Understanding the role of the extracellular matrix (ECM) in vascular morphogenesis has been possible using natural ECMs as in vitro models to study the underlying molecular mechanisms. However, little is known about vascular morphogenesis in synthetic matrices where properties can be tuned toward both the basic understanding of tubulogenesis in modular environments and as a clinically relevant alternative to natural materials for regenerative medicine. We investigated synthetic, tunable hyaluronic acid (HA) hydrogels and determined both the adhesion and degradation parameters that enable human endothelial colony-forming cells ( IntroductionGenerating a functional vascular network can potentially improve treatment for vascular disease and successful organ transplantation. 1 Since their discovery, marrow-derived circulating endothelial progenitor cells (EPCs) have been demonstrated to participate in postnatal vasculogenesis. 2,3 Putative EPCs have been proposed as a potential therapeutic tool for treating vascular disease, either through infusion to the site of vascularization [4][5][6] or via ex vivo expansion to engineer vascularized tissue constructs. [7][8][9] Research has shown that endothelial colony-forming cells (ECFCs), a subtype of EPCs recently identified from circulating adult and human umbilical cord blood, express characteristics of putative EPCs. 10,11 These ECFCs are characterized by robust proliferative potential in forming secondary and tertiary colonies, as well as de novo blood vessel formation in vivo.The complex processes of vascular regeneration and repair require EPCs to break down the extracellular matrix (ECM), migrate, differentiate, and undergo tubulogenesis. In the last decade, our understanding of the role of the ECM in vascular morphogenesis has greatly expanded because of well-defined in vitro angiogenesis models. Such natural ECMs as matrigel, collagen, and fibrin gels have allowed us to study the molecular mechanisms that regulate endothelial cell (EC) tubulogenesis, 12,13 as well as to transplant vascular progenitor cells, such as human embryonic stem (hES) cell-derived ECs, 14 ECFCs, 15 EPCs, and mesenchymal stem cells (MSCs), 8,9,16 to generate vascular networks and in vivo. However, the inherent chemical and physical properties of these natural materials have limited their manipulability for engineering vascularized tissue constructs. Moreover, problems associated with complex purification processes, pathogen transfer, and immunogenicity have hampered their clinical usage. 17 Some have suggested synthetic biomaterials, xeno-free and more clinically relevant for regenerative medicine, as an alternative. 18 Unlike natural ECMs, we can engineer these synthetic biomaterials to provide instructive microenvironments capable of recapitulating complex stages of vascular morphogenesis. 17 Although several studies have attempted to generate vascular network assembly within such synthetic biomaterials in vitro, 19,20 no report to date demonstrates highly controlled vascular morphogenes...

Endothelial progenitor cells (EPCs) in the circulatory system have been suggested to maintain vascular homeostasis and contribute to adult vascular regeneration and repair. These processes require that EPCs break down the extracellular matrix (ECM), migrate, differentiate and undergo tube morphogenesis. Evidently, the ECM plays a critical role by providing biochemical and biophysical cues that regulate cellular behaviour. Using a chemically and mechanically tunable hydrogel to study tube morphogenesis in vitro, we show that vascular endothelial growth factor (VEGF) and substrate mechanics co-regulate tubulogenesis of EPCs. High levels of VEGF are required to initiate tube morphogenesis and activate matrix metalloproteinases (MMPs), which enable EPC migration. Under these conditions, the elasticity of the substrate affects the progression of tube morphogenesis. With decreases in substrate stiffness, we observe decreased MMP expression while increased cellular elongation, with intracellular vacuole extension and coalescence to open lumen compartments. RNAi studies demonstrate that membrane type 1-MMP (MT1-MMP) is required to enable the movement of EPCs on the matrix and that EPCs sense matrix stiffness through signalling cascades leading to the activation of the RhoGTPase Cdc42. Collectively, these results suggest that coupled responses for VEGF stimulation and modulation of substrate stiffness are required to regulate tube morphogenesis of EPCs.

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Angiogenesis from endothelial progenitor cells is co‐regulated by vascular endothelial growth factor and matrix stiffness. Endothelial Progenitor Cells (EPCs) in the circulatory system have been suggested to maintain vascular homeostatis and contribute to adult vascular regeneration. These processes require that EPCs break down the extracellular matrix (ECM), migrate, differentiate into endothelial cells, and line the lumen of the blood vessels. Evidently the ECM plays a critical role by providing biochemical and biophysical cues that regulate cellular behavior. Using our controllable in vitro model of angiogenesis we show that vascular endothelial growth factor (VEGF) and matrix stiffness regulate angiogenesis from EPCs. When exposed to low levels of VEGF, EPCs proliferate independently of matrix stiffness. Exposure to high VEGF concentrations results in upregulation of MMPs, demonstrating EPCs migratory and invasive abilities. Under these conditions, the stiffness of the matrix affects the progression of angiogenesis. Immunofluorescence and TEM images confirm the formation of vacuoles, which fully develop into lumen within a chain elongated cell on the softest matrix. RNAi studies further suggest that EPCs sense matrix stiffness through signaling cascades downstream of integrin ligation leading to the activation of the RhoGTPase and Cdc42. These results suggest that dual regulation of VEGF and matrix compliances are required to modulate angiogenesis dynamics of EPCs.Grant Funding SourceAHA