The Role of Oxidative Stress in Diabetes Induced Progenitor Cell Deficits
Diabetes affects 25.8 million people in the United States (8.3% of the population), and is particularly detrimental to the vascular system. Our laboratory has delineated specific and discrete defects in a number of stem/progenitor cell populations in diabetes that may underlie impaired wound healing and neovascularization. We have also recently identified the enzyme glyoxalase 1 (Glo1) (a regulator of glycolysis-derived methylgloxal [MG] formation and production of reactive oxygen species [ROS]) as a critical regulator of diabetic vascular and end organ dysfunction. In this proposal, we build on these novel findings to test our fundamental hypothesis that diabetic progenitor cell defects are the result of MG-induced oxidative stress, and that normalization of this stress through modulation of the MG-regulating enzyme Glo1 will prevent and reverse diabetic progenitor cell defects and wound healing sequelae. This work will determine whether restoration of progenitor defects by reducing MG-derived ROS is a clinically viable strategy to treat diabetic complications.

Novel Microfluidic Approaches to Define Diabetic Progenitor Cell Dysfunction
Diabetes-induced impairments in new blood vessel growth greatly increase the severity of wound healing and ischemic processes, including coronary artery disease, cerebrovascular disease, and peripheral vascular disease. Bone marrow (BM) derived vascular progenitor cells are believed to play a critical role in ischemic neovascularization, and may have considerable therapeutic potential as cell-based vectors for the repair of vascular injury. However, the specific lineage of these cells, termed endothelial progenitor cells (EPCs), remains unclear. Early clinical trials using BM-derived cells have yielded disappointing and discordant results, suggesting the need for further characterization. Mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) are considered the two most likely candidate sources, but overlap among definitions, as well as considerable heterogeneity intrinsic to these populations, has complicated their evaluation. We have previously demonstrated in vivo and in vitro dysfunction of specific MSC populations using murine models of diabetes. However, overcoming the heterogeneity inherent to such populations will ultimately require transcriptional analysis of individual cells. To achieve this we have developed a novel method for high-throughput single cell analysis employing microfluidic technology. This proposal seeks to apply that technique to further characterize HSC and MSC populations, using diabetes as a phenotypic marker of disease, to evaluate vasculogenic potential and identify specific diabetes-induced alterations. In Specific Aim 1, we develop a quantitative measure of population heterogeneity and apply it to evaluate HSCs and MSCs from the BM of wild-type (WT) and diabetic mice. A novel partitive analysis is applied in Specific Aim 2 to detect discrete MSC subpopulations and generate characteristic expression profiles. Specific Aim 3 extends these studies to evaluate the transcriptional response of individual subpopulations to peripheral tissue ischemia.

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