Junctional Adhesion Molecule-a, Suppresses Integrin αvβ3 Activation to Control Neovascularization

Abstract

Introduction: Neovascularization, the formation of new blood vessels from the preexisting vessels, is a complex process. It is strictly regulated in healthy adults. Disruption of endothelial cell-cell junction is critically important in inflammation and pathological angiogenesis. Growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2), two potent angiogenic factors, have been shown to disrupt endothelial cell (EC) junctions. VEGF expression by EC and thus VEGF-dependent neovascularization is normally suppressed in healthy adults. The mechanism of this suppression is not known.Methods: Using Jam-A gene-trap knockout mice, we performed a variety of neovascularization assays such as Matrigel plug, aortic ring sprouting and in vivo tumor growth. Age-matched wild-type (WT) mice were used as controls. Vascular permeability was assessed by Miles assay. Level of VEGF in both the WT and Jam-A null mouse plasma were measured by Elisa assay. Real-time PCR and western blot analyses were used to assess gene expression.Results: Jam-A knockout mice showed significantly enhanced (P<0.004) vascular permeability as assessed by Miles assay. Interestingly, VEGF-, but not FGF-2-dependent neovascularization was significantly augmented (P<0.001) in the absence of Jam-A. ECs isolated from Jam-A null mouse showed significantly enhanced (P<0.05) cell migration in the response to VEGF as compared to WT ECs. We found that the plasma levels of VEGF in the Jam-A null mice were significantly (P<0.000001) and age-dependently increased compared to WT mice. We also found that both mRNA and protein levels of VEGF and its receptor VEGFR2 were significantly increased (P<0.001) in Jam-A null ECs. When tested if the expression of soluble Flt (sFlt), which is a decoy receptor for VEGF that is known to trap VEGF, is downregulated in the absence of Jam-A, we found no significant difference in sFlt mRNA expression in Jam-A null ECs compared to WT. Additionally, augmented vascular permeability observed in Jam-A null mice was completely abrogated upon inhibition of VEGFR2 using a function-blocking antibody DC101, suggesting that the VEGF/VEGFR2 signaling axis is augmented in the absence of Jam-A. In order to determine the mechanism of this upregulation of the VEGF and VEGFR2 in the absence of Jam-A, we tested the expression of hypoxia inducible factor-1α (HIF-1α and inhibitor of DNA binding 1 (Id1), two transcription factors known to upregulate VEGF and VEGFR2 gene expression respectively. Interestingly, mRNA and protein levels of both HIF-1α and Id1 were significantly augmented (P<0.02) in ECs lacking Jam-A compared to WT. Since JAM-A is known to maintain integrin αIIbβ3 in a low affinity state in resting platelets by recruiting CSK to the αIIbβ3-Src complex, we tested if lack of Jam-A will augment integrin αvβ3 signaling in ECs. We found that in fact αvβ3-dependent signaling is augmented in Jam-A null ECs or when Jam-A is inhibited using function-blocking antibody compared to WT. Consistent with this finding, the overexpression of JAM-A in HUVECs attenuated the extent of αvβ3 signaling as well as HIF-1α and Id1 expression, suggesting that Jam-A suppresses αvβ3 signaling to maintain low levels of VEGF and VEGFR2 expression in quiescent ECs.Conclusion: The results presented here suggest that Jam-A suppresses integrin αvβ3 signaling to control VEGF/VEGFR2 expression in adult quiescent ECs thus suppressing neovascularization in adults. During pathological conditions, such as ischemia and inflammation, αvβ3/Jam-A complex is dissociated or Jam-A levels are downregulated allowing αvβ3 signaling, thus supporting pathological angiogenesis. DisclosuresNo relevant conflicts of interest to declare.