Mesenchymal cells stimulate capillary morphogenesis via distinct proteolytic mechanisms

Abstract

During angiogenesis, endothelial cells (ECs) degrade their surrounding extracellular matrix (ECM) to facilitate invasion. How interactions between ECs and other cells within their microenvironment facilitate this process is only partially understood. We have utilized a tractable 3D co-culture model to investigate the proteolytic mechanisms by which pre-committed or more highly committed mesenchymal cells stimulate capillary formation. On their own, ECs invade their surrounding matrix, but do not form capillaries. However, in the presence of either mesenchymal stem cells (MSCs) or fibroblasts, ECs form polarized, tubular structures that are intimately associated with mesenchymal cells. Further, ECs upregulate gene expression of several extracellular proteases upon co-culture with either mesenchymal cell type. The administration of both broad spectrum and specific protease inhibitors demonstrated that MSC-stimulated capillary formation relied solely on membrane-type matrix metalloproteinases (MT-MMPs) while fibroblast-mediated sprouting proceeded independent of MMP inhibition unless the plasminogen activator/plasmin axis was inhibited in concert. While other studies have established a role for the ECM itself in dictating proteolysis and matrix degradation during capillary morphogenesis, the present study illustrates that heterotypic cellular interactions within the microenvironment can direct the proteolytic mechanisms required for capillary formation.

Effects of Extracellular Matrix Density and Mesenchymal Stem Cells on Neovascularization In Vivo

“…The overall size of the implants decreased over time due to the degradation of the fibrin hydrogels, a process previously shown to be required for capillary morphogenesis. 11,13 However, because the data reported here represent total numbers of vessels per field of view, rather than vessel density (i.e., numbers of vessels per implant area), the shrinking size of the implants did not impact the quantitative results.…”

“…10 However, when either normal human lung fibroblasts or MSCs were distributed within the hydrogel, they delivered proangiogenic factors closer to the HUVECs, adopted a pericyte-like phenotype, and improved the angiogenic phenotype of the HUVECs even in denser matrices. 11,13 On the basis of our prior in vitro results, the primary objective of this study was to investigate the influence of ECM density on capillary formation in vivo, and to assess the ability of MSCs to stimulate functional vessels within dense 3D fibrin matrices in vivo. To accomplish this goal, fibrin gel precursor solutions of varied protein concentrations of 5, 10, or 15 mg/mL containing mixtures of HUVECs and/or MSCs were injected subcutaneously in SCID mice.…”

Endothelial cell-by-cell profiling reveals the temporal dynamics of VEGFR1 and VEGFR2 membrane localization after murine hindlimb ischemia

“…Critical to the process of angiogenesis is the degradation of the extracellular matrix, allowing for endothelial migration. This process is mediated by several enzymes, including matrix metaloproteinases and serine proteases, through the action of the uPA-uPAR axis (28,43). Activated uPA converts plasminogen to the protease plasmin, allowing for both degradation of the extracellular matrix and activation of matrix metaloproteinases.…”

Three-Dimensional Image Quantification as a New Morphometry Method for Tissue Engineering

“…2,3 Powerful commercial suites (e.g., Imaris, Lucis, and Metamorph) capable of 3D and 4D image analysis and data processing are now available. 4,5 Unfortunately, these packages are often cost-prohibitive; hence, many computational techniques utilize and modify opensource packages such as NIH's ImageJ (Bethesda, MD). For example, plug-ins enable ImageJ users to manually trace microscope images for quantifying branch-like cellular morphologies.…”