Saptarshi Kar

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.

Accounting for oxygen in the renal cortex: A computational study of factors that predispose the cortex to hypoxia

We develop a pseudo-three-dimensional model of oxygen transport for the renal cortex of the rat, incorporating both the axial and radial geometry of the preglomerular circulation and quantitative information regarding the surface areas and transport from the vasculature and renal corpuscles. The computational model was validated by simulating four sets of published experimental studies of renal oxygenation in rats. Under the control conditions, the predicted cortical tissue oxygen tension ([Formula: see text]) or microvascular oxygen tension (µPo2) were within ±1 SE of the mean value observed experimentally. The predicted [Formula: see text] or µPo2 in response to ischemia-reperfusion injury, acute hemodilution, blockade of nitric oxide synthase, or uncoupling mitochondrial respiration, were within ±2 SE observed experimentally. We performed a sensitivity analysis of the key model parameters to assess their individual or combined impact on the predicted [Formula: see text] and µPo2 The model parameters analyzed were as follows: 1) the major determinants of renal oxygen delivery ([Formula: see text]) (arterial blood Po2, hemoglobin concentration, and renal blood flow); 2) the major determinants of renal oxygen consumption (V̇o2) [glomerular filtration rate (GFR) and the efficiency of oxygen utilization for sodium reabsorption (β)]; and 3) peritubular capillary surface area (PCSA). Reductions in PCSA by 50% were found to profoundly increase the sensitivity of [Formula: see text] and µPo2 to the major the determinants of [Formula: see text] and V̇o2 The increasing likelihood of hypoxia with decreasing PCSA provides a potential explanation for the increased risk of acute kidney injury in some experimental animals and for patients with chronic kidney disease.

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.

Diffusive shunting of gases and other molecules in the renal vasculature: physiological and evolutionary significance

Countercurrent systems have evolved in a variety of biological systems that allow transfer of heat, gases, and solutes. For example, in the renal medulla, the countercurrent arrangement of vascular and tubular elements facilitates the trapping of urea and other solutes in the inner medulla, which in turn enables the formation of concentrated urine. Arteries and veins in the cortex are also arranged in a countercurrent fashion, as are descending and ascending vasa recta in the medulla. For countercurrent diffusion to occur, barriers to diffusion must be small. This appears to be characteristic of larger vessels in the renal cortex. There must also be gradients in the concentration of molecules between afferent and efferent vessels, with the transport of molecules possible in either direction. Such gradients exist for oxygen in both the cortex and medulla, but there is little evidence that large gradients exist for other molecules such as carbon dioxide, nitric oxide, superoxide, hydrogen sulfide, and ammonia. There is some experimental evidence for arterial-to-venous (AV) oxygen shunting. Mathematical models also provide evidence for oxygen shunting in both the cortex and medulla. However, the quantitative significance of AV oxygen shunting remains a matter of controversy. Thus, whereas the countercurrent arrangement of vasa recta in the medulla appears to have evolved as a consequence of the evolution of Henles loop, the evolutionary significance of the intimate countercurrent arrangement of blood vessels in the renal cortex remains an enigma.

Modeling IL-1 induced degradation of articular cartilage

In this study, we develop a computational model to simulate the in vitro biochemical degradation of articular cartilage explants sourced from the femoropatellar grooves of bovine calves. Cartilage explants were incubated in culture medium with and without the inflammatory cytokine IL-1α. The spatio-temporal evolution of the cartilage explants extracellular matrix components is modelled. Key variables in the model include chondrocytes, aggrecan, collagen, aggrecanase, collagenase and IL-1α. The model is first calibrated for aggrecan homeostasis of cartilage in vivo, then for data on (explant) controls, and finally for data on the IL-1α driven proteolysis of aggrecan and collagen over a 4-week period. The model was found to fit the experimental data best when: (i) chondrocytes continue to synthesize aggrecan during the cytokine challenge, (ii) a one to two day delay is introduced between the addition of IL-1α to the culture medium and subsequent aggrecanolysis, (iii) collagen degradation does not commence until the total concentration of aggrecan (i.e. both intact and degraded aggrecan) at any specific location within the explant becomes ≤ 1.5 mg/ml and (iv) degraded aggrecan formed due to the IL-1α induced proteolysis of intact aggrecan protects the collagen network while collagen degrades in a two-step process which, together, significantly modulate the collagen network degradation. Under simulated in vivo conditions, the model predicts increased aggrecan turnover rates in the presence of synovial IL-1α, consistent with experimental observations. Such models may help to infer the course of events in vivo following traumatic joint injury, and may also prove useful in quantitatively evaluating the efficiency of various therapeutic molecules that could be employed to avoid or modify the course of cartilage disease states.

Systems Based Study of the Therapeutic Potential of Small Charged Molecules for the Inhibition of IL-1 Mediated Cartilage Degradation

Inflammatory cytokines are key drivers of cartilage degradation in post-traumatic osteoarthritis. Cartilage degradation mediated by these inflammatory cytokines has been extensively investigated using in vitro experimental systems. Based on one such study, we have developed a computational model to quantitatively assess the impact of charged small molecules intended to inhibit IL-1 mediated cartilage degradation. We primarily focus on the simplest possible computational model of small molecular interaction with the IL-1 system—direct binding of the small molecule to the active site on the IL-1 molecule itself. We first use the model to explore the uptake and release kinetics of the small molecule inhibitor by cartilage tissue. Our results show that negatively charged small molecules are excluded from the negatively charged cartilage tissue and have uptake kinetics in the order of hours. In contrast, the positively charged small molecules are drawn into the cartilage with uptake and release timescales ranging from hours to days. Using our calibrated computational model, we subsequently explore the effect of small molecule charge and binding constant on the rate of cartilage degradation. The results from this analysis indicate that the small molecules are most effective in inhibiting cartilage degradation if they are either positively charged and/or bind strongly to IL-1α, or both. Furthermore, our results showed that the cartilage structural homeostasis can be restored by the small molecule if administered within six days following initial tissue exposure to IL-1α. We finally extended the scope of the computational model by simulating the competitive inhibition of cartilage degradation by the small molecule. Results from this model show that small molecules are more efficient in inhibiting cartilage degradation by binding directly to IL-1α rather than binding to IL-1α receptors. The results from this study can be used as a template for the design and development of more pharmacologically effective osteoarthritis drugs, and to investigate possible therapeutic options.

Computational analysis of interactions of oxidative stress and tetrahydrobiopterin reveals instability in eNOS coupling

In cardiovascular and neurovascular diseases, an increase in oxidative stress and endothelial dysfunction has been reported. There is a reduction in tetrahydrobiopterin (BH4), which is a cofactor for the endothelial nitric oxide synthase (eNOS), resulting in eNOS uncoupling. Studies of the enhancement of BH4 availability have reported mixed results for improvement in endothelial dysfunction. Our understanding of the complex interactions of eNOS uncoupling, oxidative stress and BH4 availability is not complete and a quantitative understanding of these interactions is required. In the present study, we developed a computational model for eNOS uncoupling that considers the temporal changes in biopterin ratio in the oxidative stress conditions. Using the model, we studied the effects of cellular oxidative stress (Qsupcell) representing the non-eNOS based oxidative stress sources and BH4 synthesis (QBH4) on eNOS NO production and biopterin ratio (BH4/total biopterins (TBP)). Model results showed that oxidative stress levels from 0.01 to 1nM·s-1 did not affect eNOS NO production and eNOS remained in coupled state. When the Qsupcell increased above 1nM·s-1, the eNOS coupling and NO production transitioned to an oscillatory state. Oxidative stress levels dynamically changed the biopterin ratio. When Qsupcell increased from 1 to 100nM·s-1, the endothelial cell NO production, TBP levels and biopterin ratio reduced significantly from 26.5 to 2nM·s-1, 3.75 to 0.002μM and 0.99 to 0.25, respectively. For an increase in BH4 synthesis, the improvement in NO production rate and BH4 levels were dependent on the extent of cellular oxidative stress. However, a 10-fold increase in QBH4 at higher oxidative stresses did not restore the NO-production rate and the biopterin ratio. Our mechanistic analysis reveals that a combination of enhancing tetrahydrobiopterin level with a reduction in cellular oxidative stress may result in significant improvement in endothelial dysfunction.