Matthew B. Wolf

Menadione causes endothelial barrier failure by a direct effect on intracellular thiols, independent of reactive oxidant production

Menadione (MQ), a quinone used with cancer chemotherapeutic agents, causes cytotoxicity to endothelial cells (EC). Previous studies have suggested that MQ induces an oxidative stress and dysfunction in EC by either increasing hydrogen peroxide (H(2)O(2)) production or depleting intracellular glutathione (GSH), the main intracellular antioxidant. Since a primary function of EC is to form a barrier to fluid movement into tissues, protecting organs from edema formation and dysfunction, our aim was to see if MQ would cause a barrier dysfunction and to ascertain the mechanism. Using diffusional permeability to fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) as a measure of barrier function, we found that 15 micro M MQ incubated with a bovine pulmonary artery EC (BPAEC) monolayer for 4 h produced a profound barrier failure ( approximately 7-fold increase in permeability) with a parallel fall in glutathione, almost to depletion. These two events were highly correlated. Immunofluorescent imaging showed formation of paracellular holes consistent with a loss or rearrangement of cell-cell and cell-matrix adhesion molecules. H(2)O(2) (100 micro M), a concentration which gave about the same increase in permeability as MQ, only slightly decreased GSH concentration. Antioxidants, such as catalase (CAT) and dimethylthiourea (DMTU), which were able to block the H(2)O(2)-induced changes, had no effect on the MQ-induced permeability and GSH changes, suggesting that H(2)O(2) was not involved in MQ-induced effects. MQ caused a severe EC cytotoxicity as judged by lactate dehydrogenase (LDH) leakage from the EC, whereas H(2)O(2) caused only a minor increase. Also, MQ profoundly inhibited the activities of glucose-6-phosphate dehydrogenase (G6PDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), key thiol enzymes involved in glutathione and ATP metabolism, whereas H(2)O(2) produced only a slight decrease in these activities. We conclude that the cytotoxicity of MQ and resulting barrier dysfunction correlate with GSH depletion and inactivation of key metabolic enzymes, compromising antioxidant defenses, rather than being consistent with H(2)O(2)-mediated oxidative stress.

Effect of advanced glycation end products on oxidative stress in endothelial cells in culture : a warning on the use of cells studied in serum-free media

Abstract.Aims/hypothesis: Alterations in vascular permeability and oxidative stress are characteristics of endothelial dysfunction in diabetic vascular disease. Since AGE-proteins have been hypothesized to mediate these effects, we studied the effects of AGE-bovine serum albumin on endothelial monolayer permeability and intracellular glutathione. Methods: AGE-BSA was prepared by incubating BSA for 30 days at 37 °C with 0.5 mol/l glucose and 0.2 mol/l phosphate buffer, pH 7.4. Permeability to fluorescently labelled BSA was assessed in a bovine pulmonary artery endothelial cell monolayer preparation. Glutathione was measured by an enzymatic assay. Results: AGE-BSA concentrations greater than 3 to 4 μmol/l produced maximal increases in permeability (6–8 times basal) within 3 to 4 h of incubation with the cells. This effect persisted for at least 48 h. However, BSA incubated in the absence of glucose produced similar effects. Dialysis of the AGE-BSA showed that low molecular weight components contained the permeability-increasing activity. Phosphate buffer used to prepare the AGE-BSA, at concentrations equivalent to those present in phosphate-buffered saline and in the AGE preparation (∼ 5 mmol/l), produced similar permeability increases at equivalent incubation times. Metal chelators (0.5 mmol/l) or inclusion of fetal bovine serum (10–20 %) blocked these permeability increases. These increases in permeability were associated with a decrease in endothelial glutathione, both inhibited by 10 mmol/l N-acetylcysteine, and a loss of cell-to-cell and cell-to-matrix adhesion molecules. Conclusion/interpretation: Trace amounts of redox-active metal ions in biological buffers could induce oxidative stress and alterations in cellular functions attributed to AGE-proteins in vitro. It is important to use metal-free phosphate and bicarbonate buffers in studies on cell biology in vitro, especially in serum-free media. [Diabetologia (2001) 44: 1310–1317]

A mathematical model of blood-interstitial acid-base balance: application to dilution acidosis and acid-base status

We developed mathematical models that predict equilibrium distribution of water and electrolytes (proteins and simple ions), metabolites, and other species between plasma and erythrocyte fluids (blood) and interstitial fluid. The models use physicochemical principles of electroneutrality in a fluid compartment and osmotic equilibrium between compartments and transmembrane Donnan relationships for mobile species. Across the erythrocyte membrane, the significant mobile species Cl⁻ is assumed to reach electrochemical equilibrium, whereas Na(+) and K(+) distributions are away from equilibrium because of the Na(+)/K(+) pump, but movement from this steady state is restricted because of their effective short-term impermeability. Across the capillary membrane separating plasma and interstitial fluid, Na(+), K(+), Ca²(+), Mg²(+), Cl⁻, and H(+) are mobile and establish Donnan equilibrium distribution ratios. In each compartment, attainment of equilibrium by carbonates, phosphates, proteins, and metabolites is determined by their reactions with H(+). These relationships produce the recognized exchange of Cl(-) and bicarbonate across the erythrocyte membrane. The blood submodel was validated by its close predictions of in vitro experimental data, blood pH, pH-dependent ratio of H(+), Cl⁻, and HCO₃⁻ concentrations in erythrocytes to that in plasma, and blood hematocrit. The blood-interstitial model was validated against available in vivo laboratory data from humans with respiratory acid-base disorders. Model predictions were used to gain understanding of the important acid-base disorder caused by addition of saline solutions. Blood model results were used as a basis for estimating errors in base excess predictions in blood by the traditional approach of Siggaard-Andersen (acid-base status) and more recent approaches by others using measured blood pH and Pco₂ values. Blood-interstitial model predictions were also used as a basis for assessing prediction errors of extracellular acid-base status values, such as by the standard base excess approach. Hence, these new models can give considerable insight into the physicochemical mechanisms producing acid-base disorders and aid in their diagnoses.

Endothelin-1 induces endothelial barrier failure in the cat hindlimb

Our purpose was to see whether endothelin- (ET) 1 could produce a change in the endothelial membrane barrier to protein in skeletal muscle. Previous studies in other tissues have suggested that ET-1 affects this barrier, but the measurement methods used could not exclude vascular protein extravasation due to microvascular pressure changes or the effects of changes in perfused capillary surface area. We measured the protein sieving coefficient, a specific measure of the permeability of the membrane to protein, in the isolated, perfused cat hindlimb preparation. The integral-mass balance method determined this coefficient from the changes in hematocrit and plasma protein concentration induced by a period of transvascular fluid filtration. The data clearly indicate that ET-1 produces a dose (1-20 nM) dependent increase in permeability indicative of barrier dysfunction. Hence, elevated ET levels may contribute to the perivascular edema, hemoconcentration, and impaired tissue perfusion found in systemic inflammatory response syndromes and related diseases.

Whole body acid-base and fluid-electrolyte balance: a mathematical model.

A cellular compartment was added to our previous mathematical model of steady-state acid-base and fluid-electrolyte chemistry to gain further understanding and aid diagnosis of complex disorders involving cellular involvement in critically ill patients. An important hypothesis to be validated was that the thermodynamic, standard free-energy of cellular H(+) and Na(+) pumps remained constant under all conditions. In addition, a hydrostatic-osmotic pressure balance was assumed to describe fluid exchange between plasma and interstitial fluid, including incorporation of compliance curves of vascular and interstitial spaces. The description of the cellular compartment was validated by close comparison of measured and model-predicted cellular pH and electrolyte changes in vitro and in vivo. The new description of plasma-interstitial fluid exchange was validated using measured changes in fluid volumes after isoosmotic and hyperosmotic fluid infusions of NaCl and NaHCO3. The validated model was used to explain the role of cells in the mechanism of saline or dilutional acidosis and acid-base effects of acidic or basic fluid infusions and the acid-base disorder due to potassium depletion. A module was created that would allow users, who do not possess the software, to determine, for free, the results of fluid infusions and urinary losses of water and solutes to the whole body.

A three-pathway pore model describes extensive transport data from Mammalian microvascular beds and frog microvessels.

Objective: To show that a three‐pathway pore model can describe extensive transport data in cat and rat skeletal muscle microvascular beds and in frog mesenteric microvessels.

A comprehensive, computer-model-based approach for diagnosis and treatment of complex acid–base disorders in critically-ill patients

We have developed a computer-model-based approach to quantitatively diagnose the causes of metabolic acid–base disorders in critically-ill patients. We use an interstitial-plasma-erythrocyte (IPE) model that is sufficiently detailed to accurately calculate steady-state changes from normal in fluid volumes and electrolyte concentrations in a given patient due to a number of causes of acid–base disorders. Normal fluid volumes for each patient are determined from their sex, height and weight using regression equations derived from measured data in humans. The model inputs (electrolyte masses and volumes) are altered to simulate the laboratory chemistry of each critically-ill patient. In this process, the model calculates changes in body-fluid volumes, osmolality and yields the individual values of IPE base excess (BEIPE) attributed to changes due to: (1) fluid dilution/contraction, (2) gain or loss of Cl−, (3) hyper- or hypoalbuminemia, (4) presence of unmeasured ions, (5) gain of lactate, (6) gain or loss of phosphate, (7) gain or loss of calcium and magnesium, (8) gain or loss of potassium and (9) gain or loss of sodium. We use critically-ill patient data to show how our new approach is more informative and much simpler to interpret as compared to the approaches of Siggaard-Andersen or Stewart. We demonstrate how the model can be used at the bedside to diagnose acid–base disorders and suggest appropriate treatment. Hence, this new approach gives clinicians a new tool for diagnosing disorders and specifying fluid-therapy options for critically-ill patients.

Interaction of Endothelin-1 and Nitric Oxide in Endothelial Barrier Failure in the Cat Hindlimb

Objective: To determine the interactions of endothelin‐1 (ET‐1) and nitric oxide (NO) in the regulation of endothelial barrier function in skeletal muscle.

Simulation of human thermoregulation during water immersion : Application to an aircraft cabin water-spray system

A model was developed of transient changes in metabolic heat production and core temperature for humans subjected to cold conditions. It was modified to predict thermal effects of the upper parts of the body being sprayed with water from a system designed to reduce the smoke effects of an airplane fire. Temperature changes were computed at 25 body segments in response to water immersion, cold-air exposure, and windy conditions. Inputs to the temperature controller were:(a) temperature change signals from skin segments and (b) an integrated signal of the product of skin and head-core (hypothalamic) temperature changes. The controller stimulated changes in blood flow to skin and muscle and heat production by shivering. Two controller parameters were adjusted to obtain good predictions of temperature and heat-production experimental data in head-out, water-immersion immersion (0°–28°C) studies in humans. A water layer on the skin whose thickness decreased transiently due to evaporation was added to describe the effects of the water-spray system. Because the layer evaporated rapibly in a very cold and windy environment, its additional cooling effect over a 60-min exposure period was minimal. The largest additional decrease in rectal temperature due to the water-layer was <1°C, which was in normal conditions where total decreases were small.

Determination of the Magnitude of the Water‐Exclusive Pathway in Cat Skeletal Muscle Microvasculature

Objective: To measure the magnitude of the water‐exclusive pathway in cat skeletal muscle.