ESM1

Abstract: Rationale: The ICAP1 (integrin cytoplasmic domain–associated protein-1) is a specific intracellular binding protein of β1-integrins and the cerebral cavernous malformation (CCM) protein CCM1. ICAP1 recruits CCM1 to the cell membrane and activates CCM1 by changing its conformation. Because CCM1 plays a critical role for cardiovascular development, we hypothesized that its activator ICAP1 is involved in vascular differentiation. Objective: The objective of this study was to define the role of ICAP1 in endothelial cells. Methods and Results: Loss of ICAP1 in primary human endothelial cells causes excessive angiogenic branching and network formation in vitro (3D sprouting angiogenesis) and in vivo (xenotransplantation of ICAP1-silenced human endothelial cells). ICAP1 increases cell motility and the initial formation of capillary sprouts but prevents vessel outgrowth. ICAP1 inhibits Rho kinase activity and ERK (extracellular signal-regulated kinase) phosphorylation and induces expression of the cell cycle inhibitors p21 and p27 , leading to less endothelial proliferation. However, ICAP1 promotes endothelial survival and AKT phosphorylation. Global gene expression analyses revealed that the ICAP1 effects are mediated by strong activation of DELTA-NOTCH signaling. Active NOTCH1 or silencing of the NOTCH ligand DLL4 phenocopy the ICAP1 effects and blockade of NOTCH cleavage rescues the ICAP1-mediated defects in endothelial cells. Both ICAP1 and NOTCH1 reduce the expression of ESM1 (endothelial cell–specific molecule-1), and silencing of ESM1 disturbs vascular endothelial growth factor– or fibroblast growth factor 2–induced sprouting angiogenesis. Conclusions: In this study, we identified ICAP1 as a novel regulator to prevent excessive sprouting angiogenesis.

Abstract: Acquired resistance to antiangiogenic drugs has emerged as a potentially important issue in clinical settings; however, the underlying molecular and cellular mechanism of resistance to vascular endothelial growth factor receptor 2 (VEGFR2) tyrosine kinase inhibitor (TKI) remains largely unclear. We evaluated the cellular characteristics of human umbilical vein endothelial cell (HUVEC) clones, which are resistant to VEGFR2-TKI (Ki8751) to elucidate this mechanism of resistance to antiangiogenic drugs. Resistant HUVEC clones were 10-fold more resistant to VEGFR2-TKI than the parental cells and they exhibited an almost complete absence of VEGF-mediated cellular proliferation. The mRNA expression analysis revealed that expression of VEGFR1, VEGFR2 and VEGFR3 was lower in resistant clones, while that of several angiogenic ligands was increased. The protein expression of VEGFR2 was markedly down-regulated in two (R5 and R6 clone) out of five resistant clones. Focusing on the R5 clone, VEGF stimulation did not increase the phosphorylation of VEGFR2 or the dimerization of VEGFR2. The inhibition of phospho-AKT by VEGFR2-TKI was also weakened more than 10-fold in the R5 clone. Finally, a microarray analysis revealed that some angiogenesis-associated, and some angiogenesis-specific genes, including platelet endothelial cell adhesion molecule 1 (PECAM1)/CD31, homeobox A9 (HOXA9), and endothelial cell-specific molecule 1 (ESM1), were remarkably down-regulated in all the resistant clones compared with the parental cells. HUVEC clones resistant to VEGFR2-TKI exhibited down-regulation of VEGFR2, a decreased signal response to VEGF stimulation, and the loss of vascular endothelial markers. These results strongly suggest that an escape from VEGFR2 signaling-dependency is one of the cellular mechanisms of resistance to VEGFR2-TKI in vascular endothelial cells.

Abstract: The endothelial-to-mesenchymal transition (EndoMT) is involved in the complex pathogenesis of renal fibrosis. The soluble proteoglycan endothelial cell-specific molecule 1 (ESM1) is significantly upregulated in many tumor cells and cirrhosis-related disease. The role of ESM1 in renal fibrosis is unknown. This study investigates the role of ESM1 in renal fibrosis, using an in vivo unilateral ureteral obstruction (UUO) mouse model of renal fibrosis and in vitro mouse kidney MES 13 cells overexpressing ESM1. We observed that ESM1 overexpression significantly increased the motility and migration of MES 13 cells, independent of cell viability. In ESM1-overexpressing MES 13 cells, we also observed elevated expression of mesenchymal markers (N-cadherin, vimentin, matrix metallopeptidase 9 (MMP9)) and the fibrosis marker α-smooth muscle actin (α-SMA) and decreased expression of the endothelial marker vascular endothelial cadherin (VE-cadherin) and CD31. In a mouse model of fibrosis induced by unilateral ureter obstruction, we observed time-dependent increases in ESM1, α-SMA, and vimentin expression and renal interstitial collagen fibers in kidney tissue samples. These results suggest that ESM1 may serve as an EndoMT marker of renal fibrosis progression.