Antiphospholipid antibodies promote leukocyte–endothelial cell adhesion and thrombosis in mice by antagonizing eNOS via β2GPI and apoER2
Sangeetha Ramesh, Craig N. Morrell, Cristina Tarango, Gail D. Thomas, Ivan S. Yuhanna, Guillermina Girardi, Joachim Herz, Rolf T. Urbanus, Philip G. de Groot, Philip E. Thorpe, Jane E. Salmon, Philip W. Shaul, Chieko Mineo
Sangeetha Ramesh, Craig N. Morrell, Cristina Tarango, Gail D. Thomas, Ivan S. Yuhanna, Guillermina Girardi, Joachim Herz, Rolf T. Urbanus, Philip G. de Groot, Philip E. Thorpe, Jane E. Salmon, Philip W. Shaul, Chieko Mineo
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Research Article Hematology

Antiphospholipid antibodies promote leukocyte–endothelial cell adhesion and thrombosis in mice by antagonizing eNOS via β2GPI and apoER2

Abstract

In antiphospholipid syndrome (APS), antiphospholipid antibodies (aPL) binding to β2 glycoprotein I (β2GPI) induce endothelial cell–leukocyte adhesion and thrombus formation via unknown mechanisms. Here we show that in mice both of these processes are caused by the inhibition of eNOS. In studies of cultured human, bovine, and mouse endothelial cells, the promotion of monocyte adhesion by aPL entailed decreased bioavailable NO, and aPL fully antagonized eNOS activation by diverse agonists. Similarly, NO-dependent, acetylcholine-induced increases in carotid vascular conductance were impaired in aPL-treated mice. The inhibition of eNOS was caused by antibody recognition of domain I of β2GPI and β2GPI dimerization, and it was due to attenuated eNOS S1179 phosphorylation mediated by protein phosphatase 2A (PP2A). Furthermore, LDL receptor family member antagonism with receptor-associated protein (RAP) prevented aPL inhibition of eNOS in cell culture, and ApoER2–/– mice were protected from aPL inhibition of eNOS in vivo. Moreover, both aPL-induced increases in leukocyte–endothelial cell adhesion and thrombus formation were absent in eNOS–/– and in ApoER2–/– mice. Thus, aPL-induced leukocyte–endothelial cell adhesion and thrombosis are caused by eNOS antagonism, which is due to impaired S1179 phosphorylation mediated by β2GPI, apoER2, and PP2A. Our results suggest that novel therapies for APS can now be developed targeting these mechanisms.

Authors

Sangeetha Ramesh, Craig N. Morrell, Cristina Tarango, Gail D. Thomas, Ivan S. Yuhanna, Guillermina Girardi, Joachim Herz, Rolf T. Urbanus, Philip G. de Groot, Philip E. Thorpe, Jane E. Salmon, Philip W. Shaul, Chieko Mineo

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Figure 3

eNOS antagonism by aPL and resulting changes in monocyte adhesion require β2GPI.

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eNOS antagonism by aPL and resulting changes in monocyte adhesion requir...
(A) BAECs cultured in the presence or absence of serum for 18 hours were pretreated with NHIgG or aPL (100 μg/ml, 15 minutes), and eNOS activity stimulated by VEGF (100 ng/ml, 15 minutes) was measured. n = 4; *P < 0.05 versus no aPL. (B) BAECs were pretreated with control mouse IgG1 (10 μg/ml) or mouse monoclonal antibody to β2GPI (FC1, 10 μg/ml) for 15 minutes, and basal and VEGF-stimulated eNOS activity was measured. n = 4; *P < 0.05 versus basal, †P < 0.05 versus control. (C) BAECs were pretreated with NHIgG or aPL, or antibodies to β2GPI (3F8 or 2aG4, 10 μg/ml) for 15 minutes, and basal and VEGF-stimulated eNOS activity was measured. n = 6, *P < 0.05 versus basal, †P < 0.05 versus NHIgG. (D) BAECs were treated with vehicle (control) or LPS (100 ng/ml) with or without control IgG (BBG, 10 μg/ml) or 3F8, in the presence or absence of the NO donor SNAP (NO, 20 μmol/l) for 18 hours, and monocyte adhesion was evaluated. n = 4; *P < 0.05 versus control, †P < 0.05 versus no NO treatment. (E) BAECs were treated with vehicle or LPS with or without Ach (10 μmol/l), in the presence of NHIgG, aPL, BBG, 3F8, or 2aG4, and monocyte adhesion was evaluated. n = 4; *P < 0.05 versus no treatment, †P < 0.05 versus LPS alone. (F) BAECs were pretreated with 3F8, monomeric β2GPI (1.5 μg/ml), or β2GPI dimer (1.5 μg/ml) for 15 minutes, and basal and VEGF-stimulated eNOS activity was measured. n = 6; *P < 0.05 versus basal, †P < 0.05 versus VEGF alone.