Karel Tyml

iNOS expression requires NADPH oxidase-dependent redox signaling in microvascular endothelial cells

Redox regulation of inducible nitric oxide synthase (iNOS) expression was investigated in lipopolysaccharide and interferon‐γ (LPS + IFNγ)‐stimulated microvascular endothelial cells from mouse skeletal muscle. Unstimulated endothelial cells produced reactive oxygen species (ROS) sensitive to inhibition of NADPH oxidase (apocynin and DPI), mitochondrial respiration (rotenone) and NOS (L‐NAME). LPS + IFNγ caused a marked increase in ROS production; this increase was abolished by inhibition of NADPH oxidase (apocynin, DPI and p47phox deficiency). LPS + IFNγ induced substantial expression of iNOS protein. iNOS expression was prevented by the antioxidant ascorbate and by NADPH oxidase inhibition (apocynin, DPI and p47phox deficiency), but not by inhibition of mitochondrial respiration (rotenone) and xanthine oxidase (allopurinol). iNOS expression also was prevented by selective antagonists of ERK, JNK, Jak2, and NFκB activation. LPS + IFNγ stimulated activation/phosphorylation of ERK, JNK, and Jak2 and activation/degradation of IκB, but only the activation of JNK and Jak2 was sensitive to ascorbate, apocynin and p47phox deficiency. Ascorbate, apocynin and p47phox deficiency also inhibited the LPS + IFNγ‐induced DNA binding activity of transcription factors IRF1 and AP1 but not NFκB. In conclusion, LPS + IFNγ‐induced NFκB activation is necessary for iNOS induction but is not dependent on ROS signaling. LPS + IFNγ‐stimulated NADPH oxidase activity produces ROS that activate the JNK‐AP1 and Jak2‐IRF1 signaling pathways required for iNOS induction. Since blocking either NFκB activation or NADPH oxidase activity is sufficient to prevent iNOS expression, they are separate targets for therapeutic interventions that aim to modulate iNOS expression in sepsis. J. Cell. Physiol. 217: 207–214, 2008.

Septic impairment of capillary blood flow requires nicotinamide adenine dinucleotide phosphate oxidase but not nitric oxide synthase and is rapidly reversed by ascorbate through an endothelial nitric oxide synthase-dependent mechanism.

Objective:To determine the roles of nitric oxide synthase (NOS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the impairment of capillary blood flow in sepsis and in the reversal of this impairment by ascorbate. Design:Prospective, controlled laboratory study. Setting:Animal laboratory in research institute. Subjects:Adult male wild type (WT), neuronal nitric oxide synthase (nNOS)−/−, inducible NOS (iNOS)−/−, endothelial NOS (eNOS)−/−, and gp91phox−/− mice. Interventions:Sepsis was induced by feces injection into peritoneum (FIP). A bolus of ascorbate or NADPH oxidase inhibitor apocynin was injected intravenously at 6 hrs post-FIP. Alternatively, NOS cofactor (6R)-5,6,7,8-tetrahydro-l-biopterin (BH4) or nitric oxide donor S-nitroso-N-acetylpenicillamine was superfused on the surface of the extensor digitorum longus muscle. Measurements and Main Results:Capillary blood flow impairment and NOS activity in the extensor digitorum longus muscle were measured by intravital microscopy and by enzymatic assay, respectively. Sepsis at 6 hrs impaired flow in WT mice. Apocynin, and knockout of gp91phox but not of any NOS isoforms, rescued this impairment. Constitutive NOS activity was unaffected by sepsis, but it was abolished by nNOS knockout (iNOS activity was negligible in all mice). Ascorbate rapidly (10 mins) rescued impaired flow in WT, nNOS−/−, iNOS−/− but not eNOS−/− mice. Ascorbate also improved survival of WT mice after FIP. BH4 and SNAP rescued flow in WT mice, while BH4 failed to rescue it in eNOS−/− mice. Conclusion:Capillary blood flow impairment in septic skeletal muscle requires NADPH oxidase but not NOS, and it is rapidly reversed by ascorbate and BH4 through an eNOS-dependent mechanism.

Critical role for oxidative stress, platelets, and coagulation in capillary blood flow impairment in sepsis.

Please cite this paper as: Tyml (2011). Critical Role for Oxidative Stress, Platelets, and Coagulation in Capillary Blood Flow Impairment in Sepsis. Microcirculation18(2), 152–162.

Ascorbate Uptake by Microvascular Endothelial Cells of Rat Skeletal Muscle

Objective: In several systems, exogenous ascorbate (reduced vitamin C) has been shown to protect against microvascular injury induced by reactive oxygen species. Since skeletal muscle is relatively resistant to oxidative injury, it is possible that under physiological conditions endogenous ascorbate in the muscle microvasculature affords such protection. To examine the ability of microvascular endothelium to accumulate ascorbate, we aimed (1) to develop an in vitro model of microvascular endothelial cells derived from rat hindlimb skeletal muscles and (2) to investigate the uptake and steady‐state concentration of ascorbate in these cells.

Delayed ascorbate bolus protects against maldistribution of microvascular blood flow in septic rat skeletal muscle.

Objective:Although early administration of ascorbate has been shown to protect against the microvascular dysfunction in sepsis, it is not clear if a delayed introduction of ascorbate also yields beneficial effects. The main objective was to determine the therapeutic window for treatment of an animal model of sepsis with bolus injection of ascorbate. We also determined if sepsis per se affects urinary excretion of ascorbate. Design:Prospective, controlled laboratory study. Setting:Animal laboratory in a university-affiliated research institute. Subjects:Male Sprague-Dawley rats, 300–400 g of body weight. Interventions:Rats were made septic by cecal ligation and perforation (CLP) and volume resuscitated by continuous saline infusion. Ascorbate bolus (7.6 mg/100 g of body weight) or saline vehicle was injected intravenously at 1, 6, or 24 hrs after CLP. Measurements and Main Results:At 24 hrs post-CLP, sepsis caused antidiuresis and decreased plasma ascorbate concentration, but it did not affect urinary excretion of ascorbate in rats that received only saline. Sepsis also caused maldistribution of capillary blood flow in skeletal muscle. This maldistribution of flow was prevented by ascorbate injected at 6 hrs post-CLP. At 48 hrs post-CLP, in addition to the flow maldistribution, sepsis caused systemic arterial hypotension and fever that were prevented by both immediate (1 hr post-CLP) and delayed injections of ascorbate (24 hrs post-CLP). Conclusion:Despite volume resuscitation, the present model of sepsis resulted in maldistribution of capillary blood flow within 24 hrs and hypotension within 48 hrs. Our finding that intravenous bolus of ascorbate can protect against these deficits even if delayed 6–24 hrs after the septic insult shows, for the first time, that ascorbate can reverse microcirculatory dysfunction after the onset of sepsis.

Ascorbate inhibits iNOS expression in endotoxin- and IFNγ-stimulated rat skeletal muscle endothelial cells

Lipopolysaccharide endotoxin and interferon‐γ induced inducible nitric oxide synthase (iNOS) protein expression and nitrite/nitrate formation in microvascular endothelial cell cultures (ECs) derived from rat skeletal muscle. Pretreatment of ECs with ascorbate accumulated a large amount of ascorbate inside the cells and consequently decreased both intracellular oxidant level and iNOS induction. These effects of ascorbate were abolished in the presence of exogenous superoxide generated by xanthine oxidase/xanthine plus catalase but were not altered when N‐nitro‐L‐arginine methyl ester was applied to inhibit nitric oxide synthesis. Ascorbate also attenuated the activation of transcription factor IRF‐1 but not NFκB. These results indicate that ascorbate inhibits iNOS expression in ECs by an antioxidant mechanism independent of both NFκB activation and the reported negative feedback effect of nitric oxide.

Endotoxin increases intercellular resistance in microvascular endothelial cells by a tyrosine kinase pathway.

Gap junction communication between microvascular endothelial cells has been proposed to contribute to the coordination of microvascular function. Septic shock may attenuate microvascular cell‐to‐cell communication. We hypothesized that lipopolysaccharide (LPS) attenuates communication between microvascular endothelial cells derived from rat hindlimb skeletal muscle. Endothelial cells grown in monolayers expressed mRNA for connexin 37, 40, and 43. The expression of connexin 43 protein was confirmed, but connexin 40 protein was not detected by immunocytochemistry or immunoblot analysis. Intercellular resistance between cells of the monolayer, calculated using a Bessel function model, was increased from 3.3 to 5.3 MΩ by LPS. The effect was seen after 1 h of exposure and required a minimum concentration of 10 ng/ml. Intercellular resistance returned to normal 1 h following removal of LPS. Neither the response to LPS, nor its reversal, was blocked by the protein synthesis inhibitor cycloheximide (10 μg/ml). Pretreatment of monolayers with the tyrosine kinase inhibitors PP‐2 (10 nM), lavendustin‐C (1 μM), and geldanamycin (200 nM) prevented this LPS response; geldanamycin was also able to reverse the response. Inhibitors of MAP kinases, PD 98059 (5 μM) and SB 202190 (5 μM), and PKC (500 nM bisindolylmaleimide I) were unable to block the LPS response. We propose that LPS attenuates cell‐to‐cell communication through a signaling pathway that is tyrosine kinase dependent. J. Cell. Physiol. 185:117–125, 2000.

Lipopolysaccharide-induced reductions in cellular coupling correlate with tyrosine phosphorylation of connexin 43

We have previously shown in cultured rat microvascular endothelial cells (RMEC) that lipopolysaccharide (LPS) stimulates a protein tyrosine kinase (PTK)‐dependent reduction in cellular coupling. We hypothesized that connexin 43 (Cx43) becomes phosphorylated following exposure to LPS. Cx43 was immunoprecipitated from control and LPS‐treated RMEC monolayers. Tyrosine phosphorylation of Cx43, detected by immunoblot, was found only in the LPS treatment. To verify these results, Cx43 was radiolabeled with [32P]‐orthophosphate. Radiolabeled Cx43 exhibited a slight increase in phosphorylation in response to LPS; phosphoamino acid analysis displayed equivalent amounts of phosphoserine in control and LPS treatments, but detected phosphotyrosine only in the LPS treatment. The PTK inhibitors PP‐2 (10 nM) and geldanamycin (200 nM) were found to block the response to LPS in terms of Cx43 tyrosine phosphorylation and cellular coupling. The phosphatase inhibitor BpV (1 μM) accentuated the effect of LPS, while the putative phosphatase activator C6‐ceramide prevented it. When measuring cell communication, phosphatase inhibition also blocked the reversal of the LPS response following LPS washout. We conclude that Cx43 is tyrosine phosphorylated following exposure to LPS and suggest that the LPS‐induced increase in intercellular resistance may be mediated by tyrosine phosphorylation of this connexin. Altering tyrosine kinase and phosphatase activities can modulate the LPS‐induced tyrosine phosphorylation of Cx43 and reductions in cellular coupling. J. Cell. Physiol. 193: 373–379, 2002.

Sepsis inhibits reduction of dehydroascorbic acid and accumulation of ascorbate in astroglial cultures: intracellular ascorbate depletion increases nitric oxide synthase induction and glutamate uptake inhibition

Sepsis is associated with oxidative stress and impaired glutamatergic transmission in brain. We investigated whether sepsis impairs accumulation of the antioxidant, ascorbate, and uptake of glutamate by astrocytes. Bacterial endotoxin (Escherichia coli lipopolysaccharide, LPS) and the inflammatory cytokine, interferon‐γ (IFNγ), were applied to primary astrocyte cultures to model sepsis. In the absence of ascorbate, the combination of LPS and IFNγ (LPS + IFNγ) up‐regulated inducible nitric oxide synthase (iNOS) and decreased the initial rate of glutamate uptake by 50% within 24 h. Cell viability and facilitated glucose transport activity were not affected at 24 h. Pre‐treatment with ascorbate‐2‐O‐phosphate increased intracellular ascorbate concentration and attenuated the induction of iNOS and inhibition of glutamate uptake caused by LPS + IFNγ. Subsequent experiments examined the mechanisms by which cells accumulate ascorbate. LPS + IFNγ decreased slightly the initial rate of uptake of ascorbate and inhibited markedly the rate with which intracellular dehydroascorbic acid (DHAA) was reduced to ascorbate. We conclude that septic insult impairs astrocytic clearance of DHAA from the extracellular fluid and decreases intracellular ascorbate concentration. Furthermore, sepsis induces iNOS and inhibits glutamate uptake by astrocytes through mechanisms that can be modulated by intracellular ascorbate. These results indicate treatments that increase intracellular ascorbate concentration may be beneficial for patients at risk for neurologic complication in sepsis.

Capillary as a communicating medium in the microvasculature

The preceding study (Dietrich and Tyml, 1992. Microvasc. Res. 43) demonstrated that a local application of norepinephrine (NE) on a capillary in a skeletal muscle produces a temporary reduction in blood flow within this capillary. The reduction is mediated via constriction of the supplying arteriole. The objective of the present study was to address the mechanism by which the local NE stimulus is propagated from the capillary to the arteriole. Using intravital video microscopy we measured red blood cell velocity in capillaries, and diameter of supplying arterioles, in the sartorius muscle in anesthetized frogs. Velocity responses were measured following iontophoretic application of NE (3 mM in the pipette) on the capillary, with or without pretreatment with 0.9 mM tetrodotoxin (nerve-specific sodium channel blocker), 30 mM lidocaine (nonspecific sodium channel blocker), and 30 mM yohimbine (alpha 2-receptor blocker). Diameter responses were measured before and after capillary damage introduced by microcautery. Tetrodotoxin did not block the NE-induced velocity reduction (i.e., from 0.2 to 0.07 mm/sec), while lidocaine attenuated it. Yohimbine blocked it only when applied on the same site as NE. Capillary damage abolished the NE-induced arteriolar constriction (i.e., from 27.8 to 21.5 microns). We conclude that the observed responses were not due to (1) direct diffusion of NE from the capillary to the arteriole, (2) conduction along adrenergic nerves, or (3) venous-arteriolar diffusional cross-talk. We interpret our data to indicate that the capillary itself could function as a communicating medium.