Chloride intracellular channel 4 is involved in endothelial proliferation and morphogenesis in vitro

Abstract

New capillaries are formed through angiogenesis and an integral step in this process is endothelial tubulogenesis. The molecular mechanisms driving tube formation during angiogenesis are not yet delineated. Recently, the chloride intracellular channel 4 (CLIC4)-orthologue EXC-4 was found to be necessary for proper development and maintenance of the Caenorhabditis elegans excretory canal, implicating CLIC4 as a regulator of tubulogenesis. Here, we studied the role of CLIC4 in angiogenesis and endothelial tubulogenesis. We report the effects of inhibiting or inducing CLIC4 expression on distinct aspects of endothelial cell behavior in vitro. Our experiments utilized RNA interference to establish cultured human endothelial cell lines with significant reduction of CLIC4 expression, and a CLIC4-expressing lentiviral plasmid was used to establish CLIC4 overexpression in endothelial cells. We observed no effect on cell migration and a modest effect on cell survival. Reduced CLIC4 expression decreased cell proliferation, capillary network formation, capillary-like sprouting, and lumen formation. This suggests that normal endogenous CLIC4 expression is required for angiogenesis and tubulogenesis. Accordingly, increased CLIC4 expression promoted proliferation, network formation, capillary-like sprouting, and lumen formation. We conclude that CLIC4 functions to promote endothelial cell proliferation and to regulate endothelial morphogenesis, and is thus involved in multiple steps of in vitro angiogenesis.

Notch1 functions as a negative regulator of lymphatic endothelial cell differentiation in the venous endothelium

“…HdLEC were maintained in EGM-2MV BulletKit (Lonza) with 10 ng/ml VEGFC (RnD). HdLEC lines were generated by lentiviral infection using pCCL.pkg.wpre vector encoding N1IC, Hey1, Hey2 or GFP (Tung et al, 2009). Transgene expression was confirmed by quantitative (q) RT-PCR and western blotting.…”

Notch signaling functions in lymphatic valve formation

“…HdLECs were infected with an adenovirus encoding a constitutively active Notch1 intracellular domain (N1IC), a constitutively active allele of Notch4 (N4int3), lacZ or GFP (Shawber et al, 2007;Tung et al, 2009). N1IC or N4int3 induced α9 transcripts (Fig.…”

“…HdLECs were infected with adenovirus expressing constitutively active Notch1 (N1IC) or Notch4 (N4int3), or Hey1, Hey2, lacZ or GFP (Shawber et al, 2007;Tung et al, 2009). Expression verified by qRT-PCR and/or western blot analyses.…”

Tips, Stalks, Tubes: Notch-Mediated Cell Fate Determination and Mechanisms of Tubulogenesis during Angiogenesis

“…They exist as both soluble cytoplasmic proteins and membrane bound channel proteins with a putative transmembrane domain near the amino terminus, making them unique as mammalian ion channels (Redhead et al 1997;Berry and Hobert 2006). Of the six known mammalian CLICs, only CLIC1 and CLIC4 are expressed highly in endothelial cells Money et al 2007;Suh et al 2007;Ulmasov et al 2007;Tung et al 2009;Tung and Kitajewski 2010;Wegner et al 2010). In addition, CLIC5 levels are found to be low in placental endothelial cells (Money et al 2007) and high in glomerular endothelial cells (Wegner et al 2010).…”

“…CLIC4 has been found to be localized to vesicles in human umbilical vein endothelial cells (Bohman et al 2005) and large vacuoles in mouse heart endothelial cells (Ulmasov et al 2009). In vitro, CLIC4 promotes endothelial proliferation, network formation, capillary-like sprouting, and lumen formation (Tung et al 2009). Analyses of retinal vasculature in developing mice, and in adult mice challenged by an oxygen toxicity assay, reveals stunted vascular development in clic4 2/2 mice (Ulmasov et al 2009).…”