Defective Angiogenesis, Endothelial Migration and MAPK Signaling in Rap1b−/− Mice.

The small GTPase rap1 regulates multiple cellular processes. Murine rap1b-deficiency is associated with up to 85% lethality due to embryonic hemorrhage, occurring at about E13.5. Surviving rap1b-null mice have a mild platelet aggregation defect (Chrzanowska-Wodnicka et al., JCI, 115:680), but a minimal symptomatic bleeding. Because of the discrepancy between the severity of embryonic and adult phenotypes and because vascular defects are a more likely cause of embryonic hemorrhage we examined vascular patterning in rap1b−/− mice. PECAM1 whole-mount staining at E10.5, and PECAM1 and smooth muscle actin staining of multiple organs at E13.5 was normal in rap1b−/ − mice. Angiogenesis is the main mechanism through which primary vascular plexus is remodeled in embryos after mid-gestation and after birth. In the neonatal retinal model of angiogenesis, blood vessels form as a superficial primary vascular plexus originating at the optic nerve head and spreading over the inner surface of the retina. The measurement of the fluorescently-labeled isolectin-stained vascularized area of the fixed retinas provides a quantitative assessment of angiogenesis. Primary vascular plexus covered 74.3 ± 5.6% of the total retinal area of P7 rap1b +/− mice. In contrast, the vascularized area in rap1b −/ − littermates was decreased to 57.3 ± 7.6% of the total area (data from 7 litters). There was no significant reduction in the vascularized area of rap1b+/− mice when compared to wild-type (WT) littermates (73.63 ± 6.24% and 75.17 ± 4.67%, respectively; data from 9 litters). To further explore the impact of rap1b-deficiency on ex-vivo angiogenesis, we performed aortic ring assay in which isolated aortas were sectioned, embedded in Matrigel and subjected to angiogenic factors. We found a significant decrease in the average number of sprouts from rap1b−/ − rings compared to WT controls in response to bFGF (18.5 ± 7.3 in WT vs. 2.2 ± 3.5 in rap1b−/ − aortas), VEGF (4.94 ± 0.57 in WT vs. 0.86 ± 0.52 in rap1b−/ − aortas), and sphingosine-1− phosphate (3.45 ± 1.23 in WT and 0.88 ± 1.13 in rap1b−/ −). To establish if defective migration may be responsible for the defective angiogenesis in rap1b−/ − mice, we wounded monolayer cultures of lung endothelial cells isolated from rap1b−/ − and WT mice with a pipette tip and subjected them to VEGF or bFGF stimulation. Cells at the wound edge migrated into the wound, closing it over time. The progress of wound closure, a measure of the migration rate, was significantly delayed in rap1b−/ − cells compared to WT cells in response to either VEGF or bFGF (P values were 0.0234 and 0.0476, respectively; α=0.025 in a two-tailed paired t-test, n=9, cells isolated from 4 sets of mice). To begin to elucidate the molecular mechanism responsible for the defective angiogenic response of rap1b−/ − endothelial cells we analyzed signaling downstream from VEGF and bFGF receptors. We found that phosphorylation of p38 MAP kinase, a key regulator of migration, was decreased in rap1b−/ − cells in response to either angiogenic factor. Phosphorylation of ERK, another MAP kinase and a key regulator of cellular proliferation, was decreased in rap1b−/ − cells in response to VEGF treatment. As phosphorylation of these kinases is indicative of their activation, we conclude that rap1b deficiency in endothelial cells leads to defective p38 and ERK signaling downstream from the angiogenic receptors and may provide a molecular explanation for the observed defective in vivo and ex vivo angiogenesis in rap1b−/ − mice.