Heparan sulfate glycosaminoglycan regulation of vasculogenesis

Abstract

Neovascularization is an essential process to repair ischemic tissues following myocardial infarction, stroke, diabetic complications, or transplant procedures. Blood vessels are generated by distinct vasculogenic and angiogenic processes. Although multiple proangiogenic factors have been identified, limited success has been achieved translating these as clinical therapeutics. Furthermore, recent studies have shown that vasculogenesis contributes to adult neovascularization in multiple settings. Harnessing the vasculogenic potential of embryonic stem cells is an emerging concept to generate neovasculature. The differentiation of embryonic stem cells into endothelium has been well documented, however most studies focus on genetic or chemokine regulation. Limited information exists which implicates the role of the extracellular microenvironment in stem cell differentiation. Heparan sulfate glycosaminoglycans (HSGAG) are a crucial part of the dynamic extracellular matrix and have been shown to regulate multiple signaling cascades, including vasculogenic specific growth factors VEGF and FGF. The goal of this thesis is to elucidate the role of HSGAG in vasculogenesis. An embryonic stem cell embryoid body model was used to establish the necessity of sulfated HSGAG for endothelial differentiation. We identified that the chemical composition of HSGAG sulfation patterns change with differentiation. Perturbation of HSGAG structure by chemical, enzymatic, or genetic modification effectively inhibited vasculogenesis. Genetic silencing of HSGAG modifying enzyme, N-deacetylase/N-sulfotransferase-1, translated to inhibition of HSGAG sulfation and resulted in impaired blood vessel development in zebrafish embryos. Interestingly, vessel formation in both embryonic stem cell and zebrafish models was restored by the addition of exogenous HSGAG, opening the door for engineering glyco-based microenvironments for controlling vascular development. To explore novel mechanisms of vasculogenesis modulated by HSGAG perturbation, we performed a global transcriptome analysis of N-deacetylase/N-sulfotransferase-1 mutant zebrafish embryos. Several novel pathways were identified that regulate vascular differentiation, including Foxo3A and Insulin-Like Growth Factor (IGF) pathways. We explored the role of IGFs in vasculogenesis specifically and determined for the first time that IGF1 and IGF2 promote mesoderm and endothelial differentiation, mediated through HIFl[alpha] stabilization, in embryonic stem cells. In summary, we've identified several mechanisms by which HSGAG regulate neovascularization, laying the groundwork for incorporating HSGAG in strategies for ischemic tissue regeneration.