Abstract 17216: Aquaporin-1 is Required for Vascular Differentiation of Human Induced Pluripotent Stem Cells Following Exposure to Glucose-Induced Hyperosmolarity
Background and objective: Diabetic hyperglycemia increases plasma osmolarity, leading to adaptive cell reponses. Aquaporin-1 (AQP1) is induced by hyperosmolarity and plays a role in the vascular permeability. Induced pluripotent stem (iPS) cells offer a disease-relevant cellular model for the interplay of risk factors in the vascular complications of diabetes. We tested the hypothesis that glucose-induced hyperosmolarity promotes angiogenesis in human iPS cells through activation of AQP1 and downstream osmosignaling pathway, thus orchestrating cell sprouting and migration.
Methods and results: Human iPS cells were generated from skin fibroblasts by lentiviral transduction of four reprogramming factors (Oct4, SOX2, Klf4 and c-Myc). Following 3 weeks compact refractive embryonic stem cell-like colonies emerged. All the iPS cell colonies expressed OCT4, SSEA3, SSEA4 and SOX2, characteristic of successful dedifferentiation. After reprogramming, iPS cells were transfected with siRNA-AQP1 or scrambled controls, and exposed to 5.5 mmol/L glucose (normoglycemia), high glucose (HG) at 12.5, 25 and 45 mmol/L or with high mannitol (HM) at 12.5, 25 and 45 mmol/L for 24-72 hours. Exposure to either HG or HM increased expression of AQP1 and tonicity enhancer binding protein (TonEBP) while decreasing the iPS cell numbers. In iPS cells, AQP1 could be co-immunoprecipitated with β-catenin. HG and HM strongly induced the expression of β-catenin (n=3, p<0.001 vs normal glucose by ANOVA). Under these conditions, proteins co-immunoprecipitated with anti-AQP1 and β-catenin showed increased ratio of F-actin vs G-actin (n=3, p<0.01 vs normal glucose by ANOVA). iPS cells formed bundles in methylcellulose matrix and tubing networks in matrigel, especially when they were exposed to HG and HM (HG 2.8±0.2 fold; HM 3.3±0.5 fold; n=3, p<0.01 by ANOVA. SiRNA to AQP1 reverted the inducing effects of HG and HM on β-catenin expression, actin polymerization and angiogenic activities.
Conclusions: The hyperosmolarity serves as a biophysical factor that promotes angiogenesis in human iPS cells. This effect may occur through an AQP1-associated cytoskeleton remodeling. Targeting the osmosignaling pathway offers a novel strategy to reduce vascular complications of diabetes.
- © 2011 by American Heart Association, Inc.