Reactive Oxygen Species Mediated A 'Metabolic Memory' Of High Glucose Stress Signalling In Bovine Retinal Pericytes

Chapter1Consistent and selective culture of bovine retinal pericytesObjective:To establish a simplified method for consistent and selective culture of microvascular pericytes from bovine retina.Methods:1. Retinal microvessels were isolated by using limited collagenase digestion and sieving.Cell viabilities were assessed by trpyan blue dye exclusive assay and cell morphology was observed by inverted phase contrast microscope.2. The origin of these cells was comfirmed by α-SAM using immunohistochemistry.3. Cell proliferation of the passage2were assessed by Cell counting kit-8(CCK-8) and the cell growth curve was drawn.Results:1. The cell visibilities of capillary fragments after collagenase digestion was more than96%.After1days in primary culture, capillary fragments attached to the plate and produced growth of cells.After3-5days,numerous individual vascular cells fully released and migrated from the capilliary fragments.After5-7days,numerous long shuttle-shaped adhered cells formed single and big colonies.After9-10days,cells approached confluence and presented short fusiform shape.After subcultued,the cells attached to the plate within1hours and approached confluence for about5-7days characterized by their irregular or polygonal shape.2. The cells show positive stain for α-SAM in cytoplasm and the positive rate of expression is more than98%;The cells show negative stain for GFAP and vWf.3. The cell growth curve of the passage2shows that cells presented the exponential growth phase on the forth day and stationary growth phase on the tenth day.Conclusions:1. Bovine retinal pericytes can be cultrued successfully with high cell viabilities and passaged serially by using limited collagenase digestion and sieving.2. The passage2and passage3are the better chose to be used in the following research.Chapter2High glucose mediate a cellular'memory'in bovine retinal pericytesObjective:To investigate the effect of reversal of high glucose to normal glucose on cell viabilities and apoptosis in bovine retinal pericytes (BRPs)Methods:1. BRPs were incubated in high glucose(20mM,25mM or30mM) for2days followed by normal glucose(5.6mM) for2days(2d-2d),4days(2d-4d) or6days (2d-6d),or in high glucose (20mM,25mM or30mM) for4days followed by normal glucose for2days(4d-2d) or4days(4d-4d).BRPs incubated in continous normal or high glucose for2,4,6,8days served as control.The cell viabilities were assessed by Cell Counting Kit-8(CCK-8)kit.2. BRPs were incubated in high glucose(30mM) for4days followed by normal glucose(5.6mM) for4days(4d-4d).BRPs incubated in continous normal or high glucose for4,8days served as control.The cell apotosis were assessed by Cell death ELISA Kit and protein levels of cleaved caspase-3were quantified by western blot。Results:1. Four days of normal glucose that followed2days of high glucose (20mM or25mM) can reverse the decreasing of cell viability. However, when the concentration of the initial exposure to high glucose change to30mM,which was follwed by normal glucose extended to6days can then reverse the decreasing of cell viability.2. When the period of exposure of high glucose(20mM or25mM) was extended to4days,which was then follwed by normal glucose for4days had partially benefical effect on the decreasing of cell viabilty.However, when the concentration of the initial exposure to high glucose change to30mM,which was then follwed by normal glucose for4days had no benefical effect on the decreasing of cell viability3. Four days of normal glucose that followed4days of high glucose (30mM),the cell apopotosis remained elevated.Conclusions:1. Four days of normal glucose that followed4days of high glucose (30mM) failed to reverse the decreasing of cell viability arid cell apopotosis remained elevated, and the retinal perictyes experience metabolic memory phenomenon.2. The duration and concentration of the initial exposure to high glucose, and the duration of normal glucose that follows high glucose are critical in determining the outcome of the metabolic memory.Chapter3Reactive Oxygen Species mediated a'Metabolic Memory 'of high glucose stress signalling in Bovine Retinal PericytesObjective:To investigate the role of reactive oxygen species (ROS) and antioxidant mechanism in bovine retinal pericytes (BRPs) of hyperglycemia memory.Methods:BRPs were incubated in high glucose (30mM) for4days followed by normal glucose (5.6mM) for4days; BRPs incubated in continuous normal or high glucose for4or8days as controls. The ROS of mitochondria, the activity of MnSOD and CAT were determined. Futhermore, the mRNA and protein expression of MnSOD were also determined.Results:1. Continuous high glucose exposure for4days significantly elevated the ROS of mitochondria, and significantly decreased the activity of MnSOD and CAT. Four days of nomal glucose that follows provides no beneficial effect to reverse the metabolic abnormalities.2. Continuous high glucose exposure for4days,mRNA and protein expression of MnSOD increased to accommodate ROS production compensatively. The compensative mechanism disappeared when high glucose exposure was extended to8days or followed by normal glucose for4days.Conclusions:1. Hyperglycemia induced elevations in the ROS of mitochondria and decreased in the activity of MnSOD and CAT in BRPs resist reversal after reinstitution of normal glucose.2. creased protein and gene expression of MnSOD is important in the hyperglycemia memory and the regulation antioxidant machinery could help reverse the hyperglycemia memory in BRPs.Chapter4The recombinant adeno-associated viruses of the serotypes2mediated overexpression of MnSOD protects against high glucose-induced metabolic memory in bovine retinal pericytesObjective:To investigate the influence of overexpression of MnSOD by the recombinant adeno-associated virues of the serotypes2(AAV2) mediated gene-delivery for high glucose-induced metabolic memory in bovine retinal pericytes (BRPs).Methods:1. BRPs were infected using rAAV2-MnSOD at a multiplicity of infection(MOI) of5000particles per cell,and controls were infected with AAV-GFP. Four or eight days after AAV infecion,the expression and activity of MnSOD in BRPs were examined. 2. BRPs were pretreated with rAAV2-MnSOD and subsequently incubated in high glucose (30mM) for4days followed by normal glucose (5.6mM) for4days.Pericytes were unpretreated with rAAV2-MnSOD and incubated in continuous normal or high glucose for8days as controls. The reactive oxygen species(ROS) of mitochondria,the cell apoptosis and protein levels of cleaved caspase-3were determined.Results:1. Four days after BRPs infected with AAV, the expression of MnSOD was increased approximately threefold and MnSOD activity was increased approximately twofold compared with cells treated with AAV-GFP, and lasted over eight days.2. BRPs were pretreated with rAAV2-MnSOD and subsequently incubated in high glucose (30mM) for4days followed by normal glucose (5.6mM) for4days, and the ROS of mitochondria,the cell apoptosis and protein levels of cleaved caspase-3were significantly decreased compared with cells which unpretreated with rAAV2-MnSOD or incubated in continuous high glucose for8days.Conclusions:Delivery of the antioxidant gene can reverse the hyperglycemia memory in BRPs by reducing the ROS of mitochondria and cell apoptosis,and suggesting that overexpression of MnSOD may be a potential therapeutic strategy to reverse hyperglycemic memory and treat diabetic retinopathy.