The Effect of Hypoxia Mimetic and Laminar Flow on Islet of Langerhans Beta-Cell Function and Endothelial Cell Morphology

Pancreatic islets are highly vascularized in vivo, ensuring proper blood glucose sensing and insulin secretion directly into the blood stream. Islet vascularity is determined by several factors including blood flow, endocrine tissue, and endothelial cells (ECs). However, once islets are isolated and cultured under static conditions (petri dish), their microenvironment is disrupted, and vascularity is quickly lost. Therefore, we designed and manufactured a microfluidic device to mimic nutrient flow in ex vivo islet culture for short- and long-term (Chapter 2). We found that culturing islets in the device resulted in twice the maintained EC-density and -connected length compared to static culture. This positive effect was due to the penetration of serum albumin, an anti-apoptotic signal for ECs, into the tissue. We then observed maintained function of islet beta-cells via two-photon NAD(P)H response and insulin secretion. In chapter 3, we investigated the combined effect of the hypoxia mimetic, cobalt chloride (CoCl2) and microfluidic flow. We found that CoCl2 induced hypoxia did not affect beta cell metabolism, insulin secretion, or survival. CoCl2 stimulated HIF-1α, PDK-1, and LDHA mRNA and also stimulated VEGF expression and secretion. We observed a modest switch to the less oxidative isoform of COX4 (isoform 1 to 2); which was mirrored in the glucose-stimulated cytoplasmic NAD(P)H response. EC morphology and survival were greater in CoCl2 treated islets compared to exogenous VEGF-A in both static and microfluidic flow culture. The EC response appeared to be mechanistically independent from survival induced by the penetration of serum albumin. Lastly, in chapter 4, we conducted preliminary experiments to investigate the role of CoCl2 induced angiogenic factors regulation of VE-cadherin, as a possible mechanism in islet EC maintenance. We found several angiogenic factors and their receptor mRNA present in islets and focused on VEGF-A and FGFs as possible candidates. However, results around their regulation of VE-cadherin were inconclusive. Future studies are required to determine the mechanism of the CoCl2 induced EC maintenance. Taken altogether, these studies are important for long-term ex vivo studies to understand the function of islets and they are also important for the clinical implications of islet transplantation.Ph.D