Angiogenesis and the Cdc42 protein
The vascular network comprises three major types of cells: endothelial cells, pericytes, and smooth cell muscle cells [1, 2]. An intricate machinery takes care of cell proliferation, migration, and apoptosis, keeping vessel growth under control and avoiding vascular malformations. A key player is Cdc42, a GTPase with multiple functions across different cell types and species . Cdc42 regulates actin cytoskeleton, polarization, cell junctions, cell migration, and tube formation in endothelial cells. However, it is still unclear which of these functions are directly linked to angiogenesis, because the results coming from different biological models can differ in its relevance in vivo. To elucidate this question, the team lead by Konstantin Gängel created a mouse model with an inducible Cdc42 deletion in endothelial cells, and analyzed angiogenesis in the postnatal retina using confocal microscopy.
Cdc42 is key to functional cell migration in angiogenesis
The researchers generated an inducible Cdc42 deletion in mouse postnatal P7 retinas using Cdh5-CreERT2. An inducible deletion minimizes systemic vascular disruption from constitutive gene knockout, but can also often result in a variable proportion of chimerism. This apparent drawback provides the advantage of having both CDC42-null and Wt endothelial cells in the same sample (mosaic retina), so that phenotypes can be compared at a cellular level if the different populations can be identified. To that end, the Rosa26‑EYFP reporter was also present, allowing to faithfully follow recombinant endothelial cells in mosaic CDC42‑null / Wt retinas (Figure 1).
With this new model, the authors focused on the morphology and composition of sprouting vessels in mosaic retinas. They found that the proportion of Cdc42-null cells at the developing vascular front was significantly lower compared to Wt cells. Complementary experiments demonstrated that Cdc42-null cells migrate significantly slower than Wt endothelial cells and fail to attain their axial polarity.
The role of Cdc42 in regulating actin-driven motility and migration in endothelial cells was clear after imaging recombinant cells. The Cdc42-null cells presented abnormal cytoskeleton, with discontinuous stress fibers, altered actin accumulation, and mislocalized paxillin, responsible for the regulation of cell motility through propagation of signal from integrins and growth factors.
An assessment of the cell proliferation levels revealed that the absence of Cdc42 had no influence on this process. Together with live cell imaging and in silico simulation, these results suggested that the endothelial cells failed to spread into the vascular network, but rather accumulated densely in veins and capillaries, leading to vascular malformations (Figures 2-3).
The researchers discovered an essential role of the Cdc42 protein in angiogenesis in terms of proper axial polarity and migration of endothelial cells. During normal retinal development, the proliferation of these cells in veins and capillaries is significantly higher than in arteries. A combination of normal proliferation and impaired migration in the absence of Cdc42 leads initially to a local accumulation of endothelia cells in veins and capillaries, which subsequently develop into vascular malformations (Figure 4). The findings explain why arteries still appear normal in defective retinas, and also aligns with the notion that arteries originate from vein-derived tip cells .
The proposed mouse model serves as a flexible platform for future research of the vascular function of other proteins. For example, recent reports suggest a link between Cdc42 and the Hippo protein kinase in tip cell migration and angiogenesis [5, 6]. Considering that Cdc42 vascular defects are very similar to those observed in animal models of cerebral cavernous malformations, it is of high interest to investigate if those similarities occur at the molecular level.
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- Melendez J, Grogg M, Zheng Y. Signaling Role of Cdc42 in Regulating Mammalian Physiology. Journal of Biological Chemistry 2011.
- Xu C, Hasan SS, Schmidt I, Rocha SF, Pitulescu ME, Bussmann J, Meyen D, Raz E, Adams RH, Siekmann AF. Arteries are formed by vein-derived endothelial tip cells. Nature Communications 2014.
- Kim J, Kim YH, Kim J, Park DY, Bae H, Lee D-H, Kim KH, Hong SP, Jang SP, Kubota Y, et al. YAP/TAZ regulates sprouting angiogenesis and vascular barrier maturation. J Clin Invest. 2017.
- Sakabe M, Fan J, Odaka Y, Liu N, Hassan A, Duan X, Stump P, Byerly L, Donaldson M, Hao J, et al. YAP/TAZ-CDC42 signaling regulates vascular tip cell migration. Proc Natl Acad Sci USA 2017.
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