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.

Using a fibrin-based angiogenesis model, we have established that there is no canonical mechanism used by ECs to degrade the surrounding extracellular matrix (ECM), but rather the set of proteases used is dependent on the mural cells providing the angiogenic cues. Mesenchymal stem cells (MSCs) originating from different tissues, which are thought to be phenotypically similar, promote angiogenesis through distinct mechanisms. Specifically, adipose-derived stem cells (ASCs) promote utilization of the plasminogen activator-plasmin axis by ECs as the primary means of vessel invasion and elongation in fibrin. Matrix metalloproteinases (MMPs) serve a purpose in regulating capillary diameter and possibly in stabilizing the nascent vessels. These proteolytic mechanisms are more akin to those involved in fibroblast-mediated angiogenesis than to those in bone marrow-derived stem cell (BMSC)-mediated angiogenesis. In addition, expression patterns of angiogenic factors such as urokinase plasminogen activator (uPA), hepatocyte growth factor (HGF), and tumor necrosis factor alpha (TNFα) were similar for ASC and fibroblast-mediated angiogenesis, and in direct contrast to BMSC-mediated angiogenesis. The present study illustrates that the nature of the heterotypic interactions between mural cells and endothelial cells depend on the identity of the mural cell used. Even MSCs which are shown to behave phenotypically similar do not stimulate angiogenesis via the same mechanisms.

Stem cell niches are composed of numerous microenvironmental features, including soluble and insoluble factors, cues from other cells, and the extracellular matrix (ECM), which collectively serve to maintain stem cell quiescence and promote their ability to support tissue homeostasis. A hallmark of many adult stem cell niches is their proximity to the vasculature in vivo, a feature common to neural stem cells, mesenchymal stem cells (MSCs) from bone marrow and adipose tissue, hematopoietic stem cells, and many tumor stem cells. In this study, we describe a novel 3D microfluidic device (MFD) as a model system in which to study the molecular regulation of perivascular stem cell niches. Endothelial cells (ECs) suspended within 3D fibrin gels patterned in the device adjacent to stromal cells (either fibroblasts or bone marrow-derived mesenchymal stem cells) executed a morphogenetic process akin to vasculogenesis, forming a primitive vascular plexus and maturing into a robust capillary network with hollow well-defined lumens. Both MSCs and fibroblasts formed pericytic associations with the ECs, but promoted capillary morphogenesis with distinct kinetics. Biochemical assays within the niche revealed that the perivascular association of MSCs required interaction between their α6β1 integrin receptor and EC-deposited laminin. These studies demonstrate the potential of this physiologically relevant ex vivo model system to study how proximity to blood vessels may influence stem cell multipotency.

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