(249c) Investigating Angiogenesis in Living Vascular Networks in Vitro

 Microvascular networks support metabolic activity and define the microenvironmental conditions within tissues during healthy and pathological processes, and provide the foundation for embryonic development.  Following its de novo synthesis, the microvasculature develops and remodels via the process of angiogenesis, the formation of new capillaries from preexisting blood vessels by the sprouting or splitting of preexisting vessels into a hierarchically branching vascular network.  In this presentation, we describe how we are studying angiogenesis by engineering living vascular networks in vitro in a microfluidic culture, using lithographic techniques to form endothelialized microvessels within a native collagen matrix.  The role of physical and chemical forces in angiogenesis is investigated experimentally and modeled mathematically.

 Angiogenesis is a tightly controlled sequence of events involving endothelial cell sprouting, migration and proliferation; degradation of tissue; new capillary vessel formation and loop formation (anastomosis).  However, none of these events occur until the onset of flow, with sufficient viscosity and hence shear stress, through the network.  We hypothesize that mechanical stresses provide the key triggering event to initiate biochemical signaling pathways that guide subsequent growth.  Further, these mechanically induced changes evolve both spatially and temporally in response to complimentary and competing effects of chemotaxis and perfusion-related hemodynamic forces.  Post-development, angiogenesis also occurs during wound healing, menstruation, in response to hypoxia (low oxygen levels), and in pathologies such as cancer.  Elucidation of the basic mechanism of angiogenesis and related triggering stimuli could, for example, lead to the clarification of mechanobiological signaling pathways, aid in the refinement of anti-angiogenic cancer therapies and form the basis of strategies to engineer vascularized tissues for regenerative medicine.

 We will present our work toward the formation of functional microvascular structures in vitro.  We will describe our strategies for the formation of endothelialized vessels within cell-remodelable matrices and our experimental methods for defining physiologically relevant perfusion stresses and biochemical gradients during extended cultures.  We will then turn to a discussion of our observations of the response of these vessels, as evaluated via live microscopy and at immunohistochemically stained end points.  We will focus on characterization of morphology, and long-term stability of endothelia, as a function of individual physical forces  (e.g., shear stress and wall tension) and different flow regimes (e.g.,  pulsatile, laminar, and oscillatory).  We will further discuss the behavior of co-cultures with perivascular cells in the bulk of the matrix.  We will conclude with a discussion of how our observations relate to observations in in vivo studies and how they inform mathematical models of angiogenic maturation.