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Coordinated regulation by Cdc42, integrin alpha2beta1, and membrane type‐1 metalloproteinase‐dependant signaling of capillary tube formation in 3D collagen matrices

Endothelial cell (EC) capillary lumen formation is one of the key steps in new blood vessel formation. Our previous work has shown that the development and coalescence of intracellular vacuoles is critical for regulating capillary tube assembly in 3D collagen matrices. Here, we show that this vacuole development and coalescence process involves a signaling pathway including integrins, RhoGTPases, and matrix metalloproteinases (MMPs). We utilize siRNA knockdown technology and time‐lapse imaging analysis to show that integrin alpha2beta1, Cdc42 RhoGTPase, and membrane type‐1 MMP (MT1‐MMP) are three key molecules required for EC capillary tube formation. Treatment of ECs with siRNAs targeting alpha2 integrin subunit, Cdc42, or MT1‐MMP markedly hinders the initiation and progression of intracellular vacuole formation, thereby inhibiting EC capillary tube network formation in 3D collagen matrices. Furthermore, we show that inhibitors of both Protein Kinase C (PKC) and tyrosine kinases strongly block these events. Also, addition of phorbol esters, which stimulate PKC, strongly enhances the lumen formation process. Our findings reveal that coordinated regulation of Cdc42, integrin alpha2beta1, and MT1‐MMP‐dependant signaling is required for intracellular lumen and tubular network formation in 3D matrices. A detailed examination is underway to elucidate how these molecules interact and signal together during EC tube morphogenesis.