Coregulation of vascular tube stabilization by endothelial cell TIMP-2 and pericyte TIMP-3

Abstract

The endothelial cell (EC)–derived tissue inhibitor of metalloproteinase-2 (TIMP-2) and pericyte-derived TIMP-3 are shown to coregulate human capillary tube stabilization following EC–pericyte interactions through a combined ability to block EC tube morphogenesis and regression in three-dimensional collagen matrices. EC–pericyte interactions strongly induce TIMP-3 expression by pericytes, whereas ECs produce TIMP-2 in EC–pericyte cocultures. Using small interfering RNA technology, the suppression of EC TIMP-2 and pericyte TIMP-3 expression leads to capillary tube regression in these cocultures in a matrix metalloproteinase-1 (MMP-1)–, MMP-10–, and ADAM-15 (a disintegrin and metalloproteinase-15)–dependent manner. Furthermore, we show that EC tube morphogenesis (lumen formation and invasion) is primarily controlled by the TIMP-2 and -3 target membrane type (MT) 1 MMP. Additional targets of these inhibitors include MT2-MMP and ADAM-15, which also regulate EC invasion. Mutagenesis experiments reveal that TIMP-3 requires its proteinase inhibitory function to induce tube stabilization. Overall, these data reveal a novel role for both TIMP-2 and -3 in the pericyte-induced stabilization of newly formed vascular networks that are predisposed to undergo regression and reveal specific molecular targets of the inhibitors regulating these events.

In vitro assays of angiogenesis for assessment of angiogenic and anti-angiogenic agents

“…Suspension of endothelial cells within a gel is technically more complicated than plating of HUVEC on Matrigel, but the CLS in gel suspension assays form in more-dense networks, and non-HUVEC endothelial cells readily form CLS in these assays. Endothelial tube formation within matrix has been used to show both angiogenic and anti-angiogenic effects ( Figure 3B; Saunders et al, 2006).…”

Fluid Shear Stress and Sphingosine 1-Phosphate Activate Calpain to Promote Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Membrane Translocation and Endothelial Invasion into Three-dimensional Collagen Matrices

“…The MT1-MMP cytoplasmic tail is phosphorylated by Src to regulate proteolytic activity and membrane localization (47), and S1P stimulates translocation of MT1-MMP to the membrane (48). MT1-MMP is clearly required for vessel outgrowth and lumen formation (38,39,41,42), but the intracellular molecular events that control MT1-MMP activation and membrane translocation following proangiogenic stimulation of ECs are defined incompletely. Here, we investigated whether calpain activation acts upstream of proangiogenic factor-induced MT1-MMP membrane translocation and activation.…”

“…1), suggesting a possible defect in matrix proteolysis. MT1-MMP plays a key role in matrix proteolysis and regulates angiogenic sprouting events (38,41,40,56). Calpain inhibition significantly attenuated invading sprout thickness (Fig.…”

EphrinB2 Reverse Signaling Protects against Capillary Rarefaction and Fibrosis after Kidney Injury

“…The assay was performed as described 5,22,38 with some modifications. In brief, HUVECs (ATCC, P3-6), were grown in Vascular Cell Basal Medium (ATCC) containing Endothelial Cell Growth Kit-BBE (ATCC).…”