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Homocysteine-mediated thrombosis and angiostasis in vascular pathobiology
Joseph Loscalzo
Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA.
Address correspondence to: Joseph Loscalzo, Department of Medicine, Brigham and Women’s Hospital, 75 Francis Street, Boston, Massachusetts 02115-2394, USA. Phone: (617) 732-6340; Fax: (617) 732-6439; E-mail: jloscalzo@partners.org.
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Published October 19, 2009 - More info
J Clin Invest. 2009;119(11):3203–3205. https://doi.org/10.1172/JCI40924.
© 2009 The American Society for Clinical Investigation
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Abstract
When plasma levels of homocysteine (HC), a thiol amino acid formed upon methionine demethylation, exceed 12 μM, individuals are at increased risk of developing large vessel atherothrombosis and small vessel dysfunction. The annexin A2 complex (termed “A2”) is the cell surface coreceptor for plasminogen and TPA and accelerates the catalytic activation of plasmin, the major fibrinolytic agent in mammals. We previously showed that HC prevents A2-mediated, TPA-dependent activation of plasminogen in vitro by disulfide derivatization of the “tail” domain of A2. We also demonstrated that fibrinolysis and angiogenesis are severely impaired in A2-deficient mice. We now report here that, although hyperhomocysteinemic mice had a normal coagulation profile and normal platelet function, fibrin accumulated in their tissues due to reduced perivascular fibrinolytic activity and angiogenesis was impaired. A2 isolated from hyperhomocysteinemic mice failed to fully support TPA-dependent plasmin activation. However, infusion of hyperhomocysteinemic mice with fresh recombinant A2, which localized to neoangiogenic endothelial cells, resulted in normalization of angiogenesis and disappearance of peri- and intravascular fibrin. We therefore conclude that hyperhomocysteinemia impairs postnatal angiogenesis by derivatizing A2, preventing perivascular fibrinolysis, and inhibiting directed endothelial cell migration. These findings provide a mechanistic explanation for microvascular dysfunction and macrovascular occlusion in individuals with hyperhomocysteinemia.
Authors
Andrew T. Jacovina, Arunkumar B. Deora, Qi Ling, M. Johan Broekman, Dena Almeida, Caroline B. Greenberg, Aaron J. Marcus, Jonathan D. Smith, Katherine A. Hajjar
Over the past 30 years, evidence from many in vitro and in vivo studies supports an association between mild to moderate hyperhomocysteinemia and vascular dysfunction and disease (1). A key mechanism that predisposes to vascular disease in hyperhomocysteinemia is endothelial dysfunction (2). Homocysteine induces endothelial dysfunction in part by promoting oxidant stress, as illustrated in cellular studies (3, 4) and in genetic animal models (2, 5, 6). The mechanisms for homocysteine-induced oxidant stress are complex and include inhibiting the translation of glutathione peroxidase-1 (7), a major antioxidant enzyme in vascular cells that regulates mitochondrial reactive oxygen species flux (8) and whose overexpression rescues the normal vascular phenotype of hyperhomocysteinemic mice (9); upregulation of NADPH oxidase expression (10); enhanced expression of inducible nitric oxide synthase (10) and uncoupling of nitric oxide synthases (11); and decreased glutathione levels owing to decreased cysteine synthesis (12), thereby shifting the redox balance of endothelial cells toward oxidant stress.
Among the specific phenotypic properties of the endothelial cell that go awry in hyperhomocysteinemia are its antithrombotic properties. Hyperhomocysteinemia produces a prothrombotic state (13), some mechanisms for which include enhanced platelet activation, enhanced coagulation (likely as a consequence of increased tissue factor expression; ref. 14), and attenuated fibrinolysis. Two recognized mechanisms for reduced fibrinolysis include posttranslational modification of fibrinogen by homocysteinylation, rendering the fibrin derived from fibrinogen relatively resistant to plasmin (15), and increased activity of thrombin-activatable fibrinolysis inhibitor (TAFI) (16).
In this issue of the JCI, Jacovina and colleagues (17) report the existence of another antifibrinolytic mechanism caused by homocysteine. In the group’s earlier work (18, 19), they demonstrated that the calcium-regulated, phospholipid-binding protein annexin A2 is an endothelial coreceptor for TPA and plasminogen that facilitates plasminogen activation by enhancing the catalytic efficiency of TPA. They next demonstrated that homocysteine forms a mixed disulfide bond with the Cys9 residue of annexin A2, which decreases the binding affinity of the receptor for TPA. As a result of this posttranslational modification, endothelial cell surface plasminogen activation is markedly attenuated (18). These observations were next confirmed in vivo using Anxa2–/– mice, which showed increased perivascular fibrin deposition and impaired fibrin clearance. Fibrin formation and plasmin-dependent remodeling are not only important in the hemostatic response to vascular injury, but also for luminal recanalization and angiogenesis during the healing phase. Consistent with this role for fibrin and plasmin, the Anxa2–/– mice showed significantly impaired angiogenesis in a range of vascular injury models (neoangiogenesis) (19). In their latest study, reported in this issue of the JCI (17), the investigators found that moderate hyperhomocysteinemia induced by feeding mice methionine also leads to impaired fibrinolysis with perivascular fibrin persistence as well as attenuated angiogenesis (angiostasis). In this animal model, they observed significantly reduced TPA–annexin A2 binding and plasminogen activation consistent with the importance of endothelial cell surface plasmin activity for normal fibrin clearance and angiogenesis. The pathophenotype was readily reversed by infusion of wild-type recombinant annexin A2 into the hyperhomocysteinemic mice (17).
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Conclusions
Thus, the current study by Jacovina and colleagues (17) adds an important new feature to the adverse vascular phenotype of hyperhomocysteinemia, namely, impaired angiogenesis or angiostasis. Furthermore, this study links angiostasis to altered endothelial cell surface plasminogen activation, supporting the importance of plasmin in the angiogenic response. In addition and importantly, their results give one pause to consider the pathobiological network that defines the interface between thrombosis and angiogenesis (see Figure 1). Taken together, these findings highlight the complexity of molecular events that conspire to produce a prothrombotic and angiostatic endothelial pathophenotype from homocysteine exposure and offer novel explanations for the epidemiological association between hyperhomocysteinemia and vascular disease.
Figure 1Direct and indirect effects of homocysteine on angiogenesis. Lines with arrows indicate permissive actions, and lines with end bars indicate inhibitory actions. Red lines indicate the contributions of the work of Jacovina and colleagues (17) to these pathways. Homocysteine (HC) induces tissue factor (TF) expression on the endothelial cell and inhibits annexin A2–dependent plasminogen (PGN) activation by TPA. In addition, homocysteine increases plasminogen activator inhibitor-1 (PAI-1) expression to impair further fibrinolysis. By activating tissue factor expression, homocysteine promotes local generation of thrombin, which converts fibrinogen (FGN) to fibrin, impairs fibrinolysis by increasing TAFI, and activates factor XIII (FXIII). FXIIIa, in turn, not only crosslinks fibrin, but also crosslinks the integrin αVβ3 to VEGFR2, which stimulates angiogenesis. Thrombin, along with factor Xa (FXa), activates MMPs (to aMMPs) to promote endothelial cell migration and angiogenesis. Offsetting these indirect angiogenic effects of thrombin are its inhibitory effects, which include factor XIIIa–mediated inhibition of thrombospondin-1 (TSP-1) and the generation of angiogenesis inhibitors endostatin and angiostatin by MMPs acting on collagen 18 and plasminogen, respectively. αVβ3-VEGFR2, factor XIIIa–crosslinked αVβ3-VEGFR2; A2, annexin A2; FDP, fibrin degradation product; Pn, plasmin.
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