Protective Effects Of Trail On High Glucose-induced Damage Of Endothelium And Its Possible Mechanisms

BackgroundWith the development of society and economy, an aging population and people’s living standards improvement, the incidence of diabetes has increased steadily, which was a serious threat to people’s health. Diabetic vascular complications, such as atherosclerosis, coronary heart disease caused by thrombotic vascular disease, and cerebrovascular and peripheral vascular disease are the main causes of the high mortality and mutilation in diabetic patients. Long-term hyperglycemia is the main risk factors of endothelial dysfunction, and endothelial dysfunction is a key event in the onset and progression of cardiovascular disease associated with diabetes. It is, therefore, thought that prevention of high glucose-induced endothelial dysfunction may have important implication for pharmacological attempts to prevent these complications.Tumor necrosis (TNF)-related apoptosis-inducing ligand (TRAIL) is expressed as a type II TNF family transmembrane protein, which plays important roles in regulating cell death, immune response, and inflammation. TRAIL interacts with five high affinity transmembrane receptors, belonging to the apoptosis-inducing TNF-receptor (R) family. TRAIL-R1(DR4) and TRAIL-R2(DR5) contain cytoplasmic death domain and transduce apoptotic signals, while TRAIL-R3(DcR1) and TRAIL-R4(DcR2), as well as osteoprotegerin (OPG) lack the intracellular death domain and apoptosis inducing capability.In recent years, accumulating experimental evidences suggest that TRAIL plays an important role in vascular physiology. Evidences from in vivo and in vitro studies demonstrate substantial cardiovascular protective effects of TRAIL. In vitro study showed that recombinant TRAIL (rTRAIL) protects HUVECs from apoptosis induced by trophic withdrawal via activating the Akt and extracellular signal-regulate kinase/mitogen-activated protein kinase pathway. Meanwhile, in vivo study suggested that administration of rTRAIL to diabetic ApoE-/-mice led to stabilize atherosclerotic plaque by increasing vascular smooth muscle cell (VSMC) content, and reduce overall lesion size. Of note, three recent in vivo studies have shown that the serum levels of TRAIL are markedly reduced in diabetic patients and patients with atherosclerosis. However, to date, there is a lack of evidence for the beneficial effects of TRAIL on endothelial dysfunction and damage in diabetes mellitus. Therefore, we hypothesized that TRAIL may protect endothelium in diabetic conditions. In the present study, we assessed whether TRAIL has protective roles on endothelium in vitro and in vivo experiments in diabetic conditions and its possible mechanisms.AimsTherefore, the aims of the present study were:(1) to determine whether TRAIL attenuate endothelial dysfunction associated with diabetes;(2) to investigate the effect of TRAIL on high glucose-induced oxidative stress,(3) and the underlying mechanisms;MethodsAnimals experiment1. Twenty rats were rendered diabetic by intraperitoneal injection of streptozocin (STZ, sigma, USA) at a dose of50mg/kg, followed by a high-fat diet for6weeks. Blood glucose levels were tested and the diagnostic criteria for diabetes mellitus (DM) based on fasting blood glucose of≥16.7mmol/L. Normal control rats (NC group) received citrate buffer alone and were processed in parallel to the diabetic rats, followed by a standard diet for6weeks. Diabetic animals were further randomized to receive rTRAIL (TRAIL group) or no treatment (no treatment group). For injection of rTRAIL, each rat received an intraperitoneal injection of rTRAIL (20ug per rat weekly) for6weeks. The no treatment and NC group rats received an intraperitoneal injection of distilled water for6weeks.2. After administration of recombinant TRAIL for6weeks, the animals were anesthetized by an intraperitoneal injection of pentobarbital sodium (60mg/kg body weight), and the thoracic aorta was excised, and endothelial function was examined by acetylcholine-induced endothelium-dependent vasorelaxation using aortic rings. Soluble TRAIL levels were assayed in serum samples using sandwich-type ELISA kits; NO concentration was measured using Griess Reaction; Activation of eNOS was detected by immnohistochemical SP method. The expression levels of eNOS and P-eNOS were measured by western blot analysis. Vitro study1. Primary HUVECs were obtained from freshly discarded umbilical cords by collagenase perfusion. Human umbilical vein endothelial cells (HUVECs) were grown on0.2%gelatin-coated tissue culture plates in M199endothelial growth medium supplemented with20%FBS,10ug/ml heparin, and50ug/ml ECGF (endothelial cell growth factor). For all experiments, cells were used between the third and fifth passage in vitro.2. The cultured HUVECs were treated with normal glucose (5.5mmol/L), high glucose (30mmol/L), high glucose+TRAIL (30mmol/L+100ng/mL) for48h respectively. To further determine the role of the PI3K-Akt-eNOS prosurvival pathway in the effect of TRAIL, the other two groups of HUVECs were pretreated with the specific PI3K inhibitors LY-294002(5uM, LY group) or eNOS inhibitors L-NAME (100uM, L-NAME group)30min before high glucose and TRAIL treatment. 3. Apoptosis of HUVECs was detected by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) and AnnexinVFITC/PI staining; Intracellular ROS production was detected using the peroxide-sensitive fluorescent probe2’,7’-dichlorofluorescin diacetate (DCFH-DA; Molecular Probes); The expression levels of superoxide dismutase (SOD) and glutathione peroxidase type1(GPx-1) were measured by quantitative real-time reverse transcriptase polymerase chain reaction; The expression levels of Akt, P-Akt, eNOS, P-eNOS were measured by western blot.Reault Animals experiment1. Serum TRAIL level in no treatment group was39.8±5.9pg/ml, which was significantly lower than that in NC group (59.3±4.7pg/ml, n=8in each, P＜0.001)2. Concentration-dependent vasorelaxation in response to ACh was significantly decreased in DM group compared with NC group (63.54±4.62%vs.93.52±2.67%relaxation at10-4mmol/L Ach, n=8in each, P＜0.001). TRAIL treatment significantly improved the vascular endothelium-dependent relaxation in response to Ach compared with the DM group (78.43±2.79%vs.63.5±4.62%at10-4mmol/L Ach, n=8in each, P＜0.001)3. Compared with NC group, NO concentration in isolated aortic in rings decreased, activation of eNOS of aorta were lower in DM group (P＜0.001).Compared with DM group, aortic NO concentration and activation of eNOS increased markedly in TRAIL group (P＜0.001).4. Compared with DM group, rTRAIL treatment significantly increased endothelial nitric oxide synthase (eNOS) phosphorylation (P＜0.01).Vitro study1. TUNEL staining:Compared with NC group, the percent of apoptotic HUVECs in HG group was significantly increased (5.9±0.8%vs.37.4±0.9%, p＜0.001), and the effect was blocked by cotreatment with rTRAIL. The percent of apoptosis in HG+TRAIL group was17.2±0.4%, which was significantly lower than that in HG group (37.4±0.9%, p<0.01). The protective action of TRAIL on endothelial apoptosis was abolished by a pretreatment with pharmacological inhibitors of the PI3K/Akt pathway (LY294002) or eNOS (L-NAME).2. AnnexinVFITC/PI staining:Compared with NG group, the apoptosis ratio in HG group was significantly increased (P＜0.01), and which is inhibited by cotreatment with rTRAIL (P＜0.01). The protective effect of TRAIL was abolished by a pretreatment with pharmacological inhibitors of the PI3K/Akt pathway (LY294002) or eNOS (L-NAME).3. TRAIL inhibits high glucose-induced the production of ROS:Compared with NC group, the formation of intracellular ROS oxidase activity in HG group was significantly increased, whereas rTRAIL markedly reduced intracellular ROS generation compared with HG group. The effect of rTRAIL on high glucose-induced ROS formation was abolished by LY-294002or L-NAME. 4. TRAIL increased the mRNA expression levels of SOD and GPX-1:The intracellular defensive factors against oxidative stress (SOD and GPX-1) were significantly increased in HUVECs cotreated with rTRAIL compared with that treated with high glucose alone. Moreover, pretreatment with LY-294002or L-NAME significantly reduced the mRNA expression levels of SOD and GPX-1compared with HG+TRAIL group.5. The effects of TRAIL on the expression of Akt, P-Akt, eNOS, P-eNOS protein levels:Incubation of HUVECs with rTRAIL induced a markedly increase in Akt and eNOS phosphorylation. However, the stimulatory effects of TRAIL on Akt and eNOS phosphorylation were blocked by PI3K and eNOS inhibitionConclusionThe present study for the first time demonstrated the two important points for the role of TRAIL in diabetic conditions. Firstly, we demonstrated that treatment with rTRAIL improved endothelial dysfunction, and increased endothelial nitric oxide synthase (eNOS) phosphorylation. Secondly, rTRAIL significantly attenuated high glucose-induced endothelial cell apoptosis and oxidative stress injury via an activation of PI3K-Akt-eNOS signaling pathways.