Isolated A2 domain of von Willebrand factor binds to stimulated endothelial cells via vimentin, promoting cell signals
ISTH Academy. Martinez-Vargas M. Jul 9, 2019; 264371; PB1180 Topic: Stroke
Marina Martinez-Vargas
Marina Martinez-Vargas
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Isolated A2 Domain of von Willebrand Factor Binds to Stimulated Endothelial Cells via Vimentin, Promoting Cell Signals

M. Martinez-Vargas1, C. Valladolid2, F. Lam1,3, S.-H. Hong4, S. Marrelli4, V. Vijayan1, M. Cruz1
1Baylor College of Medicine, Center for Translational Research on Inflammatory Diseases /Michael E. DeBakey VA Medical Center, Medicine, Houston, United States, 2Baylor College of Medicine,Center for Translational Research on Inflammatory Diseases/Michael E. DeBakey VA Medical Center, Molecular Physiology and Biophysics, Houston, United States, 3Texas Childrens Hospital, Pediatrics, Houston, United States, 4McGovern Medical School, The University of Texas Health Science Center, Neurology, Houston, United States

Main Topic: Arterial Thromboembolism
Category: Stroke

Background: We reported that the interaction of extracellular endothelial vimentin with the A2 domain of von Willebrand factor (VWF) mediates cerebral VWF strings formation. This interaction was inhibited with recombinant A2 domain (rA2), which also improved reperfusion after ischemic stroke and reduce intracerebral hemorrhage using a murine stroke model. Ischemic stroke destabilizes endothelial barrier, promoting leakage. Besides blocking VWF strings formation, we speculate that the binding of rA2 to vimentin induces signals that may result in a good outcome as seen in mice after ischemic stroke.
Aims: To test the hypothesis that the binding of rA2 to vimentin modulates signaling of endothelial cell functions related to endothelial barrier.
Methods: We use purified rA2, Human Umbilical Vein Endothelial Cells (HUVECs), Lipopolysaccharide (LPS) for cell stimulation, RT-PCR, immunoprecipitation, and immunoblots. Electrical impedance was examined to analyze the effect of rA2 in the presence or absence of LPS. In addition, we monitor the effect of rA2 in the expression of Vimentin under the same previous conditions by using RT-PCR, immunoblots, and fluorescence microscopy. Data obtained from at least three experiments in triplicates.
Results: Interestingly, rA2 reduced electrical impedance in both unstimulated and LPS-stimulated HUVECs. The rA2 did not affect the mRNA level of vimentin at steady state. Notably, the high mRNA level of vimentin induced by LPS in HUVECs was significantly attenuated by rA2. Fluorescence microscope analysis of LPS-treated HUVECs incubated with rA2 demonstrated a difference in the distribution of vimentin. Lastly, phosphorylation of PKC and Akt was decreased in HUVECs treated with LPS/rA2 compared to cells treated with LPS/saline.
Conclusions: The results demonstrate that rA2 affects the endothelial permeability independently of endothelial cells activation. The rA2 clearly reduce the mRNA expression of vimentin only on stimulated endothelial cells, and appears to modulate signaling.

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