The BTB-Kelch Protein KLEIP Controls Endothelial Migration and Sprouting Angiogenesis

Abstract

Abstract-Sprouting and invasive migration of endothelial cells are important steps of the angiogenic cascade. Vascular endothelial growth factor (VEGF) induces angiogenesis by activating intracellular signal transduction cascades, which regulate endothelial cell morphology and function. BTB-kelch proteins are intracellular proteins that control cellular architecture and cellular functions. The BTB-kelch protein KLEIP has been characterized as an actin-binding protein that interacts with the nucleotide exchange factor ECT2. We report that KLEIP is preferentially expressed in endothelial cells, suggesting that it may play a critical role in controlling the functions of migrating, proliferating, and invading endothelial cells during angiogenesis. KLEIP mRNA level in endothelial cells is strongly regulated by hypoxia which is controlled by hypoxia-inducible factor-1␣. Functional analysis of KLEIP in endothelial cells revealed that it acts as an essential downstream regulator of VEGF-and basic fibroblast growth factor-induced migration and in-gel sprouting angiogenesis. Yet, it is not involved in controlling VEGF-or basic fibroblast growth factor-mediated proliferative responses. The depletion of KLEIP in endothelial cells blunted the VEGF-induced activation of the monomeric GTPase RhoA but did not alter the VEGF-stimulated activation of extracellular signal-regulated kinase 1/2. Moreover, VEGF induced a physical association of KLEIP with the guanine nucleotide-exchange factor ECT2, the depletion of which also blunted VEGF-induced sprouting. We conclude that the BTB-kelch protein KLEIP is a novel regulator of endothelial function during angiogenesis that controls the VEGF-induced activation of Rho GTPases. (Circ Res. 2007;100:1155-1163.)Key Words: BTB-kelch protein KLEIP Ⅲ angiogenesis Ⅲ migration Ⅲ hypoxia Ⅲ G proteins A ngiogenesis, the formation of blood vessels from preexisting ones, is a crucial process during embryonic development and in several pathological conditions. Vascular endothelial growth factor (VEGF) and its receptors are major regulators of endothelial cell (EC) function and angiogenesis. 1 VEGF is upregulated under hypoxic conditions and induces several angiogenesis-related functions in ECs, such as proliferation, migration, and NO release, which are mediated by cascades of intracellular signaling pathways. [2][3][4] Genetic experiments suggest that VEGF receptor Flk-1/KDR signaling is required for proper positioning of hemangioblastic cells from the posterior primitive streak in the yolk sac. 5 Furthermore, cells lacking Flk1/KDR are unable to coalesce to form blood islands, suggesting that Flk1/KDR signaling controls the migration of ECs. 5 The signaling pathways regulating EC migration involve the phosphorylation of p38 mitogen-activated protein kinase and focal adhesion kinase, 6 activation of phosphatidylinositol 3-kinase, 7 as well as the phosphorylation of protein kinase Akt/protein kinase B, 8,9 with subsequent formation of NO by endothelial NO synthase (eNOS). 10 Yet, mechanistically, V...

The Bipartite Rac1 Guanine Nucleotide Exchange Factor Engulfment and Cell Motility 1/Dedicator of Cytokinesis 180 (Elmo1/Dock180) Protects Endothelial Cells from Apoptosis in Blood Vessel Development

“…Human umbilical vein endothelial cells (HUVECs) were isolated as described previously (38). HUVECs and bovine aortic endothelial cells (BAECs) (39) were cultivated in endothelial cell growth medium (ECGM, PromoCell) containing supplements, 10% FCS (Biochrom), and antibiotics. The following antibodies were used: goat anti-actin (I-19) (Santa Cruz Biotechnology), rabbit anti-Akt (Cell Signaling), mouse anti-pAkt (S473) (587F11) (Cell Signaling), rabbit anti-Dock180 (H-70) (Santa Cruz Biotechnology), goat anti-Elmo1 (Everest Biotech), mouse anti-Rac1 (Transduction Laboratories), mouse antipERK (E-4) (Santa Cruz Biotechnology), rabbit anti-ERK1 (K-23) (Santa Cruz Biotechnology), mouse anti human [pSer 139 ]Histone H2AX (9F3) (Enzo Life Science), Cy3-conjugated secondary antibodies (Dianova), and HRP-conjugated antibodies (DAKO).…”

“…The assay was performed as previously described (39). The spheroids were stimulated upon embedding in collagen with 25 ng/ml VEGF for 24 h. The cumulative sprouting length was set in relation to control and is displayed in percent.…”

Cerebral cavernous malformation protein CCM1 inhibits sprouting angiogenesis by activating DELTA-NOTCH signaling

“…Endothelial behavior was analyzed as described (33). Apoptosis was determined using the Caspase-Glo 3/7 Assay (Promega).…”

Inhibition of Rho-dependent kinases ROCK I/II activates VEGF-driven retinal neovascularization and sprouting angiogenesis

“…VEGF is a growth factor inducing several angiogenesis-related endothelial functions, including migration and proliferation (6). VEGF-receptor activation triggers several downstream signaling cascades, such as the extracellular signal-regulated kinase (ERK) 1/2 pathway (14), the serine/threonine kinase Akt with subsequent nitric oxide release (4,12,13), and the small GTPases Rho (19,27). Recent studies have suggested that VEGF-driven endothelial migration and angiogenesis require activation of the monomeric GTPase RhoA (19,27) and that inhibition of RhoA disrupts tube formation in threedimensional matrixes (8,16,22).…”

“…VEGF-receptor activation triggers several downstream signaling cascades, such as the extracellular signal-regulated kinase (ERK) 1/2 pathway (14), the serine/threonine kinase Akt with subsequent nitric oxide release (4,12,13), and the small GTPases Rho (19,27). Recent studies have suggested that VEGF-driven endothelial migration and angiogenesis require activation of the monomeric GTPase RhoA (19,27) and that inhibition of RhoA disrupts tube formation in threedimensional matrixes (8,16,22). Molecularly, GTP-bound RhoA activates the serine/threonine kinases ROCK I and ROCK II (7,11), which in turn promote stabilization of F-actin, phosphorylation of regulatory myosin light chain and an increase in contractility, processes involved in cell migration and metastasis (2,21).…”