethics Boards of The Hospital for Sick Children, Toronto, Ontario, and Queen's University, Kingston, Ontario, Canada. Written informed signed consent was obtained from all patients whose samples were used in this study. The study was executed in keeping with the regulations of the Declaration of Helsinki. BOECs were isolated from the peripheral blood of 3 healthy adult volunteers (control BOECs) and from 2 patients with Type 3 VWD (VWD BOECs). The VWD patients have been previously repo
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