Junctional Adhesion Molecule-a Regulates Vascular Endothelial Growth Factor-a-Dependent Permeability
Authors: Naik Meghna Ulhas , Naik Ulhas P. 
Published: October 2019

Introduction: 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), is an integral part of the tight junctions of ECs. It has been shown that a lack of Jam-A affects neutrophil extravasation and FGF-2-induced angiogenesis. Role of JAM-A in regulation of vascular permeability is not known.

Methods: Jam-A knockout (KO) and age-matched wild-type (WT) control mice of both sexes were used in this study. In vivo vascular permeability was assessed using Miles assay and quantified colorimetrically by extracting Evans blue in formamide at 56oC. The circulating plasma level of vascular endothelial growth factor A (VEGF-A) in WT and Jam-A KO mice was measured by ELISA assay. Surface expression of VEGF receptor 2 (VEGFR2) was analyzed by flow cytometry. Quantitative real- time PCR was used to determine gene transcription. Protein expression was determined by western blotting. VEGF-induced WT and Jam-A KO EC migration was analyzed by both scratch and trans-well assays.

Results: Although Jam-A resides at the endothelial tight junctions, genetic ablation of Jam-A does not affect blood vessel structure. However, basal vascular permeability was significantly enhanced (P<0.05) in Jam-A KO mice compared to the 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 effect of VEGF-A on vascular permeability in Jam-A KO mice. Upon intravenous injection of VEGF-A (100ng/ml), EC permeability was significantly increased in both WT and Jam-A KO mice. In order to determine the cause of this enhanced basal permeability, we evaluated the levels of VEGF-A in WT and Jam-A KO mice. Jam-A deficient mice showed a three-fold (P<0.004) increased level of plasma VEGF-A compared to WT mice. Additionally, VEGF-A protein level was also increased in the ECs from Jam-A KO mice compared to WT controls. Furthermore, 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-1 α (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 bothHIF-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 bothHIF-1α and Id1 proteins. Furthermore, 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.

Conclusion: Our results suggest that JAM-A positively regulates EC tight junction integrity by suppressing levels of VEGF-A and VEGFR2 through downregulation ofHIF-1α and Id1 transcription factors.

Disclosures

No relevant conflicts of interest to declare.