Tips, stalks, tubes: notch-mediated cell fate determination and mechanisms of tubulogenesis during angiogenesis.

Angiogenesis is the process of developing vascular sprouts from existing blood vessels. Luminal endothelial cells convert into "tip" cells that contribute to the development of a multicellular stalk, which then undergoes lumen formation. In this review, we consider a variety of cellular and molecular pathways that mediate these transitions. We focus first on Notch signaling in cell fate determination as a mechanism to define tip and stalk cells. We next discuss the current models of lumen formation and describe new players in this process, such as chloride intracellular channel proteins. Finally, we consider the possible medical therapeutic benefits of understanding these processes and acknowledge potential obstacles in drug development.

Chloride intracellular channel 1 functions in endothelial cell growth and migration.

BACKGROUND: Little is known about the role of CLIC1 in endothelium. These studies investigate CLIC1 as a regulator of angiogenesis by in vitro techniques that mimic individual steps in the angiogenic process. METHODS: Using shRNA against clic1, we determined the role of CLIC1 in primary human endothelial cell behavior. RESULTS: Here, we report that reduced CLIC1 expression caused a reduction in endothelial migration, cell growth, branching morphogenesis, capillary-like network formation, and capillary-like sprouting. FACS analysis showed that CLIC1 plays a role in regulating the cell surface expression of various integrins that function in angiogenesis including ß1 and α3 subunits, as well as αVß3 and αVß5. CONCLUSIONS: Together, these results indicate that CLIC1 is required for multiple steps of in vitro angiogenesis and plays a role in regulating integrin cell surface expression.

Patterned rhodopsin expression in R7 photoreceptors of mosquito retina: Implications for species-specific behavior.

Visual perception of the environment plays an important role in many mosquito behaviors. Characterization of the cellular and molecular components of mosquito vision will provide a basis for understanding these behaviors. A unique feature of the R7 photoreceptors in Aedes aegypti and Anopheles gambiae is the extreme apical projection of their rhabdomeric membrane. We show here that the compound eye of both mosquitoes is divided into specific regions based on nonoverlapping expression of specific rhodopsins in these R7 cells. The R7 cells of the upper dorsal region of both mosquitoes express a long wavelength op2 rhodopsin family member. The lower dorsal hemisphere and upper ventral hemisphere of both mosquitoes express the UV-sensitive op8 rhodopsin. At the lower boundary of this second region, the R7 cells again express the op2 family rhodopsin. In Ae. aegypti, this third region is a horizontal stripe of one to three rows of ommatidia, and op8 is expressed in a fourth region in the lower ventral hemisphere. However, in An. gambiae the op2 family member expression is expanded throughout the lower region in the ventral hemisphere. The overall conserved ommatidial organization and R7 retinal patterning show these two species retain similar visual capabilities. However, the differences within the ventral domain may facilitate species-specific visual behaviors.

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

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.

Regulation of cardiovascular development and integrity by the heart of glass-cerebral cavernous malformation protein pathway.

Cerebral cavernous malformations (CCMs) are human vascular malformations caused by mutations in three genes of unknown function: KRIT1, CCM2 and PDCD10. Here we show that the heart of glass (HEG1) receptor, which in zebrafish has been linked to ccm gene function, is selectively expressed in endothelial cells. Heg1(-/-) mice showed defective integrity of the heart, blood vessels and lymphatic vessels. Heg1(-/-); Ccm2(lacZ/+) and Ccm2(lacZ/lacZ) mice had more severe cardiovascular defects and died early in development owing to a failure of nascent endothelial cells to associate into patent vessels. This endothelial cell phenotype was shared by zebrafish embryos deficient in heg, krit1 or ccm2 and reproduced in CCM2-deficient human endothelial cells in vitro. Defects in the hearts of zebrafish lacking heg or ccm2, in the aortas of early mouse embryos lacking CCM2 and in the lymphatic vessels of neonatal mice lacking HEG1 were associated with abnormal endothelial cell junctions like those observed in human CCMs. Biochemical and cellular imaging analyses identified a cell-autonomous pathway in which the HEG1 receptor couples to KRIT1 at these cell junctions. This study identifies HEG1-CCM protein signaling as a crucial regulator of heart and vessel formation and integrity.