Chloride Intracellular Channel Protein-4 Functions in Angiogenesis by Supporting Acidification of Vacuoles Along the Intracellular Tubulogenic Pathway

Abstract

Endothelial cells form capillary tubes through the process of intracellular tubulogenesis. Chloride intracellular channel (CLIC) family proteins have been previously implicated in intracellular tubulogenesis, but their specific role has not been defined. In this study, we show that disruption of the Clic4 gene in mice results in defective angiogenesis in vivo as reflected in a Matrigel plug angiogenesis assay. An angiogenesis defect is also apparent in the retina, both in the decreased spontaneous development of retinal vasculature of unstressed mice and in the dramatically decreased angiogenic response of retinal vessels to an oxygen toxicity challenge. We found that endothelial cells derived from Clic4 ؊/؊ mice demonstrated impaired tubulogenesis in three-dimensional fibrin gels compared with cells derived from wild-type mice. Furthermore, we found that tubulogenesis of wildtype cells in culture was inhibited by both an inhibitor of CLICs and an inhibitor of the vacuolar proton ATPase. Finally, we showed that vacuoles along the endothelial tubulogenesis pathway are acidic in wildtype cells, and that vacuolar acidification is impaired in Clic4 ؊/؊ cells while lysosomal acidification is intact. We conclude that CLIC4 plays a critical role in angiogenesis by supporting acidification of vacuoles along the cell-hollowing tubulogenic pathway.

Clustered phosphatidylinositol 4,5 bisphosphate accumulation and ezrin phosphorylation in response to CLIC5A

“…It has been argued that CLICs might not function as Cl 2 channels because the better characterized inhibitors SIDS or DIDS are without effect on CLIC ion conductance (Jentsch et al, 2002). However, in keeping with CLIC-mediated Cl 2 ion conductance, a role in vacuolar and/or vesicular acidification has been reported for CLIC5B in osteoclasts (Edwards et al, 2006), for CLIC1 in macrophages (Jiang et al, 2012) and for CLIC4 in endothelial cells (Ulmasov et al, 2009). Here, we observed that the effect of CLIC5A on ERM phosphorylation was not inhibited by extracellular IAA-94 (Fig.…”

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

“…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).…”

“…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). CLIC4 may also influence vacuolar formation in endothelial cells by regulating pH (Ulmasov 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). CLIC4 may also influence vacuolar formation in endothelial cells by regulating pH (Ulmasov et al 2009). Furthermore, CLIC4 localizes to the midbody and centrosome of cultured bovine aortic endothelial cells and is enriched at cell-cell junctions in choriocarcinoma cells (Berryman and Goldenring 2003), suggesting a role in establishing or maintaining cell polarization.…”

CLIC4 mediates TGF-β1-induced fibroblast-to-myofibroblast transdifferentiation in ovarian cancer

“…Therefore, the stromal fibroblast is an attractive target for therapeutic intervention. CLIC4 promotes proliferation and vascular tubulogenesis of endothelial cells in the formation of new blood vessels (23,30). Here, we found that treatment of fibroblasts with the agents blocking CLIC4 had a marked repressive effect on the production of angiogenic factors associated with myofibroblast transdifferentiation, indicating that inhibiting the CLIC4 might have therapeutic potential targeting tumour stroma.…”