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.Angiogenesis, the formation of new capillaries from preexisting vasculature, is an essential process, both during development (46) and throughout life (5). Maladaptive angiogenesis contributes to numerous cardiovascular diseases such as atherosclerosis, brain ischemia, hypertension, and stroke (5). The process of angiogenesis is usually initiated by increased endothelial cell permeability and proliferation, followed by the proteolysis of basement membrane components. These cells then sprout and migrate toward a site of needed blood supply where they reestablish junctions, form tube structures, and subsequently stabilize into mature capillaries (37). However, the mechanisms underlying this process are only poorly understood.Rap1 is a Ras family small GTPase that serves as a molecular switch. It couples extracellular stimuli to intracellular effectors and their resulting biological responses by cycling between inactive GDP-and active GTP-bound states. In response to ligand binding to multiple cell surface receptors, Rap1 is activated by guanine nucleotide exchange factors (GEFs) and is subsequently converted back to its inactive GDP-bound state by GTPase-activating proteins (GAPs) (3, 4, 44). There exist two closely related Rap1 family members, Rap1a and Rap1b, which are encoded by separate genes but share 95% amino acid identity and may have complementary as well as distinct biological functions.Rap1 was first reported to antagonize Ras by binding to but not activating the c-Raf-1 kinase (11,27). However, in cell types that express B-Raf, Rap1 ...

Isoform-specific differences between Rap1A and Rap1B GTPases in the formation of endothelial cell junctions

“…Interestingly, we found that not all cellular events differentially utilized Rap1 isoforms. In agreement with previously published work, 36,42 we observed that loss of either isoform inhibited directed chemotactic migration of ECs Figure 3. characterization of isoform-specific knockdown approach.…”

“…41 However, in endothelial cells (both microvascular ECs and HUVECs), the consensus seems to be that both isoforms are required for effective migration in wound healing assays, 36,39,42 an observation that our migration data confirms (sup. fig.…”

“…With respect to EC junctional regulation and permeability, some studies have shown that siRNA-mediated knockdown of either Rap1A or Rap1B decreased transendothelial electrical resistance (TER), 42 or enhanced FITC-dextran permeability. 60 We found that only knockdown of Rap1A had a significant Figure 6.…”

“…For example, it has been reported that EC proliferation, EC permeability, and/or cell migration require Rap1A only, 29,41 Rap1B, 36 or both Rap1A and 1B. 39,42 The results have varied depending on cell type (epithelial vs. endothelial), subtype (microvascular endothelial vs. HUVEC) or method of knockdown (genetic knockout vs. various siRNA methods). In addition, not all of these studies looked at both isoforms, focusing instead on one or the other.…”

Translational Analysis of Mouse and Human Placental Protein and mRNA Reveals Distinct Molecular Pathologies in Human Preeclampsia

“…2E). Rapgef1 is involved in regulation of Fgf signaling (37) and its expression implies a unique role in the trophoblast, although Fgf signaling is also involved in some aspects of endothelial development (38). In contrast, Eng, Psen1, Ppap2b, Reck, and Cav1 were all detected uniquely in EC (Fig.…”