Defective angiogenesis, endothelial migration, proliferation, and MAPK signaling in Rap1b-deficient mice

Abstract

Angiogenesis is the main mechanism of vascular remodeling during late development and, after birth, in wound healing. Perturbations of angiogenesis occur in cancer, diabetes, ischemia, and inflammation. While much progress has been made in identifying factors that control angiogenesis, the understanding of the precise molecular mechanisms involved is incomplete. Here we identify a small GTPase, Rap1b, as a positive regulator of angiogenesis. Rap1b-deficient mice had a decreased level of Matrigel plug and neonatal retinal neovascularization, and aortas isolated from Rap1b-deficient animals had a reduced microvessel sprouting response to 2 major physiological regulators of angiogenesis: vascular endothelial growth factor (VEGF) and basic fibroblasts growth factor (bFGF), indicating an intrinsic defect in endothelial cells. Proliferation of retinal endothelial cells in situ and in vitro migration of lung endothelial cells isolated from Rap1b-deficient mice were inhibited. At the molecular level, activation of 2 MAP kinases, p38 MAPK and p42/44 ERK, important regulators of endothelial migration and proliferation, was decreased in Rap1b-deficient endothelial cells in response to VEGF stimulation. These studies provide evidence that Rap1b is required for normal angiogenesis and reveal a novel role of Rap1 in regulation of proangiogenic signaling in endothelial cells.

Figure 1

Decreased neovascularization of Matrigel implants in Rap1b^−/− mice in the directed in vivo angiogenesis assay. Quantitation of calcein-AM fluorescent staining of cells recovered from angioreactors supplemented with 500 ng/mL VEGF (■) or 187.5 ng/mL bFGF and 62.5 ng/mL VEGF (▨) implanted in normal (WT) and Rap1b^−/− (KO) mice. Shown are means; error bars are SEM (n = 3). Asterisks indicate the datasets that were compared in a t test for statistical significance. In both instances, there was a statistically significant decrease in neovascularization of Matrigel implants in Rap1b^−/− mice (P < .05).

Figure 2

Delayed neonatal vascularization in P7 Rap1b^−/− retinas. Retinas from P7 littermate Rap1b^−/− (A) and Rap1b^+/− pups (B); littermate Rap1b^+/− (C) and Rap1b^+/+ (D) pups; and P14 littermate Rap1b^−/− (E) and Rap1b^+/− pups (F) were isolated, fixed, mounted, and stained with fluorescent isolectin, as described in “Analysis of retinal neovascularization.” Shown is a representative image of a quarter of each retina. Decrease in vascularization of P7 Rap1b^−/− retinas is absent at the P14 stage. Lines have been inserted to indicate composite images.

Figure 3

Plasma VEGF levels in neonatal rap1b^−/− mice are not decreased. VEGF levels in blood plasma collected from 6- to 8-day-old wild-type (■) and Rap1b^−/− mice (▧) were measured fluorescently using the Bio-Plex Mouse VEGF assay. Plotted is the average value; error bars are SEM (n = 3-7 mice per condition, samples assayed in duplicate). There is a significant elevation of VEGF levels in Rap1b^−/− mice at day 6 compared with the wild-type mice (P = .09).

Figure 4

Decreased BrdU incorporation into endothelial cells in Rap1b^−/−retinas. (A) Low-magnification image (left micrograph) is a composite of confocal images comprising a quarter of the retina isolated from a P7 Rap1b^+/− mouse, which had been injected intraperitoneally with BrdU and stained with Texas red–conjugated isolectin (endothelium, red) and FITC-conjugated anti-BrdU antibody (nuclei, green). Bar represents 500 μm. Boxed area is enlarged (insert) to better show the detail of nuclear staining by BrdU. Bar represents 50 μm. Lines have been inserted to indicate sections of the composite image. (B) Quantification of all BrdU-positive nuclei in one quarter of a retina from each analyzed pup. Plotted are data from Rap1b^+/+ (WT), Rap1b^+/− (Het), and Rap1b^−/− (KO) mice. For each dataset, the box shows 25th to 75th percentile range and median. Whiskers extend to the 10th and 90th percentile, and standard error of the mean is plotted. The number of dividing endothelial cells was significantly reduced in Rap1b^−/− versus normal retinas (284 ± 75 vs 543 ± 101; n = 6; P < .05).

Figure 5

Decreased microvessel sprouting from Rap1b^−/− aortic rings. (A) Aortic rings prepared from normal (WT) or Rap1b^−/− mice were embedded in growth factor–deprived Matrigel and cultured for 4 days in the presence of 50 ng/mL bFGF (top row) or 50 ng/mL VEGF (bottom row). (B) Quantitation of the average number of sprouts per ring from normal (blue bars) or Rap1b^−/− (red bars) aortas indicates reduced ex vivo response of Rap1b^−/− aortas to both factors. Data from 6 independent experiments; 8 to 14 rings per experiment. Error bars are SEM.

Figure 6

Delayed wound healing by Rap1b-deficient lung endothelial cells. (A) Monolayer cultures of lung endothelial cells isolated from Rap1b^−/− and normal (WT) mice were wounded with a pipette tip and subjected to VEGF (A) or bFGF (B) stimulation (t = 0). Cells at the edge of the wound migrated into the wound, closing it over time. After 12 hours, the cells were photographed (t = 12 hours) and migrated distance was measured (C). Bar represents 100 μm. (C) The progress of wound closure, expressed as migrated distance, was significantly delayed in Rap1b^−/− cells (▧) compared with wild-type cells (■) in response to either VEGF or bFGF (n = 9, cells isolated from 4 sets of mice).

Figure 7

Impaired signaling in Rap1b^−/− lung endothelial cells. (A) Typical experiment showing time course of Rap1 activation in normal (WT) or Rap1b-deficient (KO) cells in response to treatment with VEGF. GTP-bound Rap1 was pulled down with RalGDS-GST and detected by an anti-Rap1 polyclonal antibody that recognizes both Rap1 isoforms (top blot). The graph depicts quantitation of fold induction of Rap1 activation by VEGF in WT cells in 5 experiments. The values were obtained by normalizing Rap1 signal to actin content in a corresponding lysate sample (bottom blot). Fold induction of Rap1-GTP loading was calculated by dividing values normalized for actin content from VEGF-treated samples by values from nontreated control samples. Fold induction values obtained from 5 experiments were averaged and are expressed as a percentage of nontreated controls; error bars are SEM. (B,C) Decreased MAPK activation in Rap1b-deficient lung endothelial cells. Western blot analysis of the phosphorylation level of p38 MAPK (B) and ERK (C) in normal (WT) or Rap1b-deficient (KO) endothelial cells cultured and serum-starved for 4 hours (SF) or serum-starved and stimulated for 10 minutes with 50 ng/mL of the indicated growth factor (top blots). Shown are representative experiments. The graphs depict quantitation of fold induction of MAPK phosphorylation by the indicated growth factor from 3 separate experiments. To normalize for protein content, the blots were stripped and reprobed with actin-specific antibody (bottom blots). Fold induction of MAPK phosphorylation was calculated by dividing values from growth factor–treated samples normalized for actin content by values from serum-free control samples. The values for fold induction of MAPK phosphorylation of knockout samples (▧) were expressed as percentage of wild-type values (■). Such derived values from individual experiments were averaged and mean fold induction was plotted with SEM as error bars. Bars represent fold induction of MAPK, which was calculated by dividing values from VEGF-treated samples normalized for actin content by values from nontreated control samples.