The Integrin Co-Activator Kindlin-2 Plays a Critical Role In Angiogenesis and Blood Vessel Integrity

Abstract 4

Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice and causes severe developmental defects in zebrafish. Our previous study demonstrated that reduction of kindlin-2 levels in human endothelial cells results in defective adhesive and migratory responses, suggesting that kindlin-2 may be implicated in angiogenesis. We tested this hypothesis in the kindlin-2^+/− mice utilizing murine RM1 prostate tumor and Matrigel implant models. Staining of tumor sections for EC with CD31 showed shorter and thinner blood vessels and reduced vascular area by 3.5-fold in tumors grown in kindlin-2^+/− mice compared to WT mice (P=0.0186, n=6). The vessels that did form in the kindlin-2^+/− mice were immature as they lacked smooth muscle cells and pericytes, had thinner basement membrane, and were leaky as evidenced by increased by 2-fold area for plasma-derived fibrin in tumor sections (P=0.0006, n=5). Consistent with the blunted angiogenic response and vascular leakiness in the kindlin-2^+/− mice, the tumors grown in kindlin-2^+/− animals had 2-fold larger necrotic core and were 2.5-fold smaller than those derived in WT mice (P=0.042, n=7). Also, the permeability of preexisting blood vessels in ear and dorsal skin of WT and kindlin-2^+/− mice was compared after injection of Evans blue dye. Baseline permeability of vasculature in ear and dorsal skin was enhanced by ∼70–100 % in kindlin-2^+/− mice as compared WT mice (P<0.01, n=6). Application of mustard oil, a proinflammatory stimulus, on ear skin or VEGF on dorsal skin, enhanced permeability in WT mice by ∼75–80 % (P<0.05, n=6) while it had a negligible effect in the kindlin-2^+/− mice (P=0.159, n=6). The impaired angiogenic response in the kindlin-2^+/− mice was confirmed in a Matrigel model where VEGF-induced angiogenesis was abnormal. CD31 staining showed that in contrast to the matrigel plugs derived from WT mice, those obtained from the kindlin-2^+/− mice contained shorter and thinner vessels, and the few thicker CD31⁺ structures that did form in the kindlin-2^+/− mice were disorganized. As a consequence of blood leakage from abnormal vessels the Matrigel implants collected from the kindlin-2^+/− mice exhibited a 3.5-fold increase in hemoglobin content as compared to WT mice (P=0.0149, n=5).Bone marrow transplant experiments were performed and showed that bone marrow-derived cells did not significantly contribute to abnormal angiogenesis and enhanced vascular permeability observed in the kindlin-2^+/− mice. Tumor vascular area, plasma-derived fibrin content, tumor growth and vascular permeability in ear and dorsal skin were similar in WT recipients, which received bone marrow from kindlin-2^+/− or WT donors. Aortic endothelial cells isolated from WT and kindlin-2^+/− mice exhibited similar expression levels of the β1 and β3 integrins as well as VEGFR2. However, FACS analysis of soluble ligand binding showed significantly reduced activation of the β3 integrins in kindlin-2^+/− ECs as compared to WT cells, whereas the β1 integrin activation was similar in both genotypes. Additionally, PMA or VEGF enhanced integrin activation in WT ECs, but failed to do so in the kindlin-2^+/− cells. Accordingly, integrin-dependent cellular functions: adhesion, migration and spreading of the EC derived from the kindlin-2^+/− mice were significantly blunted on the β3 integrin substrates but not on the β1 substrates. In control experiments, overexpression of kindlin-2 in the kindlin-2^+/− EC restored defective spreading on vitronectin while a kindlin-2 that does not bind integrins, did not, indicating specificity. Also, tube formation by kindlin-2^+/− ECs was significantly impaired compared to those developed by WT ECs. Taken together, kindlin-2 plays an important role in angiogenesis as well as in maintenance of vascular integrity.