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Biomedical subjects

T R Billiar

Publications and source records attributed to T R Billiar.

At least 127 records · Page 7Linked to original sources
PubMed1997

Inducible nitric oxide synthase suppresses the development of allograft arteriosclerosis.

In cardiac transplantation, chronic rejection takes the form of an occlusive vasculopathy. The mechanism underlying this disorder remains unclear. The purpose of this study was to investigate the role nitric oxide (NO) may play in the development of allograft arteriosclerosis. Rat aortic allografts from ACI donors to Wistar Furth recipients with a strong genetic disparity in both major and minor histocompatibility antigens were used for transplantation. Allografts collected at 28 d were found to have significant increases in both inducible NO synthase (iNOS) mRNA and protein as well as in intimal thickness when compared with isografts. Inhibiting NO production with an iNOS inhibitor increased the intimal thickening by 57.2%, indicating that NO suppresses the development of allograft arteriosclerosis. Next, we evaluated the effect of cyclosporine (CsA) on iNOS expression and allograft arteriosclerosis. CsA (10 mg/kg/d) suppressed the expression of iNOS in response to balloon-induced aortic injury. Similarly, CsA inhibited iNOS expression in the aortic allografts, associated with a 65% increase in intimal thickening. Finally, we investigated the effect of adenoviral-mediated iNOS gene transfer on allograft arteriosclerosis. Transduction with iNOS using an adenoviral vector suppressed completely the development of allograft arteriosclerosis in both untreated recipients and recipients treated with CsA. These results suggest that the early immune-mediated upregulation in iNOS expression partially protects aortic allografts from the development of allograft arteriosclerosis, and that iNOS gene transfer strategies may prove useful in preventing the development of this otherwise untreatable disease process.

Animals↗
PubMed1997

Targeting nitric oxide (NO) delivery in vivo. Design of a liver-selective NO donor prodrug that blocks tumor necrosis factor-alpha-induced apoptosis and toxicity in the liver.

We have designed a drug that protects the liver from apoptotic cell death by organ-selective pharmacological generation of the bioregulatory agent, nitric oxide (NO). The discovery strategy involved three steps: identifying a diazeniumdiolate ion (R2N[N(O)NO]-, where R2N = pyrrolidinyl) that spontaneously decomposes to NO with a very short half-life (3 s) at physiological pH; converting this ion to a series of potential prodrug derivatives by covalent attachment of protecting groups that we postulated might be rapidly removed by enzymes prevalent in the liver; and screening the prodrug candidates in vitro and in vivo to select a lead and to confirm the desired activity. Of five cell types examined, only cultured hepatocytes metabolized O2-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) to NO, triggering cyclic guanosine 3',5'-monophosphate (cGMP) synthesis and protecting the hepatocytes from apoptotic cell death induced by treatment with tumor necrosis factor-alpha (TNF alpha) plus actinomycin D. In vivo, V-PYRRO/NO increased liver cGMP levels while minimally affecting systemic hemodynamics, protecting rats dosed with TNF alpha plus galactosamine from apoptosis and hepatotoxicity. The results illustrate the potential utility of diazeniumdiolates for targeting NO delivery in vivo and suggest a possible therapeutic strategy for hepatic disorders such as fulminant liver failure.

Animals↗
PubMed1997

Nitric oxide protects cultured rat hepatocytes from tumor necrosis factor-alpha-induced apoptosis by inducing heat shock protein 70 expression.

Nitric oxide (NO) and tumor necrosis factor-alpha (TNFalpha) play important roles in the pathogenesis of liver disease during acute inflammation. The present study was designed to elucidate the effect of NO pre-exposure on TNFalpha-induced hepatotoxicity. Pretreatment of primary cultures of rat hepatocytes with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) induced the expression of heat shock protein 70 (HSP70) mRNA and protein, which was associated with thermotolerance and cytoprotection from TNFalpha+actinomycin D-induced hepatotoxicity and apoptosis. SNAP transiently changed the intracellular redox state by inducing glutathione (GSH) oxidation associated with the formation of S-nitrosoglutathione (GSNO). HSP70 mRNA was also induced by the GSH-oxidizing agent diamide and the GSH-conjugating agent N-ethylmaleimide, suggesting that NO induces HSP70 expression through GSH oxidation. The protective effect of SNAP pretreatment on TNFalpha-induced apoptosis correlated with the level of HSP70 expression. SNAP pretreatment inhibited reactive oxygen intermediate generation and lipid peroxidation effects that were reversed by blocking HSP70 expression using an antisense oligonucleotide to HSP70. Finally, endogenous NO formation, induced in hepatocytes stimulated with interferon-gamma and interleukin-1beta, led to the formation of GSNO and GSSG, induced HSP70, and attenuated TNFalpha-mediated cytotoxicity. These findings demonstrated that NO can induce resistance to TNFalpha-induced hepatotoxicity, possibly through the stimulation of HSP70 expression.

Animals↗
PubMed1997

Nitric oxide down-regulates hepatocyte-inducible nitric oxide synthase gene expression.

BACKGROUND: The expression of inducible nitric oxide synthase (iNOS) contributes to the systemic manifestations of sepsis. OBJECTIVE: To determine whether nitric oxide (NO) can exert negative feedback regulation on iNOS gene expression. SETTING: Molecular biology research laboratory of the department of surgery. STUDY DESIGN: Isolated rat hepatocytes were cultured with a cytokine mix consisting of tumor necrosis factor alpha, interleukin 1 beta, and interferon gamma in the presence or absence of the NO donor S-nitroso-N-acetyl-D,L-penicillamine. MAIN OUTCOME MEASURES: Nitrite and nitrate (NO2- and NO3-) levels were assayed. Hepatocyte iNOS messenger RNA and protein levels were assessed. Electromobility shift assays were performed for NF-kappa B DNA binding activity. Finally, iNOS enzyme activity was determined using high-performance liquid chromatography. RESULTS: Cytokine mix-induced hepatocyte iNOS mRNA and protein production and the addition of the NO donor S-nitroso-N-acetyl-D,L-penicillamine markedly attenuated iNOS mRNA and protein levels. Gel shift assays of the nuclear extracts disclosed that decreased cytokine mix-induced DNA binding activity for NF-kappa B in a concentration-dependent manner. Finally, NO failed to significantly inhibit iNOS enzyme activity. CONCLUSIONS: These data indicate that NO down-regulates iNOS gene transcription, and that the effect is mediated in part by inhibiting NF-kappa B activity. These results identify a novel negative feedback mechanism whereby NO down-regulates iNOS gene expression, possibly to limit overproduction during the septic response.

Animals↗
PubMed1997

Timing of prostaglandin exposure is critical for the inhibition of LPS- or IFN-gamma-induced macrophage NO synthesis by PGE2.

Macrophage nitric oxide (NO) synthesis is an integral component of the host defense system. We have previously found that NO and prostaglandins interact in a variety of ways. NO modulates Kupffer cell prostaglandin E2 (PGE2) production and we have recently described the inhibitory effects of PGE2 on NO synthesis in both Kupffer cells and hepatocytes. Activated macrophages produce a number of prostaglandins but studies regarding the capacity of prostaglandins to regulate macrophage NO synthesis have yielded conflicting results. We found that exogenous PGE2 decreased lipopolysaccharide (LPS)-induced NO synthesis in murine resident peritoneal macrophages and in the RAW 264.7 murine macrophage cell line. PGE2 also suppressed NO synthesis in response to interferon-gamma (IFN-gamma) alone and a combination of LPS + IFN-gamma. Inhibition of endogenous PGE2 synthesis with indomethacin or ibuprofen had no effect on NO synthesis. PGE2 added with the activating stimulus was most effective. PGE2 lost the capacity to block NO synthesis if added more than 180 min after LPS. PGE2 decreased inducible NO synthesis (iNOS) mRNA and immunoreactive iNOS protein, consistent with the hypothesis that exogenous PGE2 inhibits macrophage iNOS expression but that the inhibition depends on the time and concentration of prostaglandin exposure.

Animals↗
PubMed1997

Dedifferentiated human ventricular cardiac myocytes express inducible nitric oxide synthase mRNA but not protein in response to IL-1, TNF, IFNgamma, and LPS.

There is evidence that nitric oxide (NO) may mediate some of the functional myocardial changes caused by bacterial LPS and inflammatory cytokines. The expression of the inflammatory or inducible NO synthase (iNOS) in human cardiac myocytes, however, has not been well characterized. Therefore, we treated cultured, dedifferentiated human ventricular cardiac myocytes with the combination of TNF-alpha (500 U/ml), IL-1beta (30U/ml), IFNgamma (100 U/ml), and LPS (E.coli 0111:B4, 10 microg/ml). Northern blot analysis revealed a approximately 4.5 kb transcript for inducible NOS (iNOS) in the stimulated human heart cells but not in untreated cells. RT-PCR confirmed that iNOS mRNA was only present in stimulated cells. However, treatment of the myocytes for up to 96 h with cytokines and LPS did not result in NO synthesis as measured by nitrite + nitrate accumulation in the culture medium, and no iNOS enzymatic activity could be detected in the cell lysates. Western blot analysis failed to detect iNOS protein. Thus, despite high and persistent levels of iNOS mRNA in cytokine-treated cells, iNOS protein was absent in this experimental model. GTP-cyclohydrolase I was induced both at the mRNA and protein levels and resulted in increased biopterin levels, indicating sufficient amounts of the cofactor tetrahydrobiopterin (BH4) were present, and that the failure to express an inducible protein was specific to iNOS. To determine if the absence of iNOS protein was due to a novel cardiac iNOS gene or modified iNOS transcript in human myocytes, we cloned an iNOS cDNA from cytokine-treated myocytes. Sequencing and expression of the clone revealed a functional iNOS cDNA with >99% identity to other human iNOS cDNA clones. When human cardiac cells were transduced with a retroviral vector carrying only the coding region of the human hepatocyte iNOS cDNA, both iNOS mRNA and protein could be detected. In conclusion, these cells derived from cultured human cardiac myocytes lacked the capacity to express an endogenous iNOS protein, the basis of which appears to be a cell-specific suppression or failure of iNOS translation.

Argininosuccinate Synthase↗
PubMed1997

Chronic hepatitis C virus infection in humans: induction of hepatic nitric oxide synthase and proposed mechanisms for carcinogenesis.

Chronic inflammatory states frequently lead to the increased production of nitric oxide (NO) via inducible NO synthase (NOS-2). In addition, NO may produce mutagenesis through several mechanisms such as DNA oxidation, DNA deamination, and the formation of N-nitroso compounds. As there is a strong association between human hepatitis C virus (HCV) infection and the development of hepatocellular carcinoma (HCC), we were interested in whether human HCV hepatitis leads to induction of NOS-2 and if the mutation repair system of p53/p21 was upregulated. Reverse transcriptase-polymerase chain reaction (RT-PCR) for human NOS-2 message was performed on RNA samples from both liver biopsies and whole liver from HCV-positive and control patients (normal liver from hepatic resections for metastases). Immunohistochemistry (IHC) for p53 and Western blot analysis for p21 were also performed on the whole liver samples. From the liver biopsies, 60% of HCV-positive patients expressed NOS-2 by RT-PCR. Looking at the whole liver samples, 100% of the HCV-positive patients expressed NOS-2 vs 12.5% in the normal samples. p53 was not detected in either group but there was upregulation of p21 over baseline expression in a number of the HCV-positive patients. Human HCV hepatitis leads to consistent upregulation of hepatic NOS-2 message, but message is not predictably present in "normal" human liver. There is also induction of p21 in some patients with HCV hepatitis. Chronic expression of NO in HCV hepatitis may play a role in DNA mutagenesis and the development of HCC.

Adult↗
PubMed1997

Differential effects of nonselective nitric oxide synthase (NOS) and selective inducible NOS inhibition on hepatic necrosis, apoptosis, ICAM-1 expression, and neutrophil accumulation during endotoxemia.

The roles of nitric oxide derived from either the constitutive endothelial NO synthase (eNOS or NOS3) or the inducible NOS (iNOS or NOS2) in hepatic injury during endotoxemia remain controversial. To investigate this further, rats received a bolus of lipopolysaccharide (LPS) following implantation of osmotic pumps containing one of two nonselective NOS inhibitors (NMA or NAME), one of two inducible NOS inhibitors (NIL or AG), or saline. The inhibitors were infused continuously into the liver via the portal vein. Treatment of LPS-injected rats with NMA and NAME resulted in 106 and 227% increases, respectively, in circulating hepatic enzyme levels compared to LPS-treated control rats. In contrast, infusion of the iNOS-selective inhibitors had no effect on the LPS-induced hepatic necrosis. In rats receiving NAME, LPS induced greater neutrophil infiltration and ICAM-1 expression than in the LPS + saline group, whereas NIL infusion did not. The increased hepatic necrosis and PMN infiltration in the LPS + NAME group was partially prevented by a simultaneous infusion of a liver-selective NO donor. Inhibition of PMN accumulation using an anti-ICAM-1 antibody or by PMN depletion using vinblastine pretreatment, however, did not reverse the increased necrosis with NAME infusion during endotoxemia. In contrast to the assessment for necrosis, increased apoptosis was observed in the livers of LPS-treated rats receiving infusions of either NAME or NIL, but not with LPS alone. These data indicate that NO produced by eNOS may be adequate to prevent necrosis by a mechanism independent of PMN, while induced NO appears to prevent apoptosis.

Animals↗
PubMed1997

Granulocyte colony-stimulating factor (G-CSF) production in hemorrhagic shock requires both the ischemic and resuscitation phase.

Granulocyte colony-stimulating factor (G-CSF) is the cytokine that is critical for polymorphonuclear neutrophilic granulocyte (PMN) production as well as being a potent agonist of PMN activation. We have recently reported that in the lung and the liver of rats resuscitated after hemorrhagic shock (HS) G-CSF mRNA expression is induced. It is not known if both phases of HS, the ischemic and the reperfusion phase, are required for G-CSF mRNA induction. The present study was designed to test the hypothesis that the upregulation of G-CSF mRNA expression is the consequence of HS followed by resuscitation and that ischemia alone is insufficient to induce G-CSF mRNA expression in the affected organs. Male Sprague-Dawley rats were subjected to resuscitated and unresuscitated shock protocols of varying severity. Control animals were subjected to anesthesia and all surgical preparations except for hemorrhage. Lungs and livers were isolated and their RNA extracted. Using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we demonstrated that G-CSF mRNA was induced in the lung and liver of shock animals above the level observed in control animals. Upregulation of G-CSF mRNA relative to controls occurred only in animals undergoing resuscitated HS and not in ones subjected to unresuscitated HS. These results indicate that G-CSF production specific for the hemorrhage component of shock is dependent on resuscitation. As a consequence, the production of this cytokine may be decreased through modifications in the resuscitation protocols.

Animals↗
PubMed1997

Adenoviral transfer of the inducible nitric oxide synthase gene blocks endothelial cell apoptosis.

BACKGROUND: We have previously reported that vascular inducible nitric oxide synthase (iNOS) gene transfer inhibits injury-induced intimal hyperplasia in vitro and in vivo. One mechanism by which NO may prevent intimal hyperplasia is by preserving the endothelium or promoting its regeneration. To study this possibility we examined the effect of iNOS gene transfer on endothelial cell (EC) proliferation and viability. METHODS: An adenoviral vector (AdiNOS) containing the human iNOS cDNA was constructed and used to infect cultured sheep arterial ECs. NO production was measured, and the effects of continuous NO exposure on EC proliferation, viability, and apoptosis were evaluated. RESULTS: AdiNOS-infected ECs produced 25- to 100-fold more NO than control (AdlacZ) infected cells as measured by nitrite accumulation. This increased NO synthesis did not inhibit EC proliferation as reflected by tritiated thymidine incorporation. Chromium 51 release assay revealed that EC viability was also unaffected by AdiNOS infection and NO synthesis. In addition, prolonged exposure to NO synthesis did not induce EC apoptosis. Instead, NO inhibited lipopolysaccharide-induced apoptosis in these cells by reducing caspase-3-like protease activity. CONCLUSIONS: Vascular iNOS gene transfer, while inhibiting smooth muscle cell proliferation, does not impair EC mitogenesis or viability. Augmented NO synthesis may also protect ECs against apogenic stimuli such as lipopolysaccharide. Therefore iNOS gene transfer may promote endothelial regeneration and can perhaps accelerate vascular healing.

Adenoviridae↗
PubMed1997

Management of blunt splenic trauma: significant differences between adults and children.

BACKGROUND: Although highly successful in children, nonoperative management of blunt splenic injury in adults is less defined. The purpose of this study was to determine whether mechanism of injury, grade of splenic injury, associated injuries, and pattern of injury differ between adults and children (younger than 15 years of age). METHODS: Four hundred eleven patients (293 adults and 118 pediatric patients) with blunt splenic injury were admitted to an affiliated adult/pediatric trauma program from 1989 to 1994. Computed tomography (CT) scans were interpreted in a blinded fashion. Mechanism of injury was significantly different for adults versus children (p < 0.05): motor vehicle crash (66.9% versus 23.7%), motorcycle (8.8% versus 0.8%), sports (2.4% versus 16.9%), falls (8.8% versus 25.4%), pedestrian/automobile (4.4% versus 11.0%), bicycle (1.4% versus 9.3%), and other (7.3% versus 12.7%). RESULTS: Higher injury severity scores, lower Glasgow Coma Scales, and higher mortality indicated that the adults were more severely injured than the children. Fifty-nine percent of the adults and 7% of the children required immediate laparotomy for splenic injury. Both CT grade and quantity of blood on CT predicted the need for exploration in adults but not in children. An injury severity score above 15 and high-energy mechanisms correlated with the need for operative intervention. CONCLUSIONS: Rather than children simply being physically different, they are injured differently than adults, hence the high rate of nonoperative management.

Accidents, Traffic↗
PubMed1997

Glutathione regulates nitric oxide synthase in cultured hepatocytes.

OBJECTIVE: The authors determine the relationship between glutathione and nitric oxide (NO) synthesis in cultured hepatocytes. SUMMARY BACKGROUND DATA: Glutathione is a cofactor for a number of enzymes, and its presence is essential for maximal enzyme activity by the inducible macrophage nitric oxide synthase (iNOS), which produces the reactive nitric oxide radical. Hepatocytes contain substantial quantities of glutathione, and this important tripeptide is decreased in hepatocytes stressed by ischemia/reperfusion or endotoxemia. Endotoxemia also induces the synthesis of inflammatory cytokines that result in the production of nitric oxide from hepatocytes by iNOS, suggesting that hepatocytes may be attempting to synthesize nitric oxide at times when intracellular glutathione is reduced. METHODS: Hepatocytes were cultured with buthionine sulfoximine and 1,3-bis(chloroethyl)-1-nitrosourea (BCNU) to inhibit glutathione. After exposure to cytokines, NO synthesis was assessed by supernatant nitrite levels, cytosolic iNOS enzyme activity, and iNOS mRNA levels. RESULTS: Inhibition of glutathione synthesis with buthionine sulfoximine or inhibition of glutathione reductase activity with BCNU inhibited nitrite synthesis. Both buthionine sulfoximine and BCNU inhibited the induction of iNOS mRNA, as detected by Northern blot analysis. Exogenous glutathione increased cytokine-stimulated iNOS induction, overcame the inhibitory effects of BCNU, and increased nitrite production by intact hepatocytes, induced hepatocyte cytosol, and partially purified hepatocyte iNOS. CONCLUSIONS: In cultured hepatocytes, adequate glutathione levels are required for optimal nitric oxide synthesis. This finding is predominantly due to an effect on iNOS mRNA levels, although glutathione also participates in the regulation of iNOS enzyme activity.

Animals↗
PubMed1997

Efficacy of inhaled nitric oxide in oleic acid-induced acute lung injury.

OBJECTIVE: To assess the efficacy of inhaled nitric oxide in improving pulmonary hypertension and gas exchange following oleic acid-induced acute lung injury. DESIGN: Prospective, pharmacologic study. SETTING: Surgical research laboratory at the University of Pittsburgh, Pittsburgh, PA. SUBJECTS: Instrumented, intubated pigs weighing 16 to 27 kg. INTERVENTIONS: Intravenous oleic acid and inhaled nitric oxide. MEASUREMENTS AND MAIN RESULTS: All pigs treated with intravenous oleic acid (0.11 mL/kg) developed a severe lung injury with pulmonary hypertension, accompanied by impaired oxygenation, intrapulmonary shunting, and increased extravascular lung water (p < .05 compared with baseline). Following nitric oxide inhalation, although pulmonary hypertension decreased in a dose-dependent fashion, no amelioration in pulmonary gas exchange was observed, as reflected by PaO2 and intrapulmonary shunt. Plasma nitrite and nitrate concentrations, the stable end products of nitric oxide metabolism, did not increase following nitric oxide exposure in this model of severe lung injury. CONCLUSIONS: The effect of inhaled nitric oxide, restricted to relieving pulmonary vasoconstriction in this model of lung injury, may have limited benefit in improving pulmonary gas exchange when diffusion is impaired by severe lung injury and inflammatory thickening of the alveolar-capillary barrier. Nitric oxide inhalation may have better results when used at an earlier, less severe stage of acute lung injury.

Administration, Inhalation↗
PubMed1997

Effects of hemodilution on long-term survival in an uncontrolled hemorrhagic shock model in rats.

Prehospital guidelines for the treatment of penetrating trauma recommend rapid volume resuscitation to normal blood pressure. There is evidence, however, that fluid resuscitation to normal blood pressure in the setting of uncontrolled hemorrhagic shock (UHS) causes increased bleeding, hemodilution, and mortality. To test this hypothesis, we evaluated the effects of blood pressure and hemodilution on survival in a rat model of UHS. UHS was produced in rats by preliminary bleed of 3 mL/100 g followed by a 75% tail amputation. Experimental design consisted of three phases: a prehospital phase, with uncontrolled bleeding and resuscitation to either 40 or 80 mm Hg with lactated Ringer's solution (LR) or lactated Ringer's solution and whole blood (WB); followed by a hospital phase, with control of the bleeding and continued resuscitation to mean arterial pressure (MAP) > 80 mm Hg and hematocrit near 30%; followed by a 3-day observation phase. There were four treatment groups, n = 8 in each group: group I, MAP = 80 mm Hg with LR only; group II, MAP = 80 mm Hg with WB and LR; group III, MAP = 40 mm Hg with LR only; and group IV, MAP = 40 mm Hg with WB and LR. All group I rats died within 2.5 hours. There were no significant differences in survival among groups II, III, and IV. Base deficit, arterial pH, and lactate levels were significantly worse in the rats resuscitated to a MAP of 80 mm Hg with LR (group I). The effects of blood pressure alone, hemodilution alone, and their interaction were significantly related to base deficit and arterial pH. Hemodilution, but not blood pressure as an end point in resuscitation, was significantly related to lactate levels. The high mortality in this model of uncontrolled hemorrhage was attributable to the effects of blood pressure, hemodilution, and the interaction between the two variables, rather than simply continued blood loss from increased hydrostatic pressure.

Animals↗
PubMed1997

Physiologic and molecular characterization of the role of nitric oxide in hemorrhagic shock: evidence that type II nitric oxide synthase does not regulate vascular decompensation.

To determine the role of nitric oxide (NO) in decompensated and irreversible hemorrhagic shock, rats were subjected to hemorrhagic shock (HS) for 3 or 5 h. Lung, liver, and plasma samples were studied for evidence of NO formation using Northern analysis and immunohistochemistry for Type II NOS, as well as measurement of plasma nitrite/nitrate, cyclic GMP, and nitrosylated hemoglobin levels. Comparisons were made with similarly instrumented time-matched sham rats. Type II NOS mRNA and protein were detectable in lung and liver only in the irreversible phase of HS (5 h). A large accumulation of nitrosylated hemoglobin and nitrite/nitrate appeared in the irreversible phase. Significant accumulation of cyclic GMP or nitrite/nitrate was not detectable in the decompensation phase. Despite the hemodynamic decompensation at 3 h of HS, Type II NOS mRNA and protein expression, as well as NO metabolites were not elevated. To assess whether NO plays a physiologically significant role in decompensation, rats in the decompensation phase and sham animals were subjected to nonspecific NOS inhibition. Both groups displayed a similar magnitude and duration of blood pressure elevation. Hemodynamic decompensation in HS is not mediated by Type II NOS induction. NO production increases only after prolonged HS; significant NO production is observed only in severe, irreversible HS.

Animals↗