The antiphospholipid syndrome (APS) is an autoimmune disease characterized by a markedly increased risk of thromboses and cardiovascular diseases resulting from elevated levels of circulating antiphospholipid antibodies (aPL). Alterations in the function of vascular cells induced by aPL underlie these outcomes;however, the molecular basis of aPL action is not clear. Our recently completed studies indicate that aPL isolated from APS patients fully antagonize endothelial nitric oxide synthase (eNOS) through impaired phosphorylation of the enzyme at S1177 via the phosphatase PP2A. aPL inhibition of eNOS results in increases in endothelial-leukocyte adhesion and thrombosis. We have also discovered that the cell surface receptor, apolipoprotein E receptor 2 (apoER2) is required for induction of vascular dysfunction by aPL. The overall goals of the proposed research are first to determine the molecular basis of aPL-apoER2 actions and second to test novel interventions directed at the mechanism.
Aim 1 will determine how aPL-apoER2 induces vascular dysfunction. The requirement for the adaptor molecule Dab-1 will be tested in cultured endothelial cells and isolated platelets using loss-of-function strategy. Using intravital microscopy, the in vivo role of Dab-1 in aPL-induced leukocyte adhesion and thrombus formation will be assessed in wild-type vs. knock-in mice that express mutant apoER2 incapable of interacting with Dab-1. We will also determine how aPL activates PP2A. Our focus will be to identify and characterize the regulatory B subunits of PP2A required for aPL-mediated eNOS antagonism. Furthermore, we will test if the in vivo effect of aPL on leukocyte adhesion and thrombus formation are mediated by impaired eNOS S1177 phosphorylation by using the S1177D eNOS knock-in mouse in which eNOS is constitutively-active and not amenable to dephosphorylation by PP2A.
Aim 2 will determine the critical aPL target cells in vivo. The bone marrow reconstitution between apoER2+/+ and apoER2-/- mice will be used to assess the role of bone-marrow derived platelets and monocytes. The specific role of endothelium will be tested by crossing floxed apoER2 mice with mice expressing Cre-recombinase regulated by endothelial cadherin promoter to delete apoER2 from endothelium. In both studies, we will use intravital microscopy to assay for aPL-induced leukocyte adhesion and thrombosis.
Aim 3 will test if novel interventions directed at apoER2- and eNOS- related mechanisms prevent the aPL-induced leukocyte adhesion and thrombosis. Experiments will explore whether an engineered antibody that promotes clearance of circulating aPL will prevent aPL-induced vascular responses. A blocking antibody or a small molecule that interferes aPL interaction with target cells will also be tested. The impact of provision of exogenous NO by Molsidomine treatment will also be assessed. Leukocyte adhesion and thrombosis induced by aPL will be assayed by intravital microscopy in wild-type mice. Together these aims will determine how aPL cause leukocyte adhesion and thrombosis and test potential therapeutic strategies to prevent aPL-mediated vascular dysfunction.
The goals of the proposed research project are to elucidate the molecular basis of APS using cultured cells and a series of complementary mouse models and to further test novel interventions directly based on these pathogenic mechanisms. We anticipate that the new knowledge gained will then be rapidly translated into new prophylactic or therapeutic strategies to combat the devastating impact of APS. Considering the staggering impact of vascular diseases on the patients suffering from APS, the goals of the research project are in line with the objectives of the National Institute of Health to address these disorders through medical research.
|Ulrich, Victoria; Konaniah, Eddy S; Lee, Wan-Ru et al. (2014) Antiphospholipid antibodies attenuate endothelial repair and promote neointima formation in mice. J Am Heart Assoc 3:e001369|
|Ulrich, Victoria; Konaniah, Eddy S; Herz, Joachim et al. (2014) Genetic variants of ApoE and ApoER2 differentially modulate endothelial function. Proc Natl Acad Sci U S A 111:13493-8|
|Mineo, Chieko (2013) Inhibition of nitric oxide and antiphospholipid antibody-mediated thrombosis. Curr Rheumatol Rep 15:324|