Evaluation of Chloride Intracellular Channels 4 and 1 Functions in Developmental and Pathological Angiogenesis

Members of the chloride intracellular channel (CLIC) protein family have been implicated as regulators of tubulogenesis, a critical step in the formation of new blood vessels during angiogenesis. We sought to determine CLIC4 and CLIC1 function in angiogenesis. We hypothesized that CLIC4 and CLIC1 act in endothelial lumen formation and promote both developmental and pathological angiogenesis. Using in vitro studies, we found that CLIC4 promotes endothelial proliferation, network formation, capillary-like sprouting, and lumen formation. In vivo, Clic4 knockout mice display a mild defect in retinal vascular development and an apparent decrease in retinal macrophage content. By implanting murine tumor cells in Clic4 knockout mice, we discovered that Clic4 affects the establishment of lung metastases. Endothelial and smooth muscle cell content of tumors are comparable to wild type, but overall vessel architecture is altered. In studying CLIC1, we found that CLIC1 knockdown results in reduced endothelial proliferation, directed migration, network formation, and capillary-like sprouting in vitro. In vivo analysis revealed no apparent angiogenic phenotype in the developing retinas of Clic1 knockout mice. We developed Clic4-/-;Clic1-/- double mutant embryos, which were unable to develop beyond 9.5 dpc. Whole mount staining of Clic4-/-;Clic1-/- 9.5 dpc embryos for vasculature revealed an angiogenic defect, most notable along the intersomitic vessels and in the brain. Endothelial content is reduced in Clic4;Clic1 double knockout embryos, and double nullizygous embryos were growth retarded. Preliminary histological analysis of Clic4-/-;Clic1-/- 9.5 dpc embryos suggests altered aortic development, reduced proliferation, and increased apoptosis. I conclude that CLIC4 and CLIC1 function in endothelial proliferation and morphogenesis and that Clic4 and Clic1 are required for embryonic development. Together, our findings indicate that CLIC4 and CLIC1 are important in developmental angiogenesis and should be considered in elucidating the molecular mechanisms of tubulogenesis.