Endothelial Small GTPase RhoA: A Potential Target for Anti‐angiogenic Therapy

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ObjectiveTumor vessels display abnormal structure and function due to imbalanced angiogenesis. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are the best known pro‐angiogenic factors in this process. The goal of antiangiogenic therapy is to inhibit tumor growth by oxygen and nutrient deprivation. Current antiangiogenic therapies target VEGF/VEGFR signaling pathways. However development of resistance due to upregulation of bFGF remains a serious disadvantage of current approaches, leading to efforts of simultaneous targeting of both VEGF and FGF pathways. A small GTPase RhoA has been reported to regulate VEGF‐induced angiogenesis. In the present study we investigate the role of endothelial RhoA in bFGF‐induced angiogenesis to identify whether RhoA can be considered a target for both VEGF‐ and FGF‐induced angiogenesis.MethodsWe performed RhoA pulldown assays to identify RhoA activation downstream of bFGF treatment. We used Boyden chamber cell migration assay, 2D tube formation assay using matrigel and 3D sprouting assay with collagen to assess angiogenic potential. Endothelial RhoA was knocked down by siRNAs or blocked by pharmacological inhibitors. Biochemical assays were performed to identify the molecular mechanism of bFGF‐induced RhoA activation. In vivo, endothelial‐specific inducible RhoA‐deficient mice with a fluorescent reporter were used in a newly modified matrigel plug angiogenesis model allowing identification of endothelial RhoA excision efficiency.ResultsbFGF treatment activates RhoA in human umbilical vein endothelial Cells (HUVEC). RhoA inhibition with C3 toxin or RhoA knockdown abrogated bFGF‐induced endothelial cell migration and tube formation in vitro. In vivo, endothelial RhoA deficiency blocked bFGF‐induced angiogenesis in matrigel plug assay. Among the bFGF receptors, FGFR1 is predominantly expressed in HUVECs and FGFR1 knockdown abrogated bFGF‐induced RhoA activation and angiogenesis. RhoA inhibition partially blocked bFGF‐induced JNK phosphorylation but did not affect ERK and p38 activation. Ongoing experiments are aiming to delineate the precise role of endothelial RhoA in the FGF downstream pathway.ConclusionCollectively, our data suggest that RhoA pathway participates in bFGF‐induced angiogenesis and can be a potential target for anti angiogenic therapy.