Homocysteine inhibits neoangiogenesis in mice through blockade of annexin A2–dependent fibrinolysis
Andrew T. Jacovina, … , Jonathan D. Smith, Katherine A. Hajjar
Andrew T. Jacovina, … , Jonathan D. Smith, Katherine A. Hajjar
Published October 19, 2009
Citation Information: J Clin Invest. 2009;119(11):3384-3394. https://doi.org/10.1172/JCI39591.
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Research Article

Homocysteine inhibits neoangiogenesis in mice through blockade of annexin A2–dependent fibrinolysis

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

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Figure 3

Effect of HC on A2 binding of TPA and plasmin generation.

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Effect of HC on A2 binding of TPA and plasmin generation. (A) Lung membr...
(A) Lung membrane proteins from mice maintained on chow, Gly, or Met diets and rA2 (1 μg) were immunoblotted with rabbit anti-mouse TPA or mouse anti-A2 IgG. For ligand blotting (LB), proteins resolved under reducing (R) or non-reducing (NR) conditions were incubated with human TPA and then probed with rabbit anti-mouse TPA. GAPDH served as a loading control. (B) Left: Cryosections from mice on chow, Gly, or Met diets were incubated with biotinylated TPA and stained as described in Methods. Right: Paraffin-embedded lung sections were stained with rabbit anti–HC-A2 tail peptide IgG as described in Methods. Original magnification, ×200. (C) Purification of native A2. Reduced Coomassie blue–stained (CB) SDS-PAGE and immunoblot analysis with monoclonal anti-A2 of native A2 purified from lung tissue of mice on Gly and Met diets. Samples were run on noncontiguous lanes of the same gel. (D) TPA-dependent plasmin generation in the presence of A2 purified from lung tissue. Initial rates of hydrolysis of a fluorogenic plasmin substrate were calculated as described in Methods. RFU, relative fluorescence units. *P < 0.001 versus Gly.