iNOS Knockout Research Articles

Localization of 3-nitrotyrosine to rheumatoid and normal synovium

To determine the localization of 3-nitrotyrosine (3-NT), a footprint marker of peroxynitrite (ONOO-) and other reactive nitrogen species, to the inflamed human synovium and to compare this with normal synovial and nonsynovial tissue of human and animal origin.Monoclonal and polyclonal antibodies were used to investigate for 3-NT, inducible nitric oxide synthase (iNOS), macrophage marker CD68, and the vascular smooth muscle marker alpha-actin by avidin-biotin immunocytochemistry.In the inflamed synovium, 3-NT was found in the vascular smooth muscle and macrophages. In normal human synovium, 3-NT was present in the vascular smooth muscle and some lining cells and was not associated with immunoreactivity for iNOS. Similarly, 3-NT could be demonstrated in the vascular smooth muscle cells of normal rats and iNOS knockout mice. It was not present in the vascular smooth muscle of healthy, nonsynovial tissue.The synovial vasculature in histologically normal human and naive rodent synovium was alone among the normal tissues studied in exhibiting iNOS-independent immunoreactivity for 3-NT. These findings suggest a physiologic role for ONOO- in normal synovial vascular function.

Pivotal role of nitric oxide in delayed pharmacological preconditioning against myocardial infarction

The phenomenon of ‘ischemic preconditioning’ (IP) has been vigorously investigated during the past 15 years. As our knowledge on the possible protective mechanisms of IP has been increasingly expanded, novel approaches based on preconditioning with pharmacological agents have recently emerged. Two drugs have been used to induce delayed preconditioning against myocardial infarction caused by ischemia/reperfusion. One of the drugs was monophosphoryl lipid A (MLA) — a detoxified derivative of lipopolysaccharide from gram-negative strains; and another drug was RC552 — a novel synthetic glycolipid that mimics the chemical structure of MLA. We have shown that pretreatment of adult mice with MLA or RC552 (350 μg/kg) 24 h prior to the global ischemia and reperfusion in the isolated perfused heart attenuated myocardial injury. Infarct size was significantly reduced in MLA or RC552-treated groups as compared with the vehicle-treated group. The delayed cardioprotection was associated with a moderate but significant increase of nitric oxide level in the ischemic myocardium. Treatment with S-methylisothiourea (3 mg/kg), a selective inhibitor of inducible nitric oxide synthase (iNOS) abolished MLA or RC552-induced delayed protection. In addition, neither MLA nor RC552 reduced infarct size in iNOS knockout mice. Our findings suggest that both MLA and RC552 are able to induce delayed myocardial preconditioning via iNOS-dependent pathway.

Inducible nitric oxide synthase gene knockout mice have increased resistance to gut injury and bacterial translocation after an intestinal ischemia-reperfusion injury.

Intestinal ischemia-reperfusion after severe shock states is often associated with bacterial translocation and intestinal barrier dysfunction. Our previous studies showed that inducible nitric oxide synthase (iNOS) gene knockout mice were resistant to endotoxin-induced bacterial translocation and ileal mucosal damage. The goal of this study was to test whether iNOS mediates bacterial translocation after intestinal ischemia-reperfusion, using iNOS knockout mice (iNOS-/-) and their wild-type littermates (iNOS+/+). Prospective animal study with concurrent controls. Small animal laboratory. Thirty-eight iNOS knockout mice and 51 wild-type littermates. iNOS+/+ mice or iNOS-/- mice were subjected to a sham operation or 30 mins of superior mesenteric artery occlusion followed by reperfusion. Twenty-four hours after reperfusion, bacterial translocation to mesenteric lymph nodes, ileal villous damage, and cecal bacterial population were evaluated. Sham operation did not induce bacterial translocation, change cecal bacterial population levels, or cause ileal villous damage. Intestinal ischemia-reperfusion caused bacterial translocation in 72% of the iNOS+/+ mice but only 28% of the iNOS-/- mice. Both iNOS+/+ and iNOS-/- mice subjected to superior mesenteric artery occlusion (SMAO) in which bacterial translocation occurred had cecal bacterial population levels that were three logs higher than mice subjected to sham SMAO or mice subjected to SMAO in which bacterial translocation did not occur. The magnitude of villous injury was less in the iNOS-/- mice than the iNOS+/+ mice after SMAO, although the incidence of ileal villous damage was significantly higher in both the iNOS+/+ and iNOS-/- mice in which bacterial translocation occurred after SMAO than in the mice in which bacterial translocation did not occur after SMAO. iNOS+/+ mice subjected to SMAO had increased plasma concentrations of nitrite (NO2-) and nitrate (NO3-), and the plasma concentrations of NO2- and NO3- were highest in the mice in which bacterial translocation had occurred. iNOS knockout mice were more resistant to intestinal ischemia-reperfusion-induced bacterial translocation and mucosal injury than wild-type mice, suggesting that iNOS might play a role in intestinal ischemia-reperfusion-induced loss of gut barrier function.

Nitric oxide inhibits chondrocyte response to IGF-I: inhibition of IGF-IRbeta tyrosine phosphorylation.

Chondrocytes in arthritic cartilage respond poorly to insulin-like growth factor I (IGF-I). Studies with inducible nitric oxide synthase (iNOS) knockout mice suggest that NO is responsible for part of the cartilage insensitivity to IGF-I. These studies characterize the relationship between NO and chondrocyte responses to IGF-I in vitro, and define a mechanism by which NO decreases IGF-I stimulation of chondrocyte proteoglycan synthesis. Lapine cartilage slices, chondrocytes, and cartilage from osteoarthritic (OA) human knees were exposed to NO from the donors S-nitroso-N-acetylpenicillamine (SNAP) or (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1, 2-diolate] (DETA NONOate), by transduction with adenoviral transfer of iNOS (Ad-iNOS), or by activation with interleukin-1 (IL-1). NO synthesis was estimated from medium nitrite, and proteoglycan synthesis was measured as incorporation of (35)SO(4). IGF-I receptor phosphorylation was evaluated with Western analysis. SNAP, DETA NONOate, endogenously synthesized NO in Ad-iNOS-transduced cells, or IL-1 activation decreased IGF-I-stimulated proteoglycan synthesis in cartilage and monolayer cultures of chondrocytes. OA cartilage responded poorly to IGF-I; however, the response to IGF-I was restored by culture with N(G)-monomethyl-L-arginine (L-NMA). IGF-I receptor phosphotyrosine was diminished in chondrocytes exposed to NO. These studies show that NO is responsible for part of arthritic cartilage/chondrocyte insensitivity to anabolic actions of IGF-I; inhibition of receptor autophosphorylation is potentially responsible for this effect.

INOS gene expression modulates microvascular responsiveness in endotoxin-challenged mice.

Septic shock is characterized by vasodilation and decreased responsiveness to vasoconstrictors. Recent studies suggest this results from nitric oxide (NO) overproduction after expression of the calcium-independent isoform of NO synthase (iNOS) in smooth muscle cells. However, direct evidence linking iNOS (NOS2) expression and decreased microvascular responsiveness after septic stimuli is lacking. In the present study, we determined the effect of bacterial lipopolysaccharide (LPS, 20 mg/kg, IP) on smooth muscle contraction and endothelial relaxation in mesenteric resistance arteries from wild-type and iNOS knockout mice. Four hours after challenge with LPS or saline in vivo, concentration-dependent responses to norepinephrine (NE) and acetylcholine (NE+ACh) were measured in cannulated, pressurized vessels ex vivo. In vessels from wild-type mice, NE-induced contraction was markedly impaired after LPS, and pretreatment with the iNOS inhibitor aminoguanidine (AG) partly restored the NE contraction. In contrast, NE contraction in microvessels from iNOS knockout mice was unaffected by LPS. ACh-induced relaxation was unaffected by LPS in vessels from either genotype. These data provide direct evidence that iNOS gene expression mediates the LPS-induced decrease in microvascular responsiveness to vasoconstrictors. Moreover, the observation that AG did not fully restore NE contraction after LPS, whereas iNOS gene deficiency did, suggests that iNOS expression plays a central role in the development of the NO-independent effect of LPS on microvascular responsiveness. Finally, our data indicate that LPS or iNOS expression has little effect on endothelium-dependent relaxation, and eNOS activity does not appear to play a role in the decreased smooth muscle responsiveness after LPS in this model. The full text of this article is available at http://www.circresaha.org.

Inflammation is a component of neurodegeneration in response to Venezuelan equine encephalitis virus infection in mice

Infection with the mosquito-transmitted Venezuelan equine encephalitis virus (VEE) causes an acute systemic febrile illness followed by meningoencephalitis. In this communication we characterize the cytokine profile induced in the central nervous system (CNS) in response to virulent or attenuated strains of VEE using RNase Protection Assays. Virulent VEE causes an upregulation of multiple pro-inflammatory genes including inducible nitric oxide synthase (iNOS) and tumor necrosis factor alpha (TNF-α). To determine if iNOS and TNF-α contribute to the neuropathogenesis of VEE infection, iNOS and TNF receptor knockout mice were used in VEE mortality studies and exhibited extended survival times. Finally, CNS tissue sections labeled for VEE antigen, and adjacent sections double-labeled for an astrocyte marker and apoptosis, revealed that apoptosis of neurons occurs not only in areas of the brain positive for VEE-antigen, but also in areas of astrogliosis. These findings suggest that the inflammatory response, which is in part mediated by iNOS and TNF-α, may contribute to neurodegeneration following encephalitic virus infection.

Supplemental dietary arginine enhances wound healing in normal but not inducible nitric oxide synthase knockout mice

Background: Although generation of nitric oxide (NO) from inducible nitric oxide synthase (iNOS) has been shown to be required for cutaneous wound healing, no differences have been noted in incisional healing between iNOS knockout (iNOS-KO) and wild type (WT) mice. Because supplemental dietary arginine enhances cutaneous healing in normal rodents and is the sole substrate for NO synthesis, we studied whether arginine can enhance cutaneous wound healing in iNOS-KO mice. Methods: Twenty iNOS-KO and 20 WT mice, all on a C57BL/6 background, were divided into 4 groups of 10 animals each. Ten animals with each trait were randomized to receive either normal food and tap water or food and water each supplemented with 0.5% arginine (w/w). All animals underwent a 2.5-cm dorsal skin incision with implantation of four 20-mg polyvinyl alcohol sponges into subcutaneous pockets. On postoperative day 14 the animals were killed. The dorsal wound was harvested for breaking strength determination and the wound sponges were assayed for hydroxyproline content and total wound fluid nitrite/nitrate concentration. Results: Dietary arginine supplementation enhanced both wound breaking strength and collagen deposition in WT but not iNOS-KO mice. Wound fluid nitrite/nitrate levels were higher in WT than iNOS-KO animals but were not significantly influenced by additional arginine. Conclusions: These data demonstrate that supplemental dietary arginine enhances wound healing in normal mice. The loss of a functional iNOS gene abrogates the beneficial effect of arginine in wound healing. This suggests that the metabolism of arginine via the NO pathway is one mechanism by which arginine enhances wound healing. (Surgery 2000;128:374-8.)

Inducible Nitric Oxide Synthase Knockout Mouse Macrophages Disclose Prooxidant Effect of Interferon-γ on Low-Density Lipoprotein Oxidation

To test our hypothesis that interferon-γ (IFN-γ) has a direct prooxidant effect on macrophage-mediated LDL oxidation behind its antioxidant effect via induction of inducible nitric oxide synthase (iNOS), we incubated LDL with wild-type (iNOS+/+) or iNOS knockout mouse (iNOS−/−) macrophages preincubated with IFN-γ or IFN-γ plus lipopolysaccharide (IFN-γ/LPS) for 24 h. LDL oxidation was measured in terms of formation of thiobarbituric acid reactive substances (TBARS) and electrophoretic mobility. Thiol production, nitrite production, and superoxide production from macrophages were measured by using Ellman's assay, the Griess reagent, and the SOD-inhibitable cytochrome c reduction method, respectively. IFN-γ alone or combined with LPS induced iNOS expression and increased nitrite production in iNOS+/+ macrophages, but not in iNOS−/− macrophages. TBARS formation from LDL was suppressed in IFN-γ- and IFN-γ/LPS-treated iNOS+/+ macrophages but was increased in IFN-γ-treated iNOS−/− macrophages. In the presence of NG-monomethyl-l-arginine (l-NMMA), a NOS inhibitor, the suppressive effect of IFN-γ and IFN-γ/LPS was abolished and TBARS formation was even increased to a level above that of untreated iNOS+/+ macrophage. NOC 18, an NO donor, dose dependently inhibited macrophage-mediated LDL oxidation. IFN-γ increased superoxide and thiol productions in both types of macrophages. We conclude that IFN-γ promotes macrophage-mediated LDL oxidation by stimulating superoxide and thiol production under conditions where iNOS-catalyzed NO release is restricted.

Cytokines induce apoptosis in beta-cells isolated from mice lacking the inducible isoform of nitric oxide synthase (iNOS-/-).

Prolonged exposure of rodent beta-cells to combinations of cytokines induces the inducible form of nitric oxide synthase (iNOS) expression and Fas expression, nitric oxide (NO) production, and cell death. It also induces the expression of potential "defense" genes, such as manganese superoxide dismutase (MnSOD) and heat shock protein (hsp) 70. NO is a radical with multifaceted actions. Recent studies have shown that NO, in addition to having cytotoxic actions, may regulate gene transcription. It remains unclear whether NO mediates cytokine-induced gene expression and subsequent beta-cell death. Previous studies using NO synthase blockers yielded conflicting results, which may be due to nonspecific effects of these agents. In this study, we examined the effects of cytokines on gene expression, determined by reverse transcriptase-polymerase chain reaction (RT-PCR), and viability, determined by nuclear dyes, of pancreatic islets or fluorescence-activated cell sorter (FACS)-purified beta-cells isolated from iNOS knockout mice (iNOS-/-, background C57BL/6x129SvEv) or their respective controls (C57BL/6x129SvEv). The combination of cytokines used was interleukin-1beta (50 U/ml) plus gamma-interferon (1,000 U/ml) plus tumor necrosis factor-alpha (1,000 U/ml). The lack of cytokine-induced iNOS activity in the iNOS-/- islet cells was confirmed by RT-PCR and nitrite determination. Cytokines induced a >3-fold increase in Fas and MnSOD mRNA expression in wild-type (WT) and iNOS-/- islets. On the other hand, hsp 70 was induced in WT but not in iNOS-/- islets. Prolonged (6-9 days) exposure of WT islets to cytokines leads to an 80-90% decrease in islet cell viability, whereas viability decreased by only 10-30% in iNOS-/- islet cells. To determine the mode of cytokine-induced cell death, FACS-purified beta-cells were exposed to the same cytokines. After 9 days, the apoptosis index was similarly increased in WT (39 +/- 3%) and iNOS4-/- (33 +/- 4%) beta-cells. On the other hand, cytokines increased necrosis in WT (20 +/- 4%) but not in iNOS-/- (7 +/- 3%) beta-cells. From these data, we concluded that 1) NO is required for cytokine-induced hsp 70 mRNA expression but not for Fas and MnSOD expression, 2) cytokines induce both apoptosis and necrosis in mouse beta-cells, and 3) cytokine-induced apoptosis is mostly NO-independent, whereas necrosis requires NO formation.

Reduction of interleukin-17-induced inhibition of chondrocyte proteoglycan synthesis in intact murine articular cartilage by interleukin-4.

To investigate the role of interleukin-4 (IL-4) and IL-10 in basal and IL-1- and IL-17-mediated inhibition of chondrocyte metabolism. Cartilage explants of patellae from naive mice were incubated with IL-17 and/or IL-1 alone or were pretreated with IL-4 and IL-10. In addition, knee joints of naive mice were injected intraarticularly with IL-4 and IL-10 alone or were coinjected with IL-1. Chondrocyte proteoglycan (PG) synthesis was measured in intact murine articular cartilage. Levels of nitric oxide (NO) were measured using the Griess reagent. IL-17, a novel cytokine secreted by CD4+ activated memory T cells, inhibited chondrocyte PG synthesis in intact murine articular cartilage, although the suppressive effect was less potent than that of IL-1. The suppressive effect of IL-17 was completely abolished in the presence of L-NIO (L-NS-[1-iminoethyl]ornithine), an inhibitor of NO metabolism, and IL-17 only slightly induced inhibition of PG synthesis in cartilage explants of patellae from iNOS (inducible NO synthase) knockout mice. This indicates that the suppressive effect of IL-17 was mediated by NO. Pretreatment with IL-4, but not IL-10, significantly reduced the inhibition of chondrocyte PG synthesis induced by IL-1 or IL-17. The IL-4-induced reduction in the inhibitory effects of IL-1 and IL-17 on chondrocyte PG synthesis was accompanied by decreased NO formation in the culture supernatants. IL-17 plays a role in the inhibition of chondrocyte PG synthesis. IL-4 and IL-10 have no effect on basal chondrocyte metabolism. However, IL-4-pretreated cartilage is less sensitive to the suppressive effect of IL-1 as well as IL-17. This suggests that IL-4 is protective in T cell-driven cartilage disturbances, probably through reduction of iNOS.

Endotoxin-induced mesenteric microvascular changes involve iNOS-derived nitric oxide: results from a study using iNOS knock out mice.

The administration of endotoxin alters intestinal blood flow, increases nitric oxide (NO) production, and induces gut barrier dysfunction. Thus, we investigated the hypothesis that microvascular reactivity and permeability of the mesenteric vascular bed are altered to a lesser degree in iNOS knock out (iNOS -/-) mice than their wild-type (iNOS +/+) litter mates after an endotoxin challenge. To test this hypothesis, we compared the microvascular response of iNOS knockout (iNOS -/-) mice after a topical or systemic endotoxin challenge against that of their wild-type litter mates (iNOS +/+). Intravital microscopy was used to measure arteriolar diameter and postcapillary venular permeability in the mouse ileum. Both parameters were determined by computer-assisted image analysis. Diameter was measured in A1, A2, and A3 arterioles (1, 2, 3 = rank of deployment). Changes in microvascular permeability were measured from changes in interstitial fluorescence caused by extravasation of fluorescein isothiocyanate (FITC)-dextran 150 (molecular weight = 150 kDa) and expressed as changes in integrated optical intensity (IOI). In the first series of experiments, endotoxin (100 microg/mL) was applied topically to the ileal segment. In the second series, endotoxin (10 mg/kg) was administered intraperitoneally (i.p.). Administration of topical or i.p.. endotoxin caused vasoconstriction and was associated with an early increase in permeability in both iNOS +/+ and -/- mice, although over time the responses of the iNOS -/- and iNOS +/+ mice diverged. Twenty minutes after topical endotoxin, the increase in permeability in iNOS -/- mice had reached a plateau whereas it continued to increase in the iNOS +/+ mice, such that at 80 min post-topical endotoxin, IOI was 27+/-7 in iNOS -/- vs. 39+/-5 in iNOS +/+ (P < 0.05). A similar permeability response was observed after i.p.. endotoxin, where the increase in post-capillary venular permeability was greater in the iNOS +/+ mice (P < 0.05). Both iNOS -/- and iNOS +/+ mice had a similar transient vasoconstrictive response after topical endotoxin challenge (reduction in A2 arteriolar diameters by -17+/-4% vs. -24+/-7%), with return to baseline values by 60-80 min post-endotoxin challenge. The iNOS +/+ but not the iNOS -/- mice manifested a secondary vasodilatory response that persisted throughout the experimental period. The arteriolar vasoreactive response of the iNOS -/- and iNOS +/+ mice to i.p.. endotoxin was similar to that of topical endotoxin, but of a lesser magnitude. In conclusion, the similarity in effects between topical and systemic endotoxin indicates that endotoxin causes microvascular dysfunction in the gut by directly on the microcirculation. In addition, our data suggest that NO production by iNOS is involved in the microvascular alterations associated with gut barrier dysfunction.

Septic Mucosal Intraepithelial Lymphoid Immune Suppression: Role for Nitric Oxide not Interleukin-10 or Transforming Growth Factor-??

Recent studies indicate that sepsis induces a marked depression in the splenocyte immune response (as illustrated by decreased interleukin [IL]-2 production, interferon [IFN]-gamma production, or both) in response to T-cell mitogen. However, it is not known whether a similar depression is evident in the phenotypically distinct, small intestine intraepithelial lymphocytes (IELs) or what regulates this process during sepsis. Because the maintenance of a competent mucosal immune response is thought to be central to the animal's ability to survive sepsis, we attempted to determine whether IEL's IL-2/IFN-gamma production is suppressed and what mediates this depression. Our studies indicated that C3H/HeN mice subjected to cecal ligation and puncture (CLP) exhibited a marked decline in the ability of IELs to release IL-2/IFN-gamma at 24 hours and that this decline is associated with increased secretion of IL-10 and nitric oxide (NO). To the extent that IL-10 accounted for this loss of IL-2/IFN-gamma release, we observed that IL-10 gene deficiency neither restored the IL-2/IFN-gamma release nor suppressed the increase in NO when compared with background control, C57BL/6J mouse cells. To further study whether NO was involved in this immune suppression, iNOS knockout (iNOS -/-) were also subjected to the same procedure; however, the depression in IL-2/IFN-gamma was not seen in iNOS -/- mice when compared with background controls. Our data indicate that IL-10, which affects splenic lymphoid response, may not be a key mediator of IEL immune suppression and that the induction of NO may play a more significant role in gastrointestinal immune dysfunction seen in late sepsis.

Innate lung defenses and compromised Pseudomonas aeruginosa clearance in the malnourished mouse model of respiratory infections in cystic fibrosis.

Cystic fibrosis (CF) is characterized by dysfunction of the digestive and respiratory tracts resulting in generalized malnutrition and chronic respiratory infections. Chronic lung infections with Pseudomonas aeruginosa, intense neutrophil-dominated airway inflammation, and progressive lung disease are the major cause of high morbidity and mortality in CF. Here we investigated the effects of malnutrition in CF on innate lung defenses, susceptibility to P. aeruginosa colonization, and associated inflammation, using aerosol models of acute and chronic infections in normal, malnourished, and transgenic mice. CFTR(m1Unc-/-) knockout mice displayed body weight variations and showed variable pulmonary clearance of P. aeruginosa. This variability was not detected in bitransgenic CFTR(m1Unc-/-)(FABP-hCFTR) mice in which the intestinal defect had been corrected. Diet-induced protein calorie malnutrition in C57BL/6J mice resulted in impaired pulmonary clearance of P. aeruginosa. Tumor necrosis factor alpha (TNF-alpha) and nitrite levels detected upon exposure to P. aeruginosa aerosols were lower in the lungs of the malnourished C57BL/6J mice relative than in lungs of mice fed a normal diet. The role of TNF-alpha and reactive nitrogen intermediates in P. aeruginosa clearance was tested in TNF-alpha and inducible nitric oxide synthase (iNOS) knockout mice. P. aeruginosa clearance was diminished in transgenic TNF-alpha- and iNOS-deficient mice. In contrast to the effects of TNF-alpha and iNOS, gamma interferon knockout mice retained a full capacity to eliminate P. aeruginosa from the lung. Malnutrition also contributed to excessive inflammation in C57BL/6J mice upon chronic challenge with P. aeruginosa. The repeatedly infected malnourished host did not produce interleukin-10, a major anti-inflammatory cytokine absent or diminished in the bronchoalveolar fluids of CF patients. These results are consistent with a model in which defective CFTR in the intestinal tract leads to nutritional deficiency which in turn contributes to compromised innate lung defenses, bacterial colonization, and excessive inflammation in the CF respiratory tract.

Endothelial nitric oxide synthase protects aortic allografts from the development of transplant arteriosclerosis.

Inducible nitric oxide synthase (iNOS) is up-regulated in rejecting allografts and is protective against allograft arteriosclerosis; it suppresses neointimal smooth muscle cell accumulation and inhibits adhesion of platelets and leukocytes to the endothelium. However, the functional importance of endothelial NOS (eNOS) in the rejecting allografts remains unclear. We examined the effects of selective eNOS deficiency in aortic allografts in a murine chronic rejection model using grafts from eNOS knockout (KO) mice (C57BL/6 background; H2b) and normal C3H (H2K) as recipients. Grafts from wild-type C57BL/6 mice served as controls. Grafts from iNOS KO mice served as a second group of controls where the contribution from iNOS was eliminated but eNOS was preserved. Aortic grafts were harvested and analyzed at days 10-14, 18-22, and 26-30 after transplantation. Endothelial NOS-deficient grafts showed significantly increased intima/media ratios at days 26-30 compared to controls. Immunostaining demonstrated that in eNOS KO grafts, eNOS was not detectable whereas iNOS was expressed prominently in infiltrating recipient mononuclear cells. In control grafts, eNOS expression was preserved in the endothelium even by day 30, and associated with a decrease in intimal thickening. We further demonstrated that early overexpression of iNOS by ex vivo gene transfer completely prevented the development of arteriosclerosis associated with eNOS deficiency. We found that eNOS plays a protective role in allografts, and that in eNOS-deficient allografts, early overexpression of iNOS is capable of preventing the development of allograft arteriosclerosis. In allografts with dysfunctional vascular endothelium and impaired eNOS activity as a result of ischemia or native arteriosclerotic disease, iNOS gene therapy may serve to improve their long-term survival and function.

Role of inducible nitric oxide synthase in postoperative intestinal smooth muscle dysfunction in rodents

Background & Aims: We have shown that intestinal manipulation leads to a significant inhibition of circular muscle contraction. We hypothesized that the inflammatory mediator inducible nitric oxide (NO) plays a role in surgically induced ileus. Methods: Rats and inducible NO synthase (iNOS) knockout and wild-type mice underwent a simple intestinal manipulation. Reverse-transcription polymerase chain reaction and immunohistochemistry were used to detect and localize iNOS expression. Nitrite and NO production were measured in muscularis cultures. Spontaneous and bethanechol-stimulated jejunal circular muscle contractions were measured in an organ bath. Results: Intestinal manipulation resulted in significant iNOS messenger RNA induction in mucosa and muscularis. Immunohistochemistry localized iNOS in phagocytes within the muscularis. Nitrite and NO production increased 59.8-fold 24 hours after manipulation. L-n 6-(1-iminoethyl) lysine ( L-NIL) inhibited this response. In control rats, selective iNOS inhibition did not increase spontaneous muscle activity, but after manipulation L-NIL significantly improved spontaneous activity. iNOS knockout mice showed a significant 81% decrease in neutrophil infiltration into the muscularis after intestinal manipulation compared with wild-types. Contractile activity was normal in knockout mice after intestinal manipulation. Conclusions: These results show that leukocyte-derived inducible NO inhibits gastrointestinal motility after manipulation and plays an essential role in the initiation of intestinal inflammation. GASTROENTEROLOGY 2000;118:316-327

Neointimal Hyperplasia and Remodelling after Deep Abrasion Injury in Carotid Arteries of Enos and Inos Knockout Mice

Neointimal Hyperplasia and Remodelling after Deep Abrasion Injury in Carotid Arteries of Enos and Inos Knockout Mice

Roles of endogenous gamma interferon and macrophage microbicidal mechanisms in host response to chemotherapy in experimental visceral leishmaniasis.

In experimental visceral leishmaniasis, in which the tissue macrophage is the target, in vivo responsiveness to conventional chemotherapy (pentavalent antimony [Sb]) requires a T-cell-dependent mechanism. To determine if this mechanism involves gamma interferon (IFN-gamma)-induced activation and/or specific IFN-gamma-regulated macrophage leishmanicidal mechanisms (generation of reactive nitrogen or oxygen intermediates, we treated gene-deficient mice infected with Leishmania donovani. In IFN-gamma gene knockout (GKO) mice, Sb inhibited but did not kill intracellular L. donovani (2% killing versus 76% in controls). Sb was active (>94% killing), however, in both inducible nitric oxide synthase (iNOS) knockout (KO) and respiratory burst (phagocyte oxidase)-deficient chronic granulomatous disease (X-CGD) mice. Sb's efficacy was also maintained in doubly deficient animals (X-CGD mice treated with an iNOS inhibitor). In contrast to Sb, amphotericin B (AmB) induced high-level killing in GKO mice; AmB was also fully active in iNOS KO and X-CGD animals. Although resolution of L. donovani infection requires iNOS, residual visceral infection remained largely suppressed in iNOS KO mice treated with Sb or AmB. These results indicate that endogenous IFN-gamma regulates the leishmanicidal response to Sb and achieves this effect via a pathway unrelated to the macrophage's primary microbicidal mechanisms. The role of IFN-gamma is selective, since it is not a cofactor in the response to AmB. Treatment with either Sb or AmB permits an iNOS-independent mechanism to emerge and control residual intracellular L. donovani infection.

ENOS mediates L-arginine-induced inhibition of thick ascending limb chloride flux.

We recently reported that the rat thick ascending limb (THAL) possesses an active isoform of nitric oxide synthase (NOS) that is substrate-limited in vitro. NO produced by THAL NOS inhibits chloride flux. Protein and transcript for each of the primary NOS isoforms-endothelial (eNOS), inducible (iNOS), and neuronal (nNOS)-have been demonstrated in THALs. However, the NOS isoform that mediates NO-induced inhibition of chloride flux is unknown. We hypothesized that NO produced from eNOS in the THAL inhibits NaCl transport. THALs from male eNOS, iNOS, and nNOS knockout mice and C57BL/6J wild-type controls were perfused in vitro and the response of transepithelial chloride flux (J(Cl)) to L-arginine (L-Arg), the substrate for NOS, and spermine NONOate (SPM), an NO donor was measured. We first tested whether isolated mouse THALs could synthesize NO and whether this NO inhibits transport. Addition of 0. 5 mmol/L L-Arg to the bath decreased J(Cl) from 105.8+/-17.5 to 79. 2+/-15.8 pmol/mm per minute (P<0.01) in C57BL/6J wild-type mice, whereas addition of D-Arginine had no effects on J(Cl.) In contrast, addition of 0.5 mmol/L L-Arg to the bath did not alter J(Cl) of THALs from eNOS knockout mice. When 10 micromol/L SPM was added to the bath of eNOS knockout THALs, J(Cl) decreased from 89.1+/-8.6 to 74.8+/-7.5 pmol/mm/min (P<0.05). Thus the lack of responsiveness of eNOS knockout THALs to L-Arg was not due to an inability to respond to NO. We next evaluated the role of iNOS and nNOS in the response to L-Arg. Addition of 0.5 mmol/L L-Arg to the bath decreased J(Cl) in THALs from iNOS and nNOS knockout mice by 37.7+/-6.4% (P<0.05) and 31.8+/-8.3% (P<0.01), respectively. We conclude that eNOS is the active isoform of NOS in the THAL under basal conditions. Mouse THAL eNOS responds to exogenous L-Arg by increasing NO production, which, in turn, inhibits J(Cl).

Gene transfer of IFN-gamma into established brain tumors represses growth by antiangiogenesis.

The experiments in this paper were designed to examine the therapeutic effects of adenoviral-mediated gene transfer of IFN-gamma into a mouse model of an established metastatic brain tumor. Temperature-sensitive replication-defective adenovirus was generated for gene transfer of IFN-gamma (AdIFN) and beta-galactosidase (AdBGAL) cDNAs in vivo. In this model, treatment with AdIFN elicits prolonged survival times and brain tumor rejection. Evidence against an immune-mediated response accounting for this result include: 1) absence of a memory immune response upon challenge, 2) lack of antitumor effects at sites distal to inoculation of AdIFN, and 3) preservation of the therapeutic effects of AdIFN in scid and beige mice and in inducible NO synthase (iNOS) knockouts. High concentrations of IFN-gamma do not inhibit tumor growth in vitro making it unlikely that the antitumor effect of this treatment acts directly on the growth of the tumor cells. However, gene transfer of IFN-gamma inhibits neovascularization of the tumor in a 3LL-Matrigel assay in vivo, and AdIFN induces apoptosis of endothelial cells in vivo, supporting the idea that AdIFN represses tumor growth by inhibiting angiogenesis. The substantial non-immune-mediated therapeutic benefits of AdIFN in animals paves the way for devising novel strategies for treating human brain tumors.

Nitrotyrosine generation via inducible nitric oxide synthase in vascular wall in focal ischemia-reperfusion

Nitrotyrosine produced by NO-mediated reaction is a possible marker for cytotoxicity in brain ischemia. In this study, we aimed to determine whether iNOS is responsible for the nitrotyrosine formation and which type of cell is predominantly nitrated. Fifty-eight wild-type and 28 iNOS knockout male mice were used. Under halothane anesthesia the left middle cerebral artery was occluded for 2 h and reperfused for 0.5 or 15 h. The ratio of nitrotyrosine to total tyrosine (%NO 2-Tyr) was measured by means of a hydrolysis/HPLC. After 0.5-h reperfusion, %NO 2-Tyr in the ischemic cortex of wild-type and knockout mice amounted to 0.037±0.040% ( n=8) and 0.064±0.035% ( n=6), respectively, being significantly higher than that in the sham operation group ( n=7) ( P<0.05). After 15-h reperfusion, nitrotyrosine was detected only in wild-type mice (0.039±0.025%, n=7), not in knockout or sham-operated mice ( P<0.05). Immunohistochemical reaction for nitrotyrosine was seen predominantly in the vascular wall in the peri-infarct region of the cerebral cortex in wild-type mice after 15-h reperfusion, but not in corresponding knockout mice. Our data suggest that iNOS is responsible for nitrotyrosine formation in the later phase of reperfusion, and that vascular endothelium is the major site of this reaction, at least in the case of 15-h reperfusion.