My laboratory investigates the molecular mechanisms which regulate capillary lumen formation in 3D extracellular matrices (Davis et al., Anat. Rec., 2002; Davis and Senger, Circ. Res., 2005). In these studies, we have shown that a major mechanism controlling lumen formation is the development of intracellular vacuoles (i.e. through α 2 ß 1 integrin-dependent pinocytosis of plasma membrane) that coalesce to form an intracellular lumenal space. Previously, we showed that the Cdc42 GTPase is required for these lumen formation events. New studies show that addition of matrix metalloproteinase (MMP) inhibitors that block membrane-type MMPs (i.e. TIMP-2 or TIMP-3) markedly inhibits the vacuole and lumen formation process. siRNA suppression of the α 2 integrin subunit, Cdc42 or the membrane MMP, MT1-MMP, markedly blocks EC lumen and tube network formation in 3D collagen matrices. In addition, we have developed time lapse imaging technologies to analyze the tube morphogenesis process in 3D matrices. This allows us to quantitate EC lumen formation over time and to assess the influence of specific genes controlling this process. For example, in Cdc42 siRNA-treated ECs, ECs are unable to initiate intracellular vacuole formation and assume a “half-moon” appearance over a 48 hr time-lapse analysis of morphogenesis. Similarly, α 2 integrin subunit and MT1-MMP knockdown ECs are markedly blocked in the intracellular lumen formation process. These studies have revealed a critical requirement for Cdc42, α 2 ß 1 integrin and MT1-MMP for intracellular lumen and tubular network formation in 3D matrices. A major unresolved question concerns the molecular mechanisms that underlie the ability of pericytes (a recruited EC target cell- analogous to neuron-target interactions) (Davis et al., Anat. Rec., 2002) to stabilize newly formed vascular tubes. Key events for this stabilization response include suppression of further endothelial cell (EC) tube morphogenic events and prevention of tube regression events. Recently, we reported that EC-derived proteinases such as matrix metalloproteinase (MMP)-1 and MMP-10 control capillary tube regression in 3D collagen matrices (Saunders et al., 2005). Interestingly, co-cultures of human ECs and bovine retinal pericytes (PC) in such matrices reveal that PCs completely inhibit this EC-derived MMP-dependent tube regression process. We hypothesized that both ECs and PCs may contribute specific TIMPs to regulate vessel stabilization by interfering with EC proteinases that induce regression. ECs produce TIMP-1 and TIMP-2, but not TIMP-3 or -4 while PCs cultured alone produce high amounts of TIMP-3 but interestingly, little detectable levels of TIMP-2, -3, or -4 when placed alone in 3D collagen matrices. However, EC-PC cocultures show a marked induction of TIMP-3 by PCs suggesting that ECs induce PCs to produce TIMP-3. Importantly, using high levels of PCs (50% of total ECs) in co-cultures, regression inhibition was only overcome when TIMP-2 is suppressed in ECs and TIMP-3 is suppressed in PCs using specific siRNAs. These data strongly suggest that both EC-derived TIMP-2 and PC-derived TIMP-3 are likely required to regulate neovessel stabilization in 3D collagen matrices.
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