Junctional Adhesion Molecule‐A Maintain Vascular Endothelial Barrier Integrity by Suppressing VEGF/VEGFR2 Expression

Vascular barrier function is important for maintaining vascular integrity and is regulated by endothelial tight junctions. Breaches in the tight junction result in increased endothelial cell (EC) permeability, which leads to a variety of pathological conditions such as edema, vascular degeneration, diabetic retinopathy, and ischemia reperfusion injury. Junctional adhesion molecule A (JAM‐A), which resides at the tight junctions of ECs, is known to regulate neutrophil extravasation and FGF‐2‐induced angiogenesis. The role of JAM‐A in regulation of vascular barrier function is not known. Genetic ablation of Jam‐A does not affect blood vessel structure. However, basal vascular permeability as assessed using Miles assay was significantly enhanced ( P<0.05 ) in Jam‐A knockout (KO) mice compared to the age‐matched wild‐type (WT) controls, suggesting that a lack of Jam‐A in EC compromises vascular integrity. This was further supported by the fact that there was increased edema in the lungs of the Jam‐A KO mice compared to WT controls. Since VEGF is also known as vascular permeability factor, we evaluated the plasma levels of VEGF‐A in WT and Jam‐A KO mice using ELISA. Jam‐A deficient mice showed three‐fold ( P<0.004 ) increased levels of VEGF‐A compared to WT mice. Furthermore, quantitative real‐time PCR showed that mRNA levels of VEGFR2, but not sFlt1, were increased in the Jam‐A KO ECs compared to WT ECs. Protein levels as well as surface expression of VEGFR2 were significantly increased ( P<0.001 ) in Jam‐A KO ECs compared to WT ECs, suggesting that lack of Jam‐A results in increased levels of VEGF‐A/VEGFR2. We next evaluated the protein and mRNA levels of both hypoxia‐inducible factor‐1α (HIF‐1α), a transcription factor known to regulate expression of VEGF‐A, and inhibitor of DNA binding 1 (Id1), a transcriptional factor known to regulate expression of VEGFR2. We found that both HIF‐1α and Id1 mRNA as well as protein levels were significantly ( P<0.001 and P<0.05 respectively) augmented in Jam‐A KO ECs compared to WT ECs. Additionally, overexpression of JAM‐A in human umbilical vein endothelial cells (HUVECs) resulted in significantly reduced expression of both HIF‐1α and Id1 proteins. Furthermore, we found that ECs isolated from Jam‐A KO mouse showed significantly enhanced ( P<0.05 ) cell migration in response to VEGF‐A as compared to WT ECs, suggesting that Jam‐A ECs respond favorably to VEGF‐A due to increased VEGFR2 on their surface. To confirm that the observed augmented permeability in Jam‐A KO mice was primarily due to enhanced VEGFR2 signaling, we inhibited VEGFR2 in mice using DC101 (200μg/kg), a function‐blocking antibody. Isotype specific IgG (200μg/kg) was used as control. Inhibition of VEGFR2 completely abrogated basal as well as VEGF‐A‐induced vascular permeability in both Jam‐A KO and WT mice, suggesting that the augmented EC permeability in Jam‐A KO mice is not due to structural defect in tight junctions, but due to enhanced VEGF‐A/VEGFR2 signaling. Our results suggest that JAM‐A positively regulates EC tight junction integrity by suppressing levels of VEGF‐A and VEGFR2 through downregulation of HIF‐1α and Id1 transcription factors. Support or Funding Information NHLBI 1R01 HL063960 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .