Search

Background: We have demonstrated that inhibition of inducible nitric oxide synthase (NOS) ameliorated acute cardiac allograft rejection. This study determined the time course and cellular localization of inducible NOS expression during the histologic progression of unmodified acute rat cardiac allograft rejection., Methods: Tissue from syngeneic (ACI to ACI) and allogeneic (Lewis to ACI) transplants were harvested on postoperative days 3 through 10 and analyzed for inducible NOS mRNA expression (ribonuclease protection assay), inducible NOS enzyme activity (conversion of L-[3H]arginine to nitric oxide and L-[3H]citrulline), and nitric oxide production (serum nitrite/nitrate levels). Inducible NOS mRNA and protein expression were localized using in situ hybridization and immunohistochemistry., Results: Inducible NOS mRNA and enzyme activity were expressed in allografts during mild, moderate, and severe acute rejection (postoperative days 4 through 10), but were not detected in normals, isografts, or allografts before histologic changes of mild acute rejection (postoperative day 3). Inducible NOS expression resulted in increased serum nitrite/nitrate levels during mild and moderate rejection (postoperative days 4 through 6). Inducible NOS mRNA and protein expression localized to infiltrating mononuclear inflammatory cells in allograft tissue sections during all stages of rejection but were not detected in allograft parenchymal cells or in normals or isografts., Conclusions: Inducible NOS expression and increased nitric oxide production occurred during the early stages of acute rejection, persisted throughout the unmodified rejection process, and localized to infiltrating inflammatory cells but not allograft parenchymal cells during all stages of acute rejection.

An attractive approach to the treatment of inflammatory conditions such as osteo- and rheumatoid arthritis, inflammatory bowel disease, and sepsis is through the selective inhibition of human inducible nitric oxide synthase (hiNOS) since localized excess nitric oxide (NO) release has been implicated in the pathology of these diseases. A series of monosubstituted iminohomopiperidinium salts possessing lipophilic functionality at ring positions 3, 5, 6, and 7 has been synthesized, and series members have demonstrated the ability to inhibit the hiNOS isoform with an IC50 as low as 160 nM (7). Compounds were found that selectively inhibit hiNOS over the human endothelial constitutive enzyme (heNOS) with a heNOS/hiNOS IC50 ratio in excess of 100 and as high as 314 (9). Potencies for inhibition of hiNOS and the human neuronal constitutive enzyme (hnNOS) are comparable. Substitution in the 3 and 7 positions provides compounds that exhibit the greatest degree of selectivity for hiNOS and hnNOS over heNOS. Submicromolar potencies for 6 and 7 in a mouse RAW cell assay demonstrated the ability of these compounds to inhibit iNOS in a cellular environment. These latter compounds were also found to be orally bioavailable and efficacious due to their ability to inhibit the increase in plasma nitrite/nitrate levels in a rat LPS model.

Tumor necrosis factor (TNF-alpha) and nitric oxide (NO) are important vasoactive mediators of septic shock. This study used a well-characterized quantitative permeation method to examine the effect of TNF-alpha and NO on systemic vascular barrier function in vivo, without confounding endotoxemia, hypotension, or organ damage. Our results showed 1) TNF-alpha reversibly increased albumin permeation in the systemic vasculature (e.g., lung, liver, brain, etc.); 2) TNF-alpha did not affect hemodynamics or blood flow or cause significant tissue injury; 3) pulmonary vascular barrier dysfunction was associated with increased lung water content and impaired oxygenation; 4) TNF-alpha caused inducible nitric oxide synthase (iNOS) mRNA expression in the lung and increased in vivo NO production; 5) selective inhibition of iNOS with aminoguanidine prevented TNF-alpha-induced lung and liver vascular barrier dysfunction; 6) aminoguanidine prevented increased tissue water content in TNF-alpha-treated lungs and improved oxygenation; and 7) nonselective inhibition of NOS with NG-monomethly-L-arginine increased vascular permeation in control lungs and caused severe lung injury in TNF-alpha-treated animals. We conclude that 1) TNF-alpha reversibly impairs vascular barrier integrity through NO-dependent and -independent mechanisms; 2) nonselective NOS inhibition increased vascular barrier dysfunction and caused severe lung injury, whereas selective inhibition of iNOS prevented impaired endothelial barrier integrity and pulmonary dysfunction; and 3) selective inhibition of iNOS may be beneficial in treating increased vascular permeability that complicates endotoxemia and cytokine immunotherapy.

We previously demonstrated that inhibition of inducible nitric oxide (NO) synthase (iNOS) ameliorated acute cardiac allograft rejection. This study used a rat cardiac transplant model to characterize contractile and electrophysiological dysfunction during early acute rejection, further examine the role of NO and iNOS in this process, and determine which cells expressed iNOS during early rejection. During early acute rejection, before significant myocyte necrosis, allograft papillary muscles had reduced tension development and rates of tension development and decline during beta-adrenergic, adenylate cyclase, and calcium stimulation compared with isograft and normals [e.g., tension of 36 (allograft) vs. 73 (isograft) mN/mm2 during calcium stimulation, P < 0.001]. Allografts had resting membrane potential depolarization and reduced action potential amplitude and upstroke velocity. iNOS mRNA was expressed in infiltrating inflammatory cells but not in allograft myocytes, endothelial cells, or isografts. Corticosteroids attenuated allograft contractile and electrophysiological dysfunction and inhibited iNOS enzyme activity. Direct iNOS inhibition with aminoguanidine inhibited NO production and prevented allograft contractile and electrophysiological dysfunction (e.g., tension of 64 mN/mm2 during calcium stimulation, P < 0.001). We conclude that 1) early allograft rejection caused contractile and electrophysiological dysfunction that was largely mediated by iNOS expression in infiltrating inflammatory cells, 2) corticosteroid-mediated amelioration of allograft contractile and electrophysiological dysfunction may reflect inhibition of iNOS, and 3) iNOS inhibition may offer an alternative in management of immune-mediated myocardial dysfunction.

Background: We recently demonstrated that inhibition of inducible nitric oxide synthase (iNOS) ameliorated severe acute lung allograft rejection. This study used a rat lung transplant model to determine (1) the time course and cellular localization of iNOS expression during the histological progression of unmodified acute rejection and (2) whether inhibition of iNOS prevented impaired gas exchange function of the allograft lung and/or ameliorated the histological changes of acute rejection., Methods and Results: iNOS mRNA and enzyme activity were expressed in allograft lungs during mild, moderate, and severe acute rejection, but not in normal, isograft, or allograft lungs before histological changes of mild acute rejection. iNOS expression in allografts resulted in elevated serum nitrite/nitrate levels, indicative of increased in vivo nitric oxide (NO) production. In situ hybridization demonstrated iNOS mRNA expression in infiltrating inflammatory cells, but not in allograft parenchymal cells. Allografts had significantly impaired gas exchange, which was prevented with the selective iNOS inhibitor aminoguanidine (PaO2 of 566+/-19, 76+/-22, and 504+/-105 mmHg for isograft, allograft, and aminoguanidine-treated allograft, respectively; P<0.0002). Aminoguanidine also significantly improved the histological rejection scores., Conclusions: (1) iNOS expression and increased NO production occurred during the early stages of acute rejection, persisted throughout the unmodified rejection process, and localized to infiltrating inflammatory cells, but not allograft parenchymal cells; (2) aminoguanidine ameliorated the histological and functional changes of acute rejection; and (3) increased NO production, detected by the presence of iNOS mRNA, protein, or noninvasively by measuring serum nitrite/nitrate levels, may serve as an early marker of acute allograft rejection.

Background: We recently demonstrated that inhibition of nitric oxide (NO) production ameliorated acute pulmonary allograft rejection. This study examined whether inducible NO synthase (iNOS) was expressed in the transplanted lung during acute rejection., Methods: With a rat left lung transplant model, tissue from syngeneic (Fischer 344 to Fischer 344) and allogeneic (Brown Norway to Fischer 344) transplants were harvested on postoperative day 4 and analyzed for iNOS mRNA expression (ribonuclease protection assay), iNOS enzyme activity (conversion of L-[3H]-arginine to NO and L-[3H]-citrulline), and serum nitrite/nitrate levels., Results: The iNOS mRNA was expressed in allograft lungs but was not detected in isografts or controls. The iNOS protein was present in allograft lungs, as demonstrated by high levels of L-[3H]-citrulline production compared with minimal iNOS enzyme activity in isograft and control lungs (10.1 +/- 2.4 vs 0.6 +/- 0.2 and 0.7 +/- 0.2 pmol L-[3H]-citrulline.mg-1.min-1, respectively; n = 6, p < 0.001). Allografts had significantly elevated systemic serum nitrite/nitrate levels compared with isografts and controls (38 +/- 6 vs 18 +/- 2 and 16 +/- 1 mumol/L, respectively; n = 6; p < 0.005)., Conclusions: These results, together with our previous demonstration that iNOS inhibition ameliorated lung allograft rejection, suggest that (1) iNOS expression and increased NO production contributed to acute rejection of the transplanted lung, (2) iNOS inhibition may offer an alternative in management of acute lung allograft rejection, and (3) increased NO production, detected by the presence of iNOS mRNA or protein or noninvasively by measuring serum nitrite/nitrate levels, may serve as an early marker of acute allograft rejection.

Messenger RNA encoding inducible NO synthase (iNOS) was measured by competitive reverse transcriptase polymerase chain reaction (cRT-PCR) and ribonuclease protection assays in spinal cords from mice at varying stages of experimental allergic encephalomyelitis (EAE) and from control mice. iNOS mRNA was increased in spinal cords from mice with acute EAE. cRT-PCR assays revealed a 10-20-fold increase in iNOS mRNA in spinal cords during acute EAE compared with the level observed in normal mouse spinal cords. Functional iNOS activity, as assessed by assay of calcium-independent citrulline production, was also significantly increased in spinal cords from mice with acute EAE in comparison to normal controls. The correlation of functional iNOS expression with active disease in EAE in consistent with a pathogenic role for excess NO in this model of cell-mediated central nervous system autoimmunity.

Background: We previously demonstrated that continuous treatment with aminoguanidine, a selective inhibitor of nitric oxide production by inducible nitric oxide synthase, attenuated acute cardiac allograft rejection., Methods: A rat transplant model was used to determine (1) when inducible nitric oxide synthase was expressed in the allograft heart during unmodified acute rejection and (2) whether pulse therapy with aminoguanidine attenuated the histologic changes of established acute rejection, in comparison with the effects of pulse therapy with corticosteroids., Results: Inducible nitric oxide synthase messenger RNA and protein were expressed during early and late acute rejection. Pulse therapy with aminoguanidine inhibited nitric oxide production and attenuated the histologic changes of acute rejection, but not as effectively as corticosteroid therapy (rejection scores of 4.1 +/- 0.4, 2.5 +/- 0.9, and 1.4 +/- 0.6 on postoperative day 8, for untreated, aminoguanidine-, and dexamethasone-treated allografts, respectively (scale, 0 to 5; p < 0.05)., Conclusions: (1) Inducible nitric oxide synthase expression first occurs during early acute allograft rejection and persists throughout rejection and (2) nitric oxide is an important effector molecule in acute rejection. Inducible nitric oxide synthase inhibition may offer a therapeutic adjunct in the management of acute rejection.

NO is produced during cardiac allograft rejection by expression of inducible NO synthase (iNOS) in the rejecting heart. Recent evidence indicates that NO modulates vascular permeability under both physiological and pathophysiological conditions. The present study explored the effects of early acute cardiac allograft rejection, and specifically the effects of NO, on myocardial and systemic vascular barrier function using a quantitative double-tracer permeation method in a rat cardiac transplant model. Early allograft rejection increased albumin permeation twofold to fivefold in the allograft heart and systemic vasculature (brain, lung, sciatic nerve, diaphragm, retina, muscle, kidney, and uvea) compared with isografts and controls. There were no detectable differences in regional blood flow or hemodynamics, suggesting that increased albumin permeation resulted from increased vascular permeability. iNOS mRNA was expressed in the allograft heart and native lung and was associated with increased serum nitrite/nitrate levels. iNOS inhibition with aminoguanidine prevented or attenuated allograft heart and systemic vascular barrier dysfunction and reduced allograft serum nitrite/nitrate levels to isograft values. Aminoguanidine did not affect the mild histological changes of rejection present in allografts. These data demonstrate the novel observations that (1) endothelial barrier function is compromised in the systemic vasculature, particularly in the brain, remote from the site of allograft rejection; (2) allograft vascular barrier dysfunction is associated with increased NO production and iNOS mRNA expression in the affected tissues (eg, native lung and grafted heart); and (3) inhibition of NO production by iNOS prevents vascular barrier dysfunction in the allograft heart and systemic vasculature.

A series of 2-iminoazaheterocycles have been prepared and shown to be potent inhibitors of human nitric oxide synthase (NOS) isoforms. This series includes cyclic amidines ranging from five- to nine-membered rings, of which 2-iminopiperidine and 2-iminohomopiperidine were the most potent inhibitors, with IC50 values of 1.0 and 2.0 microM, respectively, for human inducible nitric oxide synthase. This series of cyclic inhibitors was further expanded to include analogs with heteroatoms in the 3-position of the six-membered ring. This modification was tolerated for sulfur and oxygen, but nitrogen reduced the inhibitory potency. The oral administration of 2-iminopiperidine in lipopolysaccharide (LPS)-treated rats inhibited the LPS-induced increase in plasma nitrite/nitrate levels in a dose-dependent manner, demonstrating its ability to inhibit inducible NOS activity in vivo. These cyclic amidines represent a new class of potent NOS inhibitors and the foundation for potential therapeutic agents.

This study assessed the role of de novo nitric oxide (NO) production in the pathogenesis of experimental allergic encephalomyelitis (EAE) by using aminoguanidine (AG), an inhibitor of nitric oxide synthase (NOS), which preferentially inhibits the cytokine- and endotoxin-inducible isoform of NOS versus the constitutive isoforms consisting of endothelial and neuronal NOS. The maximum clinical severity of EAE and the duration of illness were significantly reduced or totally inhibited by twice daily subcutaneous injection of 100 mg/kg body weight AG. Histochemical staining for NADPH diaphorase, which detects enzymatic activity of NOS, revealed positive reactivity in untreated EAE rats both in parenchymal blood vessel walls and in anterior horn cell neurons, while normal rats and rats with EAE treated with AG showed predominantly the neuronal positivity. Moreover, this NADPH staining pattern was further supported by the immunohistochemical findings that endothelial NOS (eNOS) expression was increased in blood vessels in the inflamed lesions of untreated EAE rats and that inducible NOS (iNOS) was detected in some inflammatory cells, while treatment with AG could significantly reduce both iNOS and eNOS production. These results suggest that: (i) both iNOS and eNOS are upregulated in inflamed areas of the rat central nervous system in EAE; (ii) increased NO production plays a role in the development of clinical signs in EAE; and (iii) selective inhibitors of iNOS and/or eNOS may have therapeutic potential for the treatment of certain autoimmune diseases.

A cytokine-inducible form of nitric oxide synthase (iNOS), capable of producing large quantities of nitric oxide (NO), can be induced in many cell types. We demonstrate that conditioned medium from encephalitogenic myelin basic protein-sensitized lymphoid cells (MBP-CM) induces the expression of iNOS in primary cultures of murine astrocytes in a time- and concentration-dependent manner. iNOS mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) as early as 3 h post-exposure. Accumulation of nitrite into the astrocyte culture medium, an indirect measure of NO, was measurable 3 h post-exposure, plateaued at 24 h, and was prevented by the simultaneous administration of the NOS inhibitors, L-N(G)-nitroarginine methyl ester, N(G)-nitro-L-arginine or aminoguanidine. Astrocyte expression of iNOS protein, detected by immunohistochemistry and immunoprecipitation/Western blot, was prevented by inhibitors of RNA or protein metabolism, consistent with its dependence on de novo protein synthesis.

We have recently demonstrated that (1) nitric oxide (NO) is produced during experimental cardiac allograft rejection by the expression of inducible nitric oxide synthase (iNOS) in the rejecting organ and that (2) inhibition of NO production by iNOS attenuated acute rejection. The present study examined the interaction of corticosteroids, iNOS gene expression, and iNOS enzyme activity in a rat cardiac transplant model. Increased NO production in rejecting allografts was demonstrated by elevated serum nitrite/nitrate levels (67 +/- 5 versus 18 +/- 2 microM for isografts; P < 0.001) that were significantly reduced by pulse therapy with dexamethasone for 2 days prior to animal sacrifice or continuous dexamethasone treatment (34 +/- 2 and 19 +/- 4 microM, respectively; P < 0.001 versus untreated allografts). Increased iNOS enzyme activity was demonstrated in the allograft heart (5.11 +/- 1.00 versus 0.3 +/- 0.05 pmol L-citrulline.mg protein-1.min-1 for isografts) and was significantly reduced with dexamethasone treatment (1.13 +/- 0.47 for 2-day pulse-treated allografts and 0.02 +/- 0.01 for continuously treated allografts). Increased allograft iNOS enzyme activity resulted from induction of iNOS mRNA expression, which was more than 99% inhibited by dexamethasone treatment. Dexamethasone pulse therapy reduced but did not eliminate the histological changes of acute rejection. Thus corticosteroid treatment results in inhibition of iNOS expression during allograft rejection. These results further demonstrate that iNOS expression during acute rejection is immune-mediated and suggest that the immunosuppressive actions of corticosteroids in the treatment of acute rejection may include inhibition of iNOS expression.

Recently, the inducible isoform of nitric oxide synthase has been shown to be an important immunomodulation molecule in allograft rejection. We have observed the production of nitric oxide during rejection and the effect of nitric oxide synthase inhibition on allograft rejection in a rat lung transplant model. Rat left lung allotransplants were performed in two strain combinations: brown Norway-to-F344 (major histocompatibility complex incompatible); and Lewis-to-F344 (minor loci incompatible) as severe and mild rejection models respectively. Syngeneic F344-to-F344 transplants were performed as a negative control. Nitric oxide production during rejection was determined by measuring the recipient's serum nitrite/nitrate levels as a stable end product of nitric oxide. The progression of rejection was evaluated radiographically and the grade of rejection was determined histologically. After operation, recipients of allotransplantation were randomly divided into two groups and received either aminoguanidine (200 mg/kg, intraperitoneal every 6 hours), a potent inducible nitric oxide synthase inhibitor, or normal saline treatment. The levels of serum nitrite and nitrate in recipients increased in the early phase of rejection in both allotransplant combinations. However, in the terminal phase of rejection, the serum nitrite/nitrate level decreased significantly compared with the peak level in the brown Norway-to-F344 recipients. The serum nitrite/nitrate levels in the syngeneic transplant recipients were normal during the entire observation period. In aminoguanidine-treated animals, serum nitrite/nitrate levels remained normal in both allograft combinations. Significant suppression of rejection in aminoguanidine-treated recipients was observed histologically and radiographically in comparison with untreated recipients in the brown Norway-to-F344 combinations. In the Lewis-to-F344 combination, aminoguanidine treatment significantly ameliorated histologic rejection but did not affect radiologic appearance. We therefore conclude nitric oxide is produced during early allograft rejection and may prove to be a marker and mediator of early rejection. The inhibition of inducible nitric oxide synthase results in significant reduction in rat lung allograft rejection.

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated inflammatory demyelinating disorder of the central nervous system (CNS) which serves as a prime animal model for the human disease multiple sclerosis. Previous studies from these laboratories demonstrated excess nitric oxide (NO) in the CNS of EAE-affected mice, and amelioration of EAE with a selective inhibitor of the inducible nitric oxide synthase (iNOS). Recent studies from other laboratories have indicated that prostaglandin PGE2 is increased in CNS tissues of EAE-affected rodents and that EAE is prevented by the inhibition of cyclooxygenase activity. The present study investigated the ability of encephalitogenic lymphoid cells to induce NOS and cyclooxygenase (COX-2) in the murine macrophage line, RAW 264.7. In order to mimic the extracellular milieu present in EAE lesions, conditioned medium (CM) of activated EAE-inducer cells was added to this macrophage line. CM caused a time-dependent increase in nitrite, indicating NO production. Reverse-transcriptase PCR demonstrated iNOS mRNA in RAW 264.7 cells, first detected at 3 h, and Western blots confirmed the induction in RAW cells of the 130-kDa iNOS protein. Production of nitrite by CM-exposed RAW 264.7 cells was blocked by inhibitors of NOS (L-N-methylarginine or aminoguanidine) or by antibodies to murine IFN-gamma or IL-1 beta. CM of activated encephalitogenic cells induced production of PGE2 by RAW 264.7 cells, as determined by ELISA, and Western blots identified the presence of the 70-80-kDa inducible COX (COX-2) protein. Induction of COX-2 could be inhibited by antibody to IFN-gamma. Thus, encephalitogenic cells are capable of inducing the expression of the inflammatory enzymes iNOS and COX-2 in a murine macrophage line via the T cell cytokine IFN-gamma, alone or in combination with IL-1 beta.