Junctional Adhesion Molecule a (JAM-A) Maintains Vascular Endothelial Barrier Integrity through c-Src/Stat3-Dependent Suppression of Inhibitor of DNA-Binding 1 (Id1)

Endothelial cells (ECs) form a monolayer that line the lumen of all blood vessels. ECs maintain vascular integrity and barrier function through tight junctions. Dysregulation of EC tight junctions result in the development of chronic conditions such as hypertension and heart diseases which are the leading cause of death globally. Cell adhesion molecules play a vital role in maintaining the EC tight junctions. Among them junctional adhesion molecule A (JAM-A), which resides at the EC tight junction is known for the regulation of trans-endothelial migration of leukocytes and α_vβ₃-dependent angiogenesis. Whether JAM-A contributes to and regulates EC barrier function is not well understood. To determine the role of JAM-A in EC function, we generated Jam-A deficient mouse by using GeneTrap strategy (Jam-A^gt/gt). ECs isolated from both the Jam-A^gt/gt had impaired cell spreading and focal adhesion formation on vitronectin compared to the WT ECs. Consequently, Jam-A deficient ECs had decreased basal cell migration (P<0.01). However, addition of vascular endothelial growth factor A (VEGF-A) but not basic fibroblast growth factor (bFGF) augmented significantly (P<0.001) cell migration compared to WT ECs, suggesting that the Jam-A null ECs are hyper responsive to VEGF-A. We found that Jam-A null mice show significantly (P<0.05) heightened vascular as well as blood-brain barrier permeability, as assessed by Miles assay compared to the age-matched WT controls, suggesting that a lack of Jam-A in EC compromises vascular integrity and renders ECs more responsive to VEGF-A. Soluble form of VEGFR2 (sFlt) is known to sequester VEGF-A and regulate its signaling. It is possible that sFlt expression may be attenuated in Jam-A null ECs. We therefore tested the sFlt mRNA expression and found no significant difference between WT and the Jam-A null ECs excluding the role for sFlt. Since VEGF-A is known as a vascular permeability factor, we next evaluated the expression of VEGFR2, the major signaling receptor of VEGF-A on ECs. We found that in Jam-A null ECs the expression of VEGFR2 was significantly (P<0.01) augmented as determined by western blot analysis. Furthermore, the surface expression of VEGFR2 as assessed by flow cytometry was also significantly enhanced (P<0.01) compared to the WT ECs suggesting that the observed hyper response of Jam-A null ECs to VEGF-A is due to augmented surface expression of VEGFR2. These results suggest that VEGF-A/VEGFR2 signaling axis is augmented in the absence of Jam-A. We next evaluated whether VEGF-A/VEGFR2 signaling axis is responsible for enhanced vascular permeability in Jam-A null mice. When both Jam-A null and WT mice were pretreated with anti-VEGFR2 (DC101; 200μg/kg) a function blocking antibody or isotype specific (200μg/kg) IgG₁ as a control, we found that the vascular permeability was significantly reduced (P<0.00003) in Jam-A null mice, compared to the WT, indicating that the observed permeability is dependent on VEGFR2. Next, we tested the expression of inhibitor of DNA-binding 1 (Id1), a transcriptional regulator known to relieve E2-2 mediated VEGFR2 gene repression allowing upregulate VEGFR2 mRNA expression. We found that Id1 protein expression was significantly increased (P<0.05) in ECs lacking Jam-A compared to WT ECs. Additionally, ectopic expression of JAM-A in human umbilical vein endothelial cells (HUVECs) attenuated the levels of Id1. Id1 expression is regulated by c-Src/Stat3 dependent pathway and JAM-A is known to be a C-terminal Src kinase (CSK)-binding protein and keeps integrin β₃ associated c-Src inactive. Interestingly, we found that inhibition of c-Src or Stat3 or overexpression of JAM-A in HUVECs attenuates Id1 expression. Our results suggest that JAM-A positively regulates vascular integrity by suppressing levels of VEGFR2 expression on ECs through downregulation of Id1 transcriptional factor thus maintaining EC barrier function.