Mahendra Kavdia

Hyperglycemia induces differential change in oxidative stress at gene expression and functional levels in HUVEC and HMVEC

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.

Analysis of Kinetics of Dihydroethidium Fluorescence with Superoxide Using Xanthine Oxidase and Hypoxanthine Assay

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 2.169xa0±xa00.059xa0×xa0103xa0M−1xa0s−1 for DHE–O2− reaction that is ~100× slower than the reported value of 2.6xa0±xa00.6xa0×xa0105xa0M−1xa0s−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.

Modeling of biopterin-dependent pathways of eNOS for nitric oxide and superoxide production.

Endothelial dysfunction is associated with increase in oxidative stress and low NO bioavailability. The endothelial NO synthase (eNOS) uncoupling is considered an important factor in endothelial cell oxidative stress. Under increased oxidative stress, the eNOS cofactor tetrahydrobiopterin (BH(4)) is oxidized to dihydrobiopterin, which competes with BH(4) for binding to eNOS, resulting in eNOS uncoupling and reduction in NO production. The importance of the ratio of BH(4) to oxidized biopterins versus absolute levels of total biopterin in determining the extent of eNOS uncoupling remains to be determined. We have developed a computational model to simulate the kinetics of the biochemical pathways of eNOS for both NO and O(2)(•-) production to understand the roles of BH(4) availability and total biopterin (TBP) concentration in eNOS uncoupling. The downstream reactions of NO, O(2)(•-), ONOO(-), O(2), CO(2), and BH(4) were also modeled. The model predicted that a lower [BH(4)]/[TBP] ratio decreased NO production but increased O(2)(•-) production from eNOS. The NO and O(2)(•-) production rates were independent above 1.5μM [TBP]. The results indicate that eNOS uncoupling is a result of a decrease in [BH(4)]/[TBP] ratio, and a supplementation of BH(4) might be effective only when the [BH(4)]/[TBP] ratio increases. The results from this study will help us understand the mechanism of endothelial dysfunction.

Endothelial NO and O2 production rates differentially regulate oxidative, nitroxidative, and nitrosative stress in the microcirculation

Endothelial dysfunction causes an imbalance in endothelial NO and O₂·⁻ production rates and increased peroxynitrite formation. Peroxynitrite and its decomposition products cause multiple deleterious effects including tyrosine nitration of proteins, superoxide dismutase (SOD) inactivation, and tissue damage. Studies have shown that peroxynitrite formation during endothelial dysfunction is strongly dependent on the NO and O₂·⁻ production rates. Previous experimental and modeling studies examining the role of NO and O₂·⁻ production imbalance on peroxynitrite formation showed different results in biological and synthetic systems. However, there is a lack of quantitative information about the formation and biological relevance of peroxynitrite under oxidative, nitroxidative, and nitrosative stress conditions in the microcirculation. We developed a computational biotransport model to examine the role of endothelial NO and O₂·⁻ production on the complex biochemical NO and O₂·⁻ interactions in the microcirculation. We also modeled the effect of variability in SOD expression and activity during oxidative stress. The results showed that peroxynitrite concentration increased with increase in either O₂·⁻ to NO or NO to O₂·⁻ production rate ratio (QO₂·⁻/QNO or QNO/QO₂·⁻, respectively). The peroxynitrite concentrations were similar for both production rate ratios, indicating that peroxynitrite-related nitroxidative and nitrosative stresses may be similar in endothelial dysfunction or inducible NO synthase (iNOS)-induced NO production. The endothelial peroxynitrite concentration increased with increase in both QO₂·⁻/QNO and QNO/QO₂·⁻ ratios at SOD concentrations of 0.1-100 μM. The absence of SOD may not mitigate the extent of peroxynitrite-mediated toxicity, as we predicted an insignificant increase in peroxynitrite levels beyond QO₂·⁻/QNO and QNO/QO₂·⁻ ratios of 1. The results support the experimental observations of biological systems and show that peroxynitrite formation increases with increase in either NO or O₂·⁻ production, and excess NO production from iNOS or from NO donors during oxidative stress conditions does not reduce the extent of peroxynitrite mediated toxicity.

Diffused Intra-Oocyte Hydrogen Peroxide Activates Myeloperoxidase and Deteriorates Oocyte Quality

Hydrogen peroxide (H2O2) is a relatively long-lived signaling molecule that plays an essential role in oocyte maturation, implantation, as well as early embryonic development. Exposure to relatively high levels of H2O2 functions efficiently to accelerate oocyte aging and deteriorate oocyte quality. However, little precise information exists regarding intra-oocyte H2O2 concentrations, and its diffusion to the oocyte milieu. In this work, we utilized an L-shaped amperometric integrated H2O2-selective probe to directly and quantitatively measure the real-time intra-oocyte H2O2 concentration. This investigation provides an exact measurement of H2O2 in situ by reducing the possible loss of H2O2 caused by diffusion or reactivity with other biological systems. This experiment suggests that the intra-oocyte H2O2 levels of oocytes obtained from young animals are reasonably high and remained constant during the procedure measurements. However, the intra-oocyte H2O2 concentration dropped significantly (40-50% reduction) in response to catalase pre-incubation, suggesting that the measurements are truly H2O2 based. To further confirm the extracellular diffusion of H2O2, oocytes were incubated with myeloperoxidase (MPO), and the diffused H2O2 triggered MPO chlorinating activity. Our results show that the generated hypochlorous acid (HOCl) facilitated the deterioration in oocyte quality, a process that could be prevented by pre-incubating the oocytes with melatonin, which was experimentally proven to be oxidized utilizing HPLC methods. This study is the first to demonstrate direct quantitative measurement of intracellular H2O2, and its extracellular diffusion and activation of MPO as well as its impact on oocyte quality. These results may help in designing more accurate treatment plans in assisted reproduction under inflammatory conditions.

A computational model for nitric oxide, nitrite and nitrate biotransport in the microcirculation: effect of reduced nitric oxide consumption by red blood cells and blood velocity

Bioavailability of vasoactive endothelium-derived nitric oxide (NO) in vasculature is a critical factor in regulation of many physiological processes. Consumption of NO by RBC plays a crucial role in maintaining NO bioavailability. Recently, Deonikar and Kavdia (2009b) reported an effective NO-RBC reaction rate constant of 0.2×10(5)M(-1)s(-1) that is ~7 times lower than the commonly used NO-RBC reaction rate constant of 1.4×10(5)M(-1)s(-1). To study the effect of lower NO-RBC reaction rate constant and nitrite and nitrate formation (products of NO metabolism in blood), we developed a 2D mathematical model of NO biotransport in 50 and 200μm ID arterioles to calculate NO concentration in radial and axial directions in the vascular lumen and vascular wall of the arterioles. We also simulated the effect of blood velocity on NO distribution in the arterioles to determine whether NO can be transported to downstream locations in the arteriolar lumen. The results indicate that lowering the NO-RBC reaction rate constant increased the NO concentration in the vascular lumen as well as the vascular wall. Increasing the velocity also led to increase in NO concentration. We predict increased NO concentration gradient along the axial direction with an increase in the velocity. The predicted NO concentration was 281-1163nM in the smooth muscle cell layer for 50μm arteriole over the blood velocity range of 0.5-4cms(-1) for k(NO-RBC) of 0.2×10(5)M(-1)s(-1), which is much higher than the reported values from earlier mathematical modeling studies. The NO concentrations are similar to the experimentally measured vascular wall NO concentration range of 300-1000nM in several different vascular beds. The results are significant from the perspective that the downstream transport of NO is possible under the right circumstances.

Impact of SOD in eNOS uncoupling: a two-edged sword between hydrogen peroxide and peroxynitrite

In endothelial cell dysfunction, the uncoupling of eNOS results in higher superoxide (O2•−) and lower NO production and a reduction in NO availability. Superoxide reacts with NO to form a potent oxidizing agent peroxynitrite (ONOO−) resulting in nitrosative and nitroxidative stresses and dismutates to form hydrogen peroxide. Studies have shown superoxide dismutase (SOD) plays an important role in reduction of O2•− and ONOO− during eNOS uncoupling. However, the administration or over-expression of SOD was ineffective or displayed deleterious effects in some cases. An understanding of interactions of the two enzyme systems eNOS and SOD is important in determining endothelial cell function. We analyzed complex biochemical interactions involving eNOS and SOD in eNOS uncoupling. A computational model of biochemical pathway of the eNOS-related NO and O2•− production and downstream reactions involving NO, O2•−, ONOO−, H2O2 and SOD was developed. The effects of SOD concentration on the concentration profiles of NO, O2•−, ONOO− and H2O2 in eNOS coupling/uncoupling were investigated. The results include (i) SOD moderately improves NO production and concentration during eNOS uncoupling, (ii) O2•− production rate is independent of SOD concentration, (iii) Increase in SOD concentration from 0.1 to 100 μM reduces O2•− concentration by 90% at all [BH4]/[TBP] ratios, (iv) SOD reduces ONOO− concentration and increases H2O2 concentration during eNOS uncoupling, (v) Catalase can reduce H2O2 concentration and (vi) Dismutation rate by SOD is the most sensitive parameter during eNOS uncoupling. Thus, SOD plays a dual role in eNOS uncoupling as an attenuator of nitrosative/nitroxidative stress and an augmenter of oxidative stress.

Contribution of membrane permeability and unstirred layer diffusion to nitric oxide–red blood cell interaction

Nitric oxide (NO) consumption by red blood cell (RBC) hemoglobin (Hb) in vasculature is critical in regulating the vascular tone. The paradox of NO production at endothelium in close proximity of an effective NO scavenger Hb in RBCs is mitigated by lower NO consumption by RBCs compared to that of free Hb due to transport resistances including membrane resistance, extra- and intra-cellular resistances for NO biotransport to the RBC. Relative contribution of each transport resistance on NO-RBC interactions is still not clear. We developed a mathematical model of NO transport to a single RBC to quantify the contributions from individual transport barriers by analyzing the effect of RBC membrane permeability (P(m)), hematocrit (Hct) and NO-Hb reaction rate constants on NO-RBC interactions. Our results indicated that intracellular diffusion of NO was not a rate limiting step for NO-RBC interactions. The extracellular diffusion contributed 70-90% of total transport resistance for P(m)>1 cm s(-1) whereas membrane resistance accounts for 50-75% of total transport resistance for P(m)<0.1 cm s(-1). We propose a narrow P(m) range of 0.21-0.44 cm s(-1) for 10-45% Hct, respectively, below which membrane resistance is more significant and above which extracellular diffusion is a dominating transport resistance for NO-RBC interactions.

Local Oxidative and Nitrosative Stress Increases in the Microcirculation during Leukocytes-Endothelial Cell Interactions

Leukocyte-endothelial cell interactions and leukocyte activation are important factors for vascular diseases including nephropathy, retinopathy and angiopathy. In addition, endothelial cell dysfunction is reported in vascular disease condition. Endothelial dysfunction is characterized by increased superoxide (O2 •−) production from endothelium and reduction in NO bioavailability. Experimental studies have suggested a possible role for leukocyte-endothelial cell interaction in the vessel NO and peroxynitrite levels and their role in vascular disorders in the arterial side of microcirculation. However, anti-adhesion therapies for preventing leukocyte-endothelial cell interaction related vascular disorders showed limited success. The endothelial dysfunction related changes in vessel NO and peroxynitrite levels, leukocyte-endothelial cell interaction and leukocyte activation are not completely understood in vascular disorders. The objective of this study was to investigate the role of endothelial dysfunction extent, leukocyte-endothelial interaction, leukocyte activation and superoxide dismutase therapy on the transport and interactions of NO, O2 •− and peroxynitrite in the microcirculation. We developed a biotransport model of NO, O2 •− and peroxynitrite in the arteriolar microcirculation and incorporated leukocytes-endothelial cell interactions. The concentration profiles of NO, O2 •− and peroxynitrite within blood vessel and leukocytes are presented at multiple levels of endothelial oxidative stress with leukocyte activation and increased superoxide dismutase accounted for in certain cases. The results showed that the maximum concentrations of NO decreased ∼0.6 fold, O2 •− increased ∼27 fold and peroxynitrite increased ∼30 fold in the endothelial and smooth muscle region in severe oxidative stress condition as compared to that of normal physiologic conditions. The results show that the onset of endothelial oxidative stress can cause an increase in O2 •− and peroxynitrite concentration in the lumen. The increased O2 •− and peroxynitrite can cause leukocytes priming through peroxynitrite and leukocytes activation through secondary stimuli of O2 •− in bloodstream without endothelial interaction. This finding supports that leukocyte rolling/adhesion and activation are independent events.

Induced peroxidase and cytoprotective enzyme expressions support adaptation of HUVECs to sustain subsequent H₂O₂ exposure

H2O2 mediates autocrine and paracrine signaling in the vasculature and can propagate endothelial dysfunction. However, it is not clear how endothelial cells withstand H2O2 exposure and promote H2O2-induced vascular remodeling. To understand the innate ability of endothelial cells for sustaining excess H2O2 exposure, we investigated the genotypic and functional regulation of redox systems in primary HUVECs following an H2O2 treatment. Primary HUVECs were exposed to transient H2O2 exposure and consistent H2O2 exposure. Following H2O2 treatments for 24, 48 and 72 h, we measured O2(-) production, mitochondrial membrane polarization (MMP), and gene expressions of pro-oxidative enzymes, peroxidase enzymes, and cytoprotective intermediates. Our results showed that the 24 h H2O2 exposure significantly increased O2(-) levels, hyperpolarized MMP, and downregulated CAT, GPX1, TXNRD1, NFE2L2, ASK1, and ATF2 gene expression in HUVECs. At 72 h, HUVECs in both treatment conditions were shown to adapt to reduce O2(-) levels and normalize MMP. An upregulation of GPX1, TXNRD1, and HMOX1 gene expression and a recovery of NFE2L2 and PRDX1 gene expression to control levels were observed in both consistent and transient treatments at 48 and 72 h. The response of endothelial cells to excess levels of H2O2 involves a complex interaction amongst O2(-) levels, mitochondrial membrane polarization and anti- and pro-oxidant gene regulation. As a part of this response, HUVECs induce cytoprotective mechanisms including the expression of peroxidase and antioxidant enzymes along with the downregulation of pro-apoptotic genes. This adaptation assists HUVECs to withstand subsequent exposures to H2O2.