Effects Of Superoxide And Hydrogen Peroxide On Microvessel Permeability In Rat Mesenteries In Vivo

Reactive oxygen species(ROS)during oxidative stress play important roles in the development of many disorders including cardiovascular disease. As first barrier of cardiovascular system,endothelial cell is vulnerable to ROS, and its dysfunction results in increased permeability.However,the effect of ROS to microvessel permeability and corresponding mechanisms in vivo has not been well defined.Objective:To study the effect of ROS released from suspended leukocytes upon fMLP stimulation on microvessel permeability and endothelial intracellular Ca²⁺ concentration([Ca²⁺]_i).To observe the individual roles of hydrogen peroxide,H₂O₂, and superoxide in microvessel permeability,endothelial[Ca²⁺]_i,endothelial NO production,cytoskeletal protein,adherent junction.And to investigate the mechanisms by which superoxide and H₂O₂ increased microvessel permeability.Methods:ROS release from activated neutrophils was quantified by measuring changes in chemiluminescence.Microvessel permeability was assessed by measuring hydraulic conductivity(Lp).Superoxide and H₂O₂-induced changes in endothelial [Ca²⁺]_i and NO production were measured in Fura-2 AM(Ca²⁺fluorescent indicator) or DAF-2 DA(NO fluorescent indicator)loaded microvessels in vivo via fluorescence microscopy.To observe the effect of superoxide or H₂O₂ on endothelial F-actin(filamentous actin),VE-Cadherin,the microvessel were stained with immunofluorescence technique and examined with confocal microscopy.Results:1.Effect of ROS released from fMLP-stimulated neutrophils on permeability in rat microvessels in vivo.When neutrophils(2×10⁶/ml)were exposed to fMLP(10μM),chemiluminescence transiently increased from 90.1±22.1 RLU to a peak value of 617.4±32.7 RLU(P＜0.01).Perfusing individual microvessels with fMLP-stimulated neutrophils suspension transiently increased hydraulic conductivity(Lp)to 3.6±0.6 times the control value(P＜0.01)and increased endothelial[Ca²⁺]_i from 90.44±6.17nM to a mean peak value of 296.87±10.61 nM.In contrast,perfusing vessels with fMLP alone did not affect normal Lp.Application of superoxide dismutase(1500 U/ml)abolished increases in Lp.and endothelial[Ca²⁺]_i.2.Effects of superoxide and H₂O₂ on microvessel permeability in Rats in vivo.Perfusing microvessels with superoxide generated by hypoxanthine(0.5 mM) and xanthine oxidase(10 mU/ml)induced immediate increases in microvessel Lp. The mean peak value,occurred in 5 min of HY/XO exposure,was 3.8±0.3 times that of the control(P＜0.01).In contrast,no immediate increases in Lp were observed with H₂O₂(100 and 500μM)perfusions,but at 30 min,500μM H₂O₂ perfusion increased Lp to 4.0±0.6 times the control value(P＜0.01),and at 60 min 100μM and 500μM H₂O₂ perfusions increased Lp to 6.1±0.9 and 12.7±3.7 times of the control, respectively(P＜0.01,P＜0.05).3.Effects of superxide and H₂O₂ on the endothelial[Ca²⁺]_i in rat microvessels in vivo.Superoxide can transiently increase endothelial[Ca²⁺]_i,from 105.80±5.60 nM to a peak value of 397.20±80.01 nM(P＜0.01).H₂O₂(100μM)can induce a prolonged increase in endothelial[Ca²⁺]_i.At 15 min,30 min,60 min perfusion, endothelial[Ca²⁺]_i was 154.50±13.11,231.33±31.64,714.58±144.70 nM(P＜0.01), respectively.Calcium channel blocker,Lanthanum Chloride(LaCl₃),inhibited the increase in microvessel permeability and endothelial[Ca²⁺]_i induced by superoxide and H₂O₂.4.Effects of superoxide and H₂O₂ on the endothelial NO production in rat microvessels in vivo.When microvessels were exposed to superoxide and H₂O₂,the endothelial NO production were immediatedly increased,the mean maximum value was 254.9%±44.2%,1034.3%±44.3%of baseline value(P＜0.05,P＜0.01). NOS inhibitor,L-NAME or AP-Cav-1,can inhibit increased microvessel permeability induced by superoxide or H₂O₂,but have no effect on increased endothelial[Ca²⁺]_i.5.Effects of superoxide and H₂O₂ on the endothelial cytoskeleton and adherent junction in rat microvessels in vivo. Superoxide or H₂O₂ can change actin cytoskeletal architecture.F-actin arranged disorderly and irregularly.Formation of stress fiber was observed in the middle part of cell.Superoxide and H₂O₂ can also restructure VE-Cadherin.Detachment of adherent junction and intercellular gaps formation was observed.Conclusion:1.Superoxide release from fMLP-stimulated neutrophils increased endothelial[Ca²⁺]_i and microvessel permeability and in rats2.Superoxide generated by hypoxanthine and xanthine oxidase can transiently increase microvessel permeability.H₂O₂ induced sustained increases in microvessel permeability.3.H₂O₂ can sustained increase endothelial[Ca²⁺]_i in microvessel. Superoxide induced endothelial[Ca²⁺]_i increases are immediate and transient. Calcium blocker,LaCl₃,can inhibit the permeability increase induced by superoxide and H₂O₂,which suggested that increased endothelial[Ca²⁺]_i was involved in H₂O₂ or superoxide induced increases in microvessel permeability.4.Superoxide and H₂O₂ can increase endothelial NO production.NOS inhibitor can abolish permeability increase induced by superoxide or H₂O₂, but didn't prevent increase in endothelial[Ca²⁺]_i.These results indicated that NO production increase is associated with an increase in microvessel permeability induced by superoxide and H₂O₂.5.Superoxide and H₂O₂ can restructure endothelial F-actin,induce formation of stress fiber;change the structure of VE-Cadherin,lead to detachment of adherent junction,formation of intercellular gaps,which is main causes for permeability increases.