Oxidative Injury To Vascular Endothelial Cells By Advanced Glycation End Products Was Regulated By NADPH Oxidase

BACKGROUNDThere is a large body of evidence linking advanced glycation end products (AGEs) to an increased risk of coronary artery disease. The main pathological mechanism by which AGEs may contribute to the development and progression of atherosclerosis is the induction of intracellular reactive oxygen species (ROS). ROS can then damage the endothelial cells (ECs). This will cause endothelial dysfunction. A more specific alteration in endothelial function that is also implicated in the pathophysiology of several conditions is endothelial activation, which refers to regulated changes in endothelial phenotype characterized by the expression of cell-surface adhesion molecules and other proteins involved in cell–cell interactions. Therefore inhibition of the generation of ROS will effectively reduce the oxidative injury to ECs and against the pathological potency of AGEs in atherosclerosis.Several potential sources of ROS are implicated in endothelial physiology and pathophysiology, including the mitochondrial electron transport chain, xanthine oxidase, cytochrome P-450 enzyme, uncoupled nitric oxide synthase (NOS), the phagocytic myeloperoxidase system and NADPH oxidase. Recent studies indicated that NADPH oxidase was the most important one for the generation of ROS in ECs by numbers of stimuli. It is unknown whether the increasing intracellular ROS in ECs by AGEs is also generated through NADPH oxidase. This is one of the objectives in our research.Once the highly specific regulating role of the NADPH oxidase could be observed, it will become an effective target point for blocking to reduce the EC injury by AGEs. But this may result in many untoward reactions, because NADPH oxidase has a wide distribution in the body, especially in the phagocytes where NADPH oxidase plays an essential role in non-specific host defence against microbial organisms. Systemic blockage of this oxidase will then lead to a disaster. How to inhibit the endothelial NADPH oxidase specifically? Broadly comparable enzymes have been reported to exist in numerous non-phagocytic cell types, such as ECs, smooth muscle cells, cardiomyocytes and fibroblasts. The molecular composition of these non-phagocytic enzymes has begun to be clarified in the last decade. They are several homologues of the gp91phox catalytic subunit. These homologues are now designated Noxs (NADPH oxidases), with gp91phox now also called Nox2. Other members of the Nox family comprise Nox1, Nox3, Nox4 and Nox5, as well as larger and more complex homologues termed Duox1 and Duox2. Their distribution appears to be highly tissue specific. Nox4 has been identified as the predominant catalytic component of endothelial NADPH oxidase. But its precise pathophysiological function remains unknown.The importance of oxidative stress in the development of endothelial activation and dysfunction is now well recognized, as is the long-term significance for future cardiovascular morbidity and mortality. However, this knowledge has so far not resulted in the introduction of new therapies. Clinical trials that have tested various antioxidants (e.g. vitamin C, vitamin E and the carotenoids) for the prevention of cardiovascular end points have generally been unsuccessful. With a better understanding of the roles that oxidative stress may play in disease pathogenesis, it seems likely that a therapeutic approach based on blanket scavenging of free radicals is probably flawed. Instead, it may be more appropriate to focus on the inhibition of specific ROS-generating enzymes. The NADPH oxidases may be especially important in this regard in view of their highly specific regulation and involvement in ROS production.OBJECTIVES1. To developed a method to isolate human coronary artery ECs in vivo from patients. These cells may be used for subsequent cellular functional analyses and help to understand mechanisms of coronary artery diseases.2. To analysis the relationships between the serum concentrations of AGEs and the severity of coronary artery lesions or oxidative dysfunction of coronary artery ECs.3. To observe the dose and time effects of AGEs on the oxidative injury to vascular ECs.4. To evaluate the contributions of several oxidases in AGEs induced oxidative injury to vascular ECs and clarify which one is the key regulator.5. To investigate the role of Nox4 in the generation of ROS and the activation of ECs by AGEs.METHODS1. Isolation and characterization of human coronary artery ECs in vivo from patients. Coronary guide wires were collected to obtain ECs samples from coronary arteries in patients undergoing percutaneous coronary interventions. Cells were eluted from wires tips and purified by immunomagnetic beads. von Willebrand factor (vWF) and CD31 were used as immunocytochemical markers to identify cells as endothelium. Cell viability was evaluated as following: (1) A simultaneous double-staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) was performed to assess the membrane integrity; (2) The uptake of fluorescent DiI-labeled acetylated low-density lipoprotein was performed to test the metabolic function; (3) Double staining with FITC-labeled annexin V and PI was used to detect apoptosis. We also attempted to culture the isolated ECs in vitro.2. Analysis the relationships between the serum concentrations of AGEs and the severity of coronary artery lesions or oxidative dysfunction of coronary artery ECs. The coronary artery lesions were quantified according to Gensini's scoring system. The concentrations of serum AGEs were measured by an enzyme linked immunosorbent assay (ELISA) kit. Intracellular ROS of isolated coronary artery ECs were evaluated by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Blood samples from coronary sinus were obtained and the concentrations of serum nitric oxide (NO) were measured by NO assay kit to detect the endothelial function. Then the correlations were assessed among them.3. Dose and time effects of AGEs on the oxidative injury to vascular ECs. AGEs were prepared. Human umbilical vein endothelial cells (HUVECs) were cultured and identified by vWF. HUVECs were treated with different concentration of AGEs (100μg/mL, 200μg/mL, 300μg/mL, 400μg/mL, 500μg/mL, 600μg/mL, 700μg/mL and 800μg/mL) for different time course (1h, 2h, 4h, 8h, 16h and 24h). Intracellular ROS were evaluated by DCFH-DA and the expression of intercellular adhesion molecule-1 (ICAM-1) was determined by flow cytometric analysis.4. Evaluate the contributions of several oxidases in AGEs induced oxidative injury to vascular ECs and clarify which one is the key regulator. We used following inhibitors of oxidases: rotenone (a selective inhibitor of mitochondrial complex I), thenoyltrifluoroacetone (TTFA, a selective inhibitor of mitochondrial complex II), antimycin A (a selective inhibitor of mitochondrial complex III), allopurinol (a selective inhibitor of xanthine oxidase), Nω-Nitro-L-arginine methyl ester (L-NAME, a selective inhibitor of nitric oxide synthase), and diphenylene iodonium (DPI, a selective inhibitor of NADPH oxidase). Inhibitors were added to HUVECs 30 minutes before addition of AGEs and remained present throughout AGEs (600μg/mL) incubation. Intracellular ROS were evaluated by DCFH-DA after 16h of incubation.5. The role of Nox4 in the generation of ROS and the activation of ECs by AGEs. Nox4 mRNA expression was detected by RT-PCR, while immunofluorescence staining and western blot were performed for the assessment of Nox4 protein. Nox4 Pre-designed siRNA was transfected into vascular ECs through Lipofectamine 2000 to silence Nox4. The intracellular ROS generation and membrane ICAM-1 expression under the stimulation of AGEs with or without Nox4 Pre-designed siRNA tranfection were detected.RESULTS1. Isolation and characterization of human coronary artery ECs in vivo from patients. 37 coronary guide wires were collected to sample human coronary artery ECs in the selected 37 patients. After Wright staining, cells isolated by immunomagnetic beads displayed a round or oval morphology, more than 20μm in diameter, with a granular pink cytoplasm and an oval red-purple nucleus. The cells count per slide averaged 9.6 (range of 3–14, estimated total of 96 ECs per subject). All the cells showed expression of vWF and CD31 antigens. After FDA-PI double staining, viable cells fluoresced bright green, while nonviable cells showed bright red nuclei. Viable cells also displayed the ability to uptake DiI-labeled acetylated low-density lipoprotein. Of the total cells, about 96% showed good membrane integrity and metabolic activity. After stained by FITC-labeled annexin V and PI, a small numbers of cells exhibited an early apoptotic labeling pattern (annexin V–positive but PI–negative, about 7%) as well as a late apoptotic (or necrotic) labeling pattern (annexin V–positive and PI–positive, about 2%). Cells were plated into a single well of a 96 well plate for culture. After 4 h of incubation, the primary cells began to attach. Two days later these cells changed to a cobblestone morphology. Cells started to proliferate after 3–5 days and became a semiconfluent monolayer after 7–9 days in cultur. After this, most cells began to die gradually. The cells cultured showed positive staining for von Willebrand factor. 2. Analysis the relationships between the serum concentrations of AGEs and the severity of coronary artery lesions or oxidative dysfunction of coronary artery ECs. Gensini's scores of Patients were 56.5±41.1. The concentrations of serum AGEs were 62.3±14.9 U/mL. The concentrations of serum AGEs significantly correlated with the severity of coronary artery lesions (γ=0.416,P<0.01). Intracellular ROS fluorescence intensities of isolated coronary artery ECs were 64.6±13.7 and significantly correlated with the concentrations of serum AGEs (γ=0.588,P<0.01). The concentrations of serum NO in coronary sinus blood samples were 57.0±5.6μmol/L and significantly negatively correlated with the concentrations of serum AGEs (γ=-0.608,P<0.01).3. Dose and time effects of AGEs on the oxidative injury to vascular ECs. Intracellular ROS had a baseline production in cultured HUVECs and were significantly increased after AGEs treatment in a concentration-dependent manner. Significant increase in ROS was observed after 2 hours of incubation and reaching a plateau at 16 hours, remaining stable thereafter. EC membrane ICAM-1 also had a baseline expression and significantly increased after AGEs stimulation. It could reach about 4.5 folds of the baseline expression.4. Evaluate the contributions of several oxidases in AGEs induced oxidative injury to vascular ECs and clarify which one is the key regulator. DPI almost completely inhibited the generation of ROS. No significant effect was observed in rotenone, TTFA, antimycin A or allopurinol. While L-NAME increased the ROS level slightly.5. The role of Nox4 in the generation of ROS and the activation of ECs by AGEs. Nox4 mRNA and protein was detected in HUVECs. AGEs stimulation caused a significantly increasing of Nox4 mRNA and protein expression. Nox4 Pre-designed siRNA transfection significantly decreased the expression of both Nox4 mRNA and Nox4 protein. At the same time, intracellular ROS generation and membrane ICAM-1 expression were both decreased.CONCLUSIONS1. Viable human coronary ECs could be obtained by guide wires combined with immunomagnetic beads during routine procedures of percutaneous coronary interventions. These cells may be used for advanced cellular functional analyses such as immunocytochemistry and molecular biology. Such information could aid in understanding mechanisms of coronary artery diseases.2. The concentrations of serum AGEs significantly correlated with the severity of coronary artery lesions and oxidative dysfunction of coronary artery ECs. This indicates that AGEs may participate in the oxidative injury to ECs and the progression of atherosclerosis.3. AGEs manifests dose and time effects on the oxidative injury to vascular ECs.4. Increasing intracellular ROS production in vascular ECs by AGEs is generated through the pathway of NADPH oxidase.5. As the predominant catalytic component of endothelial NADPH oxidase, Nox4 possesses the capability of highly specific regulation on AGEs induced ROS generation in vascular ECs. It may be a key target for blocking to reduce the EC injury.