The Effects And Mechanism Of Advanced Glycation End Products On Cardiac Microvascular Endothelial Cells And Diabetic Myocardial Ischemia/reperfusion Injury

BackgroundDiabetes mellitus (DM) is a major risk factor for cardiovascular disease, withvascular complications as the leading etiology of morbidity and mortality in thediabetic population. Despite interventional technique and medication therapyadvance, the diabetic condition portends an adverse outcome followingrevascularization, such as severe cardiac microvascular endothelial cell (CMECs)injury evidenced by high morbidity of “no reflow” and cardiomyocytes deathevidenced by poor heart function recovery. These evidences indicate that thediabetic CMECs and cardiomyocytes are more susceptible to the ischemic injury.Therefore, to elucidate the mechanism by which diabetic CMECs andcardiomyocytes are more susceptible to the ischemic injury may provide newtargets for treatment of the ischemia-induced CMECs and cardiomyocytes injury.AGEs are non-enzymatically modified proteins or lipids that become glycatedand oxidized after contact with sugars. AGEs form in vivo during aging, or inhyperglycemic environments, and are contributive to the pathophysiology ofvascular disease in diabetes. It is well known that the interaction of AGEs with itsreceptor (RAGE) increases intracellular reactive oxygen species (ROS) generation and result in the NF-κB activation followed by increased ICAM-1andVCAM-1expression, causing endothelial cell damage. However the effect andmechanism of AGEs/RAGE on the diabetic CMECs and cardiomyocytesischemic injury remain to be elucidated.Ubiquitously expressed in living cells, thioredoxin-1(Trx-1) is a small proteinwith many protective biological functions. Trx-1not only exerts cytoprotectivefunctions against oxidative stress, but also regulates cell survival signalingpathways. In addition to its upregulated or downregulated expression at the genelevel, thioredoxin activity is regulated by posttranslational modification. Recentstudy demonstrated that Trx-1can be modified at the tyrosine residue by nitration,resulting in loss of its cardioprotective action. However, the upstream moleculesand mechanisms causing increased nitrative Trx inactivation unidentified. Moreimportantly, the role of nitrative thioredoxin inactivation in the CMECs ischemicinjury also remains unknown.Objectives1. To investigate the effect of AGEs/RAGE on the ischemia/reperfusion injuryof CMECs and cardiomyocytes in the diabetic heart.2. To elucidate whether AGEs/RAGE induced CMECs and cardiomyocytesinjury is involved in the nitrative thioredoxin inactivation.3. To provide the potential targets for the treatment of CMECs andcardiomyocytes injury in diabetic populationsMethods1. The effect and mechanism of AGEs/RAGE on the ischemia/reperfusion injuryin CMECs1.1Preparation of AGE proteins in vitro non-enzymatically1.2CMECs culture and identification: The CMECs were isolated enzymatically from left ventricle of rats and identified by characteristic morphology, expressionof factor Ⅷ and uptaking of Dil-AC-LDL.1.3Simulated ischemia/reperfusion model: the oxygen-glucose deprivation injuryoccurred by placing cells in a hypoxic environment (1%O2/5%CO2/94%N2)maintained by incubator in the presence of glucose-free DMEM medium for4h,at which time the medium was exchanged with oxygenated and normal glucoseDMEM medium in an incubator at37°C to simulate the reperfusion condition for6h.1.4Experimental protocol: Cells were assigned to one of the following treatments:BSA (100ug/ml as control), AGE-BSA (100ug/ml), AGE-BSA+EUK134(7uM,a peroxynitrite decomposition catalyst), AGE-BSA+hTrx-1(1ug/ml), orAGE-BSA+sRAGE (4ug/ml, a RAGE decoy). After48h incubation, cells weresubjected to either sham simulated ischemia/reperfusion (SI/R,10h ofnormoxia/normal-glucose environment) or SI/R.1.5SI/R-induced CMECs injury evidenced by LDH and caspase-3wasdetermined by enzyme activity assay and ELISA, respectively.1.6superoxide production and nitrotyrosine content were determined bylucigenin-enhanced chemiluminescence and ELISA, respectively.1.7Trx-1, iNOS, RAGE, and gp91phox were determined by Western blot1.8Trx activity and Trx-1nitration were tested by insulin disulfide reductionassay and immunoprecipitation, respectively.2. The effect and mechanism of AGEs/RAGE on the ischemia/reperfusion injuryin diabetic heart.2.1Mice diabetes mellitus model: The diabetic state was induced byintraperitoneal injection of40mg/kg STZ for5consecutive days. Diabetes onsetwas confirmed by hyperglycemia exceeding300mg/dl10days after initial STZ administration2.2Mice MI/R model: MI was induced by temporarily exteriorizing the heart viaa left thoracic incision, and placement of a6-0silk suture slipknot around the leftanterior descending coronary artery. After30minutes of MI, the slipknot wasreleased, and the myocardium was reperfused for3hours or24hours reperfusion2.3In vitro nitration of Trx-1: Purifed human Trx-1was incubated with SIN-1at37°30minutes.2.4Intramyocardial injections: RAGE siRNA or scrambled siRNA was deliveredvia three separate intramyocardial injections, temporarily blanching the leftventricular free wall.2.4Experimental protocol: Hearts were subjected myocardialischemia/reperfusion (MI/R)48hours after siRNA injection and murine solubleRAGE (500μg/day) was administered via intraperitoneal (IP) injection for3daysduration prior to MI/R.10min before reperfusion, the non-RAGE siRNA andnon-sRAGE treated diabetic mice were randomized to receive vehicle (PBS, pH7.5) or reduced human Trx (2mg/kg), EUK-134(5mg/kg), nitratively modifiedhTrx (N-hTrx),1400W(a selective iNOS inhibitor,2mg/kg), apocynin (aselective NADPH oxidase inhibitor,5mg/kg) via IP injection.2.5Cardiac function and myocardial infarct size were determined byechocardiography and TTC2.6Myocardial apoptosis was determined by TUNEL.2.7CML and nitrotyrosine content were determined by ELISA.2.8Trx nitration and Trx activity were determined by insulin disulfide reductionassay and immunoprecipitation.2.9iNOS, RAGE, and gp91phox were determined by Western blot. Results1. The effect and mechanism of AGEs/RAGE on the ischemia/reperfusion injuryin CMECs1.1AGE-BSA increases the SI/R injury in CMECs: SI/R induced a significantLDH release and caspase-3activation. Compared with cells pre-cultured in BSA,cells pre-cultured in AGE-BSA had increased SI/R-induced LDH release andcaspase-3activity.1.2AGE-BSA promotes the SI/R-induced oxidative/nitrative stress in CMECs:SI/R increase oxidative stress, evidenced by enhanced superoxide generation andincreased nitrative stress as well, evidenced by greater iNOS expression, total NOproduction and nitrotyrosine production. Furthermore, AGE-BSA additionallyamplified SI/R-induced superoxide generation, iNOS expression, total NOproduction and nitrotyrosine production in CMECs.1.3AGE-BSA promotes SI/R-induced Trx-1inactivation and nitration: SI/Rdecreased Trx-1activity in both control and AGE-BSA group, compared to sham.This observed decrease in Trx-1activity occurred despite increased expression ofTrx-1protein in both groups. Furthermore, AGE-BSA additionally amplifiedSI/R-induced Trx-1inactivation (but had no effect on Trx-1expression in sham orSI/R conditions). Moreover, AGE-BSA further enhanced SI/R-induced Trxnitration1.4Preventing Trx-1nitration or treatment with exogenous Trx-1attenuates SI/Rinjury and RAGE expression in cells pre-cultured with AGE-BSA: EUK134orhTrx-1significantly attenuated SI/R-induced injury, as evidenced by mitigatedLDH release and caspase-3activity. EUK134or hTrx-1dramatically attenuatedboth nitrotyrosine content and Trx nitration, and recovered Trx-1activity.Compared to vehicle, EUK134or hTrx-1significantly decreased RAGE expression.1.5Blockade of RAGE attenuated SI/R injury in cells cultured with AGE-BSA:sRAGE recovered Trx-1activity, while attenuating CMECs LDH release,caspase-3activity, Trx-1nitration, and gp91phox expression.2. The effect and mechanism of AGEs/RAGE on the ischemia/reperfusion injuryin diabetic heart2.1MI/R induced myocardial injury is exacerbated in diabetic mice: Diabeticmice manifest much larger I/R-induced infarct size by TTC and poorer cardiacfunction by echocardiography compared to control (infarct size:45.2+3.06vs.22.90+2.10%, P<0.01; cardiac function:60.60+5.38vs.41.10+5.15%P<0.01).2.2MI/R induced AGE/RAGE expression and nitrative thioredoxin inactivationwere increased in diabetic mice: Compared to control, diabetic mice harboredsignificantly elevated Nε-carboxymethyl-lysine (CML, the prevailing AGEsubtype, P <0.05) and RAGE (P<0.05) in sham group. After MI/R, both CMLcontent (P<0.01) and RAGE expression (P<0.01) were amplified in diabetic micecompared to control in MI/R group. The diabetic condition increasednitrotyrosine content (the well accepted footprint of protein nitration, comparedto control, sham conditions, P<0.05), Trx nitration (compared to control, shamconditions, P<0.01), and attenuated Trx activity (compared to control, shamconditions, P<0.05,). Compared to control, MI/R further increased nitrotyrosinecontent (P<0.001,), Trx nitration levels (P<0.001) and decreased Trx activity(P<0.001,) to greater extent in diabetic mice.2.3MI/R-induced myocardial injury was attenuated by sRAGE in diabetic mice:sRAGE attenuated I/R-induced infarct size (versus vehicle, P<0.05) andpreserved cardiac function post I/R (versus vehicle, P<0.05).2.4RAGE siRNA attenuated MI/R-induced oxidative/nitrative stress and nitrative Trx inactivation: RAGE siRNA significantly decreased oxidative/nitrative stress,evidenced by decreased superoxide production (versus vehicle, P<0.01),decreased nitrotyrosine content (versus vehicle, P<0.05). Most importantly, wedemonstrated for the first time that RAGE siRNA decreased the MI/R-inducedTrx nitration (versus vehicle, P<0.01), and restored I/R-induced diminished Trxactivity (versus vehicle, P<0.05).2.5MI/R-induced nitrative thioredoxin inactivation was attenuated by iNOS orNADPH oxidase inhibitor in diabetic mice: Apocynin or a1400W significantlydecreased MI/R nitrotyrosine production (versus vehicle, all P<0.05), attenuatedthe thioredoxin nitration (versus vehicle, P<0.01, P<0.05, respectively) andrestored the thioredoxin activity (versus vehicle, all P<0.05). MI/R-induced iNOS(versus vehicle, P<0.05) and gp91phox (a major component of NADPH oxidase)expression (versus vehicle, P<0.05) were significantly attenuated by RAGEknockdown or sRAGE.2.6EUK134or reduced hTrx, but not nitrated hTrx, attenuated MI/R-inducedmyocardial apoptosis in diabetic mice: Reduced hTrx and EUK134bothattenuated I/R-induced myocardial apoptosis (versus vehicle, P<0.05) andcaspase-3activity. Administration of nitratively modified hTrx had no effect uponmyocardial apoptosis (versus vehicle, P>0.05).2.7EUK134or reduced hTrx, but not nitrated hTrx, attenuated MI/R-inducedAGEs/RAGE expression in diabetic mice: Reduced hTrx and EUK134dramatically attenuated MI/R-induced CML production (versus vehicle, allP<0.05) and RAGE expression (versus vehicle, all P<0.05). However,supplementation of nitrated hTrx had no effect upon I/R-induced CMLproduction and RAGE expression in the diabetic heart. Conclusions1. AGEs/RAGE is a key factor that exacerbates the ischemia/reperfusion injury ofCMECs and cardiomyocytes in the diabetic mice.2. In diabetic state, the AGEs/RAGE expression is increased, which results in theTrx nitrative inactivation. Conversely, nitrative modification of Trx blocked itsinhibitory effect upon RAGE overexpression in the diabetic heart, ultimatelyforming a vicious crosstalk between RAGE overexpression and nitrative Trxinactivation. The vicious crosstalk is responsible for the increased susceptibilityto the ischemic injury of CMECs and cardiomyocytes in the diabetic mice.2. Our findings suggest that interventions interfering with their interaction maybe novel means of mitigating diabetic MI/R injury.