Modeling Endothelial Dysfunction Induced By High Glucose And Screening Drugs For Diabetic Vascular Complication Using Cell-based Impedance Sensing

The main etiology for death and for a great percent of morbidity with diabetes is vascular disease. Microvascular disease is the hallmark of retinopathy, neuropathy, and nephropathy, whereas macroangiopathy in diabetes is manifested by accelerated atherosclerosis, which affects vital organs. Endothelial dysfunction has been documented to be the initial lesion and pathophysiological basis for diabetic angiopathy, and results from integral impacts of a variety of factors on different taches. As a common characteristic, hyperglycemia is an independent factor contributing to diabetic endothelial dysfunction. Thus, in parallel with controlling blood glucose concentration, treatment of vascular complications is also important.In this study, cell-based impedance sensing, which reflects cell biological conditions in real-time and label-free, was introduced into study on endothelial barrier function. Model of endothelial dysfunction induced by high glucose and method assessing endothelial dysfunction dynamically were established, which could be meaningful to study on mechanisms of diabetic vascular injury and screening of drugs for diabetic vascular complications.Using RT-CES (real-time cell electrical sensing) system, endothelial barrier dysfunction induced by high glucose (27.8 mM) was monitored in real-time. The whole process of electrode impedance alteration from cell adhesion, spreading, proliferation, confluence and injury induced by high glucose was detected, indicating dynamic changes in endothelial barrier function. Morphology study through permeabilty test and cell cytoskeletal staining was conducted. Meanwhile, experiments with mononuclear-endothelial cell adhesion, nitric oxide release and calcium oscillations were also conducted. Thus, model of endothelial dysfunction and dynamic method detecting endothelial function injury were established based on cell impedance analysis.Applying dynamic detecting model of endothelial dysfunction to drug screeningfor endothelial protection, astragaloside IV, a saponin isolated from Astragalus membranaceus (Fisch) Bge was found improving endothelial barrier dysfunction and inhibiting electrode impedance reduction induced by high glucose. Results from permeability tests and F-actin staining also supported the protective effect of astragaloside IV which prevented increase in endothelial permeability to FITC-dextran and F-actin redistribution.Protein kinase C (PKC) is one of important pathway involved in diabetic vascular complications. In our endothelia dysfunction model induced by high glucose, PKC was activated. Results from ELISA and fluorescent probe to PKC indicated that the quantity and activity in the membranous fractions increased. Further investigation of mechanisms of effects of astragaloside IV on endothelial dysfunction was conducted and activation of PKC was inhibited significantly by astragaloside IV. Western blot analysis revealed that incubation with serial doses of astragaloside IV significantly reduced PKC a expression in the membranous fraction, from 254 ± 17.3% to 130 ± 23.1%, 97 ± 9.9% and 94 ± 11% versus Normal group, respectively. Astragaloside IV of lower concentration (0.5 ug/mL) did not significantly reduce PKC p2 expression, while inhibited PKC P2 expression significantly at 5.0 and 50 ug/mL, from 270 ± 25.1% to 190 ± 9.1% and 125 ± 19.3% versus Normal group, respectively.Our study manifested that the method based on cell impedance analysis could monitor endothelial dysfunction dynamically and the real-time detecting model could screen and assess drugs for diabetic complications. Astragaloside IV, screened out from our model, can improve endothelial dysfunction induced by high glucose and inhibit PKC a and p2 activation, indicating the feasibility of development of drugs for diabetic vascular complications through screening selective PKC inhibitors. The model based on cell impedance of endothelial dysfunction induced by high glucose analysis and our study on protein kinase C pathway will helpful to related drug screening, exploitation and mechanism investigation.