Junctional Adhesion Molecule-1 Is Involved in bFGF-Induced Angiogenesis In Vivo.

Neovascularization is a multistep process that occurs in the body in both physiological and pathological conditions. We have recently shown that Junctional Adhesion Molecule-1 (JAM-1), a member of the Ig superfamily of molecules, is involved in endothelial cell adhesion and migration, leading to angiogenesis. In quiescent endothelial cells, JAM-1 is located at the cell-cell junctions where it forms a complex with integrin αvβ3. Upon treatment of the cells with growth factors, such as bFGF, JAM-1 dissociates from its complex with αvβ3 and redistributes to the cell surface. Blockage of the extracellular domain of JAM-1 inhibits bFGF-induced endothelial cell morphology, proliferation and angiogenesis. Additionally, functional knock-down of JAM-1 using the RNAi technique in endothelial cells showed decreased adhesion and migration of these cells, indicating a possible role for JAM-1 in angiogenesis. In this report, we show that JAM-1 has an important role in bFGF-induced angiogenesis in vivo. Here we present for the first time the generation JAM-1 knock-out mice, using the gene trap strategy. We have successfully confirmed the JAM-1 −/− genotype via Southern, Northern, and Western blot analyses. JAM-1 −/− mice are viable and do not seem to have any external abnormalities, except that they appear to be smaller in size. Retinal fluorescein angiogram revealed no evidence for morphological defects in the vasculature of JAM-1 −/− mice. To evaluate the role of JAM-1 in angiogenesis, we performed an aortic ring assay with both wild type and JAM-1−/− mice. Mouse thoracic aortas were harvested, cross-sectioned into rings of 1-mm thickness, and cultured in a three-dimensional Matrigel supplied with 50 ng/ml bFGF. Vascular sproutings were counted every other day for a period of 7 days at which time they were stained with crystal violet and photographed. Aortic rings from WT mice treated with bFGF showed a 2.8-fold increase in microvessel growth, compared to WT controls with no supplementation of bFGF. In contrast, microvessel sproutings in bFGF treated aortic rings from JAM-1 −/− mice were no more than the vessels in the WT control mice. These results suggest that JAM-1 may be important for bFGF induced angiogenesis. To further confirm the role of JAM-1 in angiogenesis, WT and JAM-1 −/− mice were injected in their flank region with Matrigel containing 80 ng/ml bFGF and 60 U/ml heparin. Two weeks after injection, Matrigel plugs were excised, embedded in paraffin, and the presence of blood vessels was visualized by H&E staining. Matrigel plugs from control WT mice that were not treated with bFGF showed no vascularization, while bFGF supplied Matrigel plugs from WT mice showed a robust vessel growth. Interestingly, bFGF-treated Matrigel plugs form JAM-1−/− mice failed to produce any blood vessels. These ex vivo and in vivo studies using JAM-1−/− mice suggest that JAM-1 has a unique and essential role in bFGF-induced angiogenesis.