Rap1a is a key regulator of fibroblast growth factor 2-induced angiogenesis and together with Rap1b controls human endothelial cell functions

Abstract

Angiogenesis, the formation of new blood vessels from existing vasculature, is regulated primarily by endothelial cell activity. We show herein that the Ras family GTPase Rap1 has a key role in the regulation of angiogenesis by modulating endothelial cell functions. Blood vessel growth into fibroblast growth factor 2 (FGF2)-containing Matrigel plugs was absent from rap1a(-/-) mice, and aortic rings derived from rap1a(-/-) mice failed to sprout primitive tubes in response to FGF2, when the tissue was embedded in Matrigel. Knocking down either rap1a or rap1b, two closely related rap1 family members, in human microvascular endothelial cells (HMVECs) by utilizing siRNA confirmed that Rap1 plays key roles in endothelial cell function. The rap1a or rap1b knockdown resulted in decreased adhesion to extracellular matrices and impaired cell migration. HMVEC monolayers lacking Rap1 had increased permeability, and Rap1-deficient endothelial cells failed to form three-dimensional tubular structures when they were plated on Matrigel in vitro. Finally, the activation levels of extracellular signal-regulated kinase (ERK), p38, and Rac, which are important signaling molecules in angiogenesis, were all reduced in response to FGF2 when either of the Rap1 proteins was depleted. These observations place Rap1 centrally in the human angiogenic process and suggest that both the Rap1a and Rap1b proteins are required for angiogenesis and that Rap1 is a critical mediator of FGF-induced ERK activation.

FIG. 1.

rap1a knockout mice had an impaired angiogenic response to FGF2. (A) Matrigel plugs containing 20 U/ml heparin plus either PBS or 600 ng/ml FGF2 were injected subcutaneously into wild-type and rap1a^−/− mice. The Matrigel plugs were recovered at day 7, and images were taken. (B) Hemoglobin content of recovered Matrigel plugs, reflecting new blood vessel formation. Bars show means ± standard errors. ***, P < 0.001 (n = 8). (C) Endothelial cells migrated into Matrigel plugs were revealed by CD31 staining. Images are representative of experiments from five mice of each genotype. (D) Aortic rings of 1-mm thickness from either the wild-type or the rap1a^−/− mice were embedded in Matrigel supplemented with 20 U/ml heparin and 2% FBS, with or without 25 ng/ml FGF2. The outgrowth of aortic tubes was observed at day 7, and representative images are shown. (E) Quantification of aortic tube outgrowth and branching. Bars show means ± standard deviations. ***, P < 0.001 (n = 5).

FIG. 2.

Suppression of Rap1 expression decreased HMVEC migration, as shown in a wound healing assay, and adhesion to extracellular matrix proteins. (A) siRNAs selectively knocked down endogenous Rap1a or Rap1b expression in HMVECs and exogenous Rap1a or Rap1b in 293T cells. (Upper panel) Endogenous total Rap1, Rap1b, and GAPDH levels in HMVECs; (middle panel) GST-Rap1a and HA-Rap1b levels in 293T cells; (bottom panel) total Rap1 level and GAPDH from an additional set of siRNAs. (B) HMVECs transfected with two distinct sets of siRNAs were cultured until cells were confluent. Representative data are shown for set I siRNAs. Cells were removed in one direction by a razor blade, and a small incision was made in the plastic to mark the starting point of migration. The distance that cells migrated was recorded after 24 h. Filled arrows indicate the starting point of migration; open arrows indicate the end point of migration. Scale bar = 100 μm. (C) Quantification of cell migration. Bars show means ± standard deviations (SD). ***, P < 0.001 (n = 3). (D) HMVECs transfected with different siRNAs (set I) were plated on type I collagen or fibronectin matrices in 96-well plates for 30 min. Adherent cells were quantitated by staining with crystal violet and measuring absorbance at 600 nm. Bars show means ± SD. ***, P < 0.001 (n = 4).

FIG. 3.

Knockdown of Rap1 expression increased HMVEC junction permeability. HMVECs transfected with control, rap1a, rap1b, or rap1a plus rap1b siRNAs were plated on 0.4-μm filters and cultured until confluent. Transendothelial resistance was measured as an indicator of endothelial monolayer integrity. Bars show means ± standard deviations. ***, P < 0.001 (n = 8).

FIG. 4.

Reducing Rap1 expression abolished HMVEC tube formation on Matrigel. HMVECs transfected with two distinct sets of control, rap1a, rap1b, or rap1a plus rap1b siRNAs were plated on Matrigel in vitro in the presence of 25 ng/ml FGF2, and tubular structures were allowed to develop for 12 h. (A) Representative images of HMVEC tube formation. Scale bar = 500 μm. (B) Quantification of total tube length. Bars show means ± standard deviations. ***, P < 0.001 (n = 3).

FIG. 5.

Loss of Rap1 decreased HMVEC proliferation. HMVECs transfected with control, rap1a, rap1b, or rap1a plus rap1b siRNAs were cultured for 72 h in EBM-2 with different concentrations of FGF2 as indicated. Cell proliferation was measured and expressed as the increase in absorbance versus the control. *, P < 0.05 on siRNA control versus that of rap1 siRNA-treated cells (n = 3).

FIG. 6.

Rap1 depletion abolished Rac activation in HMVECs. (A) HMVECs were starved overnight and stimulated with 25 ng/ml FGF2 for 3 to 60 min. Immunoblotting for Rap1-GTP and total Rap1 levels was performed. (B) Densitometric quantification of protein levels. Bars show means ± standard deviations (SD). *, P < 0.05 (n = 4). (C) HMVECs transfected with control, rap1a, rap1b, or rap1a plus rap1b siRNAs were starved overnight and stimulated with 25 ng/ml FGF2 and 10 μg/ml heparin for 10 min. Immunoblot assays for Rac-GTP and total Rac level were performed. (D) Densitometric quantification of protein levels. Bars show means ± SD; **, P < 0.01; n = 4.

FIG. 7.

Rap1 mediated ERK1/2 and p38 activation by FGF2 in HMVECs. (A) HMVECs were starved overnight and stimulated with 25 ng/ml FGF2 for 3 to 60 min. Immunoblot assays for phosphorylated ERK1/2 and total ERK1/2 are shown. (B) HMVECs transfected with control, rap1a, rap1b, or rap1a plus rap1b siRNAs were starved overnight and incubated with or without 25 ng/ml FGF2 for 10 min. Immunoblot assays for phosphorylated and total ERK1/2 are shown. (C) Densitometric quantification of protein levels. (D) HMVECs were starved overnight and stimulated with 25 ng/ml FGF2 for 3 to 60 min. Immunoblot assays for phosphorylated p38 and total p38 are shown. (E) HMVECs were treated as described for panel B. Immunoblot assays for phosphorylated and total p38 are shown. (F) Densitometric quantification of protein levels. Bars in panels C and F show means ± standard deviations. *, P < 0.05 (n = 3).