BackgroundEndothelial dysfunction precedes pathogenesis of vascular complications in diabetes. In recent years, the mechanisms of endothelial dysfunction were investigated to outline strategies for its treatment. However, the therapies for dysfunctional endothelium resulted in multiple clinical trial failures and remain elusive. There is a need for defining hyperglycemia-induced endothelial dysfunction with both generic and specific dysfunctional changes in endothelial cells (EC) using a systems approach. In this study, we investigated hyperglycemia-induced endothelial dysfunction in HUVEC and HMVEC. We investigated hyperglycemia-induced functional changes (superoxide (O2‾), and hydrogen peroxide (H2O2) production and mitochondrial membrane polarization) and gene expression fingerprints of related enzymes (nitric oxide synthase, NAD(P)H oxidase, and reactive oxygen species (ROS) neutralizing enzymes) in both ECs.MethodGene expression of NOS2, NOS3, NOX4, CYBA, UCP1, CAT, TXNRD1, TXNRD2, GPX1, NOX1, SOD1, SOD2, PRDX1, 18s, and RPLP0 were measured using real-time PCR. O2‾ production was measured with dihydroethidium (DHE) fluorescence measurement. H2O2 production was measured using Amplex Red assay. Mitochondrial membrane polarization was measured using JC-10 based fluorescence measurement.ResultsWe showed that the O2‾ levels increased similarly in both ECs with hyperglycemia. However, these endothelial cells showed significantly different underlying gene expression profile, H2O2 production and mitochondrial membrane polarization. In HUVEC, hyperglycemia increased H2O2 production, and hyperpolarized mitochondrial membrane. ROS neutralizing enzymes SOD2 and CAT gene expression were downregulated. In contrast, there was an upregulation of nitric oxide synthase and NAD(P)H oxidase and a depolarization of mitochondrial membrane in HMVEC. In addition, ROS neutralizing enzymes SOD1, GPX1, TXNRD1 and TXNRD2 gene expression were significantly upregulated in high glucose treated HMVEC.ConclusionOur findings highlighted a unique framework for hyperglycemia-induced endothelial dysfunction. We showed that multiple pathways are differentially affected in these endothelial cells in hyperglycemia. High occurrences of gene expression changes in HMVEC in this study supports the hypothesis that microvasculature precedes macrovasculature in epigenetic regulation forming vascular metabolic memory. Identifying genomic phenotype and corresponding functional changes in hyperglycemic endothelial dysfunction will provide a suitable systems biology approach for understanding underlying mechanisms and possible effective therapeutic intervention.

Nitric oxide (NO) plays many important physiological roles, including the regulation of vascular smooth muscle tone. In response to hemodynamic or agonist stimuli, endothelial cells produce NO, which can diffuse to smooth muscle where it activates soluble guanylate cyclase (sGC), leading to cGMP formation and smooth muscle relaxation. The close proximity of red blood cells suggests, however, that a significant amount of NO released will be scavenged by blood, and thus the issue of bioavailability of endothelium-derived NO to smooth muscle has been investigated experimentally and theoretically. We formulated a mathematical model for NO transport in an arteriole to test the hypothesis that transient, burst-like NO production can facilitate efficient NO delivery to smooth muscle and reduce NO scavenging by blood. The model simulations predict that 1) the endothelium can maintain a physiologically significant amount of NO in smooth muscle despite the presence of NO scavengers such as hemoglobin and myoglobin; 2) under certain conditions, transient NO release presents a more efficient way for activating sGC and it can increase cGMP formation severalfold; and 3) frequency-rather than amplitude-dependent control of cGMP formation is possible. This suggests that it is the frequency of NO bursts and perhaps the frequency of Ca(2+) oscillations in endothelial cells that may limit cGMP formation and regulate vascular tone. The proposed hypothesis suggests a new functional role for Ca(2+) oscillations in endothelial cells. Further experimentation is needed to test whether and under what conditions in silico predictions occur in vivo.

Administration of hemoglobin-based oxygen carriers (HBOCs) frequently results in vasoconstriction that is primarily attributed to the scavenging of endothelium-derived nitric oxide (NO) by cell-free hemoglobin. The ensuing pressor response could be caused by the high NO reactivity of HBOC in the vascular lumen and/or the extravasation of hemoglobin molecules. There is a need for quantitative understanding of the NO interaction with HBOC in the blood vessels. We developed a detailed mathematical model of NO diffusion and reaction in the presence of an HBOC for an arteriolar-size vessel. The HBOC reactivity with NO and degree of extravasation was studied in the range of 2-58 x 10(6) M(-1) x s(-1) and 0-100%, respectively. The model predictions showed that the addition of HBOC reduced the smooth muscle (SM) NO concentration in the activation range (12-28 nM) for soluble guanylate cyclase, a major determinant of SM contraction. The SM NO concentration was significantly reduced when the extravasation of HBOC molecules was considered. The myoglobin present in the parenchymal cells scavenges NO, which reduces the SM NO concentration.

Nitric oxide (NO) plays an important role in autocrine and paracrine manner in numerous physiological processes, including regulation of blood pressure and blood flow, platelet aggregation, and leukocyte adhesion. In vascular wall, most of the bioavailable NO is believed to derive from endothelial cell NO synthase (eNOS). Recently, neuronal NOS (nNOS) has been identified as a source of NO in the vicinity of microvessels and has been shown to participate in vascular function. Thus NO can be produced and transported to the vascular smooth muscle cells from 1). endothelial cells and 2). perivascular nerve fibers, mast cells, and other nNOS-containing sources. In this study, a mathematical model of NO diffusion-reaction in a cylindrical arteriolar segment was formulated. The model quantifies the relative contribution of these NO sources and the smooth muscle availability of NO in a tissue containing an arteriolar blood vessel. The results indicate that a source of NO derived through nNOS in the perivascular region can be a significant contributor to smooth muscle NO. Predicted smooth muscle NO concentrations are as high as 430 nM, which is consistent with reported experimental measurements ( approximately 400 nM). In addition, we used the model to analyze the smooth muscle NO availability in 1). eNOS and nNOS knockout experiments, 2). the presence of myoglobin, and 3). the presence of cell-free Hb, e.g., Hb-based oxygen carriers. The results show that NO release by nNOS would significantly affect available smooth muscle NO. Further experimental and theoretical studies are required to account for distribution of NOS isoforms and determine NO availability in vasculatures of different tissues.

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Superoxide (O2−) is an important reactive oxygen species (ROS), and has an essential role in physiology and pathophysiology. An accurate detection of O2− is needed to better understand numerous vascular pathologies. In this study, we performed a mechanistic study by using the xanthine oxidase (XOD)/hypoxanthine (HX) assay for O2− generation and a O2− sensitive fluorescent dye dihydroethidium (DHE) for O2− measurement. To quantify O2− and DHE interactions, we measured fluorescence using a microplate reader. We conducted a detailed reaction kinetic analysis for DHE–O2− interaction to understand the effect of O2− self-dismutation and to quantify DHE–O2− reaction rate. Fluorescence of DHE and 2-hydroethidium (EOH), a product of DHE and O2− interaction, were dependent on reaction conditions. Kinetic analysis resulted in a reaction rate constant of 2.169±0.059×103 M−1s−1 for DHE-O2− reaction that is ~ 100× slower than the reported value of 2.6±0.6×105 M−1s−1. In addition, the O2− self-dismutation has significant effect on DHE-O2− interaction. A slower reaction rate of DHE with O2− is more reasonable for O2− measurements. In this manner, the DHE is not competing with superoxide dismutase and NO for O2−. Results suggest that an accurate measurement of O2− production rate may be difficult due to competitive interference for many factors; however O2− concentration may be quantified.

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1), a receptor for oxidized-LDL, is up-regulated in activated endothelial cells, and it plays a role in atherothrombosis. However, its role in platelet aggregation is unclear. Both aspirin and HMG CoA reductase inhibitors (statins) reduce LOX-1 expression in endothelial cells. In this study, we investigated the effect of aspirin and pravastatin on LOX-1 expression on platelets. After ADP stimulation, mean fluorescence intensity of LOX-1 expression on platelets increased 1.5-to 2.0-fold. Blocking LOX-1 inhibited ADP-induced platelet aggregation in a concentration-and time-dependent manner. We also established that LOX-1 is important for ADP-stimulated inside-out activation of platelet ␣ IIb ␤ 3 and ␣ 2 ␤ 1 integrins (fibrinogen receptors). The specificity of this interaction was determined by arginine-glycine-aspartate-peptide inhibition. Furthermore, we found that LOX-1 inhibition of integrin activation is mediated by inhibition of protein kinase C activity. In other experiments, treatment with aspirin (1-10 mM) and pravastatin (1-5 M) reduced platelet LOX-1 expression, with a synergistic effect of the combination of aspirin and pravastatin. Aspirin and pravastatin both reduced reactive oxygen species (ROS) released by activated platelets measured as malonyldialdehyde (MDA) release and nitrate/nitrite ratio. Aspirin and pravastatin also enhanced nitric oxide (NO) release measured as nitrite/nitrite ϩ nitrate (NOx) ratio in platelet supernates. Small concentrations of aspirin and pravastatin had a synergistic effect on the inhibition of MDA release and enhancement of nitrite/NOx. Thus, LOX-1 is important for ADP-mediated platelet integrin activation, possibly through protein kinase C activation. Furthermore, aspirin and pravastatin inhibit LOX-1 expression on platelets in part by favorably affecting ROS and NO release from activated platelets.

Nitric oxide (NO), superoxide (O(2)(-)), and peroxynitrite (ONOO(-)) interactions in pathophysiologic conditions such as cardiovascular disease, hypertension, and diabetes have been studied extensively in vivo and in vitro. A reduction in bioavailability of NO is a common event that is known as the endothelial dysfunction in these conditions. Despite intense investigation of NO biotransport and O(2)(-) and ONOO(-) biochemical interactions in vasculature, we have very little quantitative knowledge of distributions and concentrations of NO, O(2)(-), and ONOO(-) under normal physiologic and pathophysiologic conditions. Based on fundamental principles of mass balance, vessel geometry, and reaction kinetics, we developed a mathematical model of these free radicals transport in and around an arteriole during oxidative stress. We investigated the role of O(2)(-) and ONOO(-) in inactivating vasoactive NO. The model predictions include (a) NO interactions with oxygen, O(2)(-), and ONOO(-) have relatively little effect on the NO level in the vascular smooth muscle under physiologic conditions; (b) superoxide diffuses only a few microns from its source, whereas peroxynitrite diffuses over a larger distance; and (c) reduced superoxide dismutase levels significantly increase O(2)(-) and peroxynitrite concentrations and decrease NO concentration. Model results indicate that the reduced NO bioavailability and enhanced peroxynitrite formation may vary depending on the location of oxidative stress in the microcirculation, which occurs at diverse vascular cell locations in diabetes, aging, and cardiovascular diseases. The results will have significant implications for our understanding of these free radical interactions in physiologic and pathophysiologic conditions resulting from endothelial dysfunction.