The Role and Use of Heterogeneity in Microvascular Instability

Adult angiogenesis offers the potential for functional recovery following injury, whether due to cutaneous wounds, peripheral ischemia, or myocardial infarction. However, clinical interventions to harness this capability have largely failed in their inability to create stable perfused vasculatures. More efficacious therapies require a deeper understanding of vessel stabilization, which is believed to be dependent on recruitment and investment of perivascular cells, including smooth muscle cells and pericytes. Explorations into vessel stabilization have been challenged by the following: First, existing in vitro models of angiogenesis cannot fully recapitulate the in vivo cross-talk between multiple cells types. Second, in vivo angiogenesis models do not permit serial time-lapse analysis at the single-cell resolution needed to understand angiogeneic recruitment. Similarly, current studies are unable to relate cellular biochemical responses to the biomechanical cues from blood flow velocity in vivo. Third, both endothelial and perivascular cells are highly heterogeneous populations. Recent evidence suggests that pericytes exhibit pluripotency, which may undermine their stable investment, yet the genetic programs underpinning their differentiation remain unclear. Thus, this thesis makes three primary contributions. First, I have developed a novel dual-modality, high-resolution intravital imaging technique that, when applied to fluorescent-reporting and lineage-tracing mice, is able to uniquely observe angiogenesis at over time in a single network. Using intravital confocal microscopy, I dynamically tracked the maturity of individual endothelial states in a cornea angiogenesis model; then, these images were co-registered with photoacoustic microscopy images to non-invasively quantify shear stress through the vessels. Second, I used intravital confocal imaging in a Myh11-pericyte lineage tracing method to quantify pericyte migration and investment during angiogenesis, with and without pluripotent Oct4 expression. Finally, prior work in our lab demonstrated that adipose-derived stem cells are able to invest the perivascular niche in the diabetic retina to prevent microvascular drop out. In order to extend my considerations of vascular heterogeneity into potential therapies, I studied how diabetes effects the therapeutic potential (in vitro) and efficacy (in vivo) of adipose-derived stem cells to stabilize the retinal microvessels of diabetic mice. In short, this dissertation provides a deeper understanding of endothelial and pericyte heterogeneity, how these heterogeneities may lead to pathologic angiogenic capabilities and microvascular instability during diabetes.