L-arginine supplementation improves responses to injury and inflammation in dextran sulfate sodium colitis

Inflammatory bowel disease (IBD), consisting of Crohn's disease and ulcerative colitis (UC), results in substantial morbidity and is difficult to treat. New strategies for adjunct therapies are needed. One candidate is the semi-essential amino acid, L-arginine (L-Arg), a complementary medicine purported to be an enhancer of immunity and vitality in the lay media. Using dextran sulfate sodium (DSS) as a murine colonic injury and repair model with similarities to human UC, we assessed the effect of L-Arg, as DSS induced increases in colonic expression of the y(+) cationic amino acid transporter 2 (CAT2) and L-Arg uptake. L-Arg supplementation improved the clinical parameters of survival, body weight loss, and colon weight, and reduced colonic permeability and the number of myeloperoxidase-positive neutrophils in DSS colitis. Luminex-based multi-analyte profiling demonstrated that there was a marked reduction in proinflammatory cytokine and chemokine expression with L-Arg treatment. Genomic analysis by microarray demonstrated that DSS-treated mice supplemented with L-Arg clustered more closely with mice not exposed to DSS than to those receiving DSS alone, and revealed that multiple genes that were upregulated or downregulated with DSS alone exhibited normalization of expression with L-Arg supplementation. Additionally, L-Arg treatment of mice with DSS colitis resulted in increased ex vivo migration of colonic epithelial cells, suggestive of increased capacity for wound repair. Because CAT2 induction was sustained during L-Arg treatment and inducible nitric oxide (NO) synthase (iNOS) requires uptake of L-Arg for generation of NO, we tested the effect of L-Arg in iNOS(-/-) mice and found that its benefits in DSS colitis were eliminated. These preclinical studies indicate that L-Arg supplementation could be a potential therapy for IBD, and that one mechanism of action may be functional enhancement of iNOS activity.     

Striatal Malonate Lesions Are Attenuated in Neuronal Nitric Oxide Synthase Knockout Mice

Abstract: Intrastriatal administration of the reversible succinate dehydrogenase inhibitor malonate produces both energy depletion and striatal lesions by a secondary excitotoxic mechanism. To investigate the role of nitric oxide (NO^•) in the pathogenesis of the lesions we examined malonate toxicity in mice in which the genes for neuronal nitric oxide synthase (nNOS) or endothelial nitric oxide synthase (eNOS) were disrupted. Malonate striatal lesions were significantly attenuated in the nNOS mutant mice, and they were significantly increased in the eNOS mutant mice. Malonate-induced increases in levels of 2,3- and 2,5-dihydroxybenzoic acid/salicylate, markers of hydroxyl radical generation, were significantly attenuated in the nNOS knockout mice. Malonate-induced increases in 3-nitrotyrosine, a marker for peroxynitrite-mediated damage, were blocked in the nNOS mice, whereas a significant increase occurred in the eNOS mice. These findings show that NO^• produced by nNOS results in generation of peroxynitrite, which plays a role in malonate neurotoxicity.     

Suppressed apoptosis in the inflamed gastric mucosa of Helicobacter pylori-colonized iNOS-knockout mice

Deregulated cell turnover in Helicobacter pylori (H. pylori)-colonized gastric mucosa has been suggested to be linked to the gastric carcinogenesis pathway. We previously reported attenuation of apoptosis and enhancement of cellular proliferation in the H. pylori-colonized gastric mucosa of Mongolian gerbils as compared to that in mice, which might reflect a specific link between H. pylori colonization and carcinogenesis in the Mongolian gerbils; the difference between the two strains could be attributable to differences in the host genetic background. Inducible-type nitric oxide synthase (iNOS) is thought to participate in not only the inflammatory response, but also in the regulation of gastric mucosal cell turnover in H. pylori-colonized gastric mucosa. Thus, the present study was designed to examine gastric leukocyte activation and epithelial cell apoptosis in the gastric mucosa following H. pylori inoculation in iNOS-knockout mice. Methods: iNOS-knockout mice (iNOS^−/−) and their iNOS^+/+ littermates were orally inoculated with the Sydney strain of H. pylori (SS1, 10⁸ colony-forming units [CFU]). H. pylori infection was confirmed by microaerobic bacterial culture. The stomach of each mouse was evaluated 14 weeks and 30 weeks after the inoculation. Gastric mucosal accumulation of polymorphonuclear leukocytes (PMN) was assessed by determining the myeloperoxidase (MPO) activity and histological score based on the updated Sydney system. The level of apoptosis was determined by estimation of the cytoplasmic levels of mono- and oligonucleosomes and by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method. Results: The SS1-inoculated mice showed persistent H. pylori colonization for 12 weeks. While gastric mucosal PMN infiltration increased following SS1 inoculation in both iNOS^+/+ and iNOS^−/−strains, enhanced DNA fragmentation was observed in only SS1-colonized iNOS^+/+ mice, and not in the iNOS^−/− mice. In conclusion, although the recruitment of PMN in response to H. pylori was evoked even in the gastric mucosa of iNOS^−/− mice, epithelial cell apoptosis induced by H. pylori was attenuated in this strain. These data suggest that iNOS may play an important role in promoting apoptosis in the H. pylori-infected inflamed gastric mucosa, and that persistent inflammation without apoptosis in iNOS^−/− mice with H. pylori infection may be linked to preneoplastic transformation.     

B-vitamin deficiency is protective against DSS-induced colitis in mice

Vitamin deficiencies are common in patients with inflammatory bowel disease (IBD). Homocysteine (Hcys) is a thrombogenic amino acid produced from methionine (Met), and its increase in patients with IBD indicates a disruption of Met metabolism; however, the role of Hcys and Met metabolism in IBD is not well understood. We hypothesized that disrupted Met metabolism from a B-vitamin-deficient diet would exacerbate experimental colitis. Mice were fed a B(6)-B(12)-deficient or control diet for 2 wk and then treated with dextran sodium sulfate (DSS) to induce colitis. We monitored disease activity during DSS treatment and collected plasma and tissue for analysis of inflammatory tissue injury and Met metabolites. We also quantified Met cycle activity by measurements of in vivo Met kinetics using [1-(13)C-methyl-(2)H(3)]methionine infusion in similarly treated mice. Unexpectedly, we found that mice given the B-vitamin-deficient diet had improved clinical outcomes, including increased survival, weight maintenance, and reduced disease scores. We also found lower histological disease activity and proinflammatory gene expression (TNF-α and inducible nitric oxide synthase) in the colon in deficient-diet mice. Metabolomic analysis showed evidence that these effects were associated with deficient B(6), as markers of B(12) function were only mildly altered. In vivo methionine kinetics corroborated these results, showing that the deficient diet suppressed transsulfuration but increased remethylation. Our findings suggest that disrupted Met metabolism attributable to B(6) deficiency reduces the inflammatory response and disease activity in DSS-challenged mice. These results warrant further human clinical studies to determine whether B(6) deficiency and elevated Hcys in patients with IBD contribute to disease pathobiology.     

Regulation of eNOS in normal and diabetes-impaired skin repair: implications for tissue regeneration.

An important role of inducible nitric oxide (NO) synthase for epithelial action during skin repair has been well established. Although a delayed healing of skin wounds has been recently described for eNOS-deficient mice, a participation of endothelial-type NO synthase (eNOS) in skin repair largely remains unclear. In this study we determined the expression pattern of eNOS during wound healing in healthy and in diabetic mice. Remarkably, normal repair in healthy animals was characterized by a moderate induction of eNOS at the mRNA and protein level, whereas diabetes-impaired healing was associated with a clearly reduced eNOS protein expression. Immunohistochemistry revealed the endothelial lining of blood vessels within the granulation tissue, and also keratinocytes of the wound margins, the developing neo-epithelium, and the hair follicles to express eNOS protein. Keratinocyte-derived expression of eNOS could be confirmed at the mRNA level in vitro for human primary keratinocytes and the keratinocyte cell line HaCaT. Furthermore, eNOS enzymatic activity most likely contributes to epithelial regeneration, as eNOS-deficient (eNOS -/-) animals exhibited reduced wound margin epithelia associated with reduced keratinocyte proliferation.     

Inhibition of compensatory lung growth in endothelial nitric oxide synthase-deficient mice

Pneumonectomy results in rapid compensatory growth of the remaining lung and also leads to increased flow and shear stress, which are known to stimulate endothelial nitric oxide synthase (eNOS). Nitric oxide is an essential mediator of vascular endothelial growth factor-induced angiogenesis, which should necessarily occur during compensatory lung growth. Thus our hypothesis is that eNOS is critical for compensatory lung growth. To test this, left pneumonectomy was performed in eNOS-deficient mice (eNOS-/-), and compensatory growth of the right lung was characterized throughout 14 days postpneumonectomy and compared with wild-type pneumonectomy and sham controls. Compensatory lung growth was severely impaired in eNOS-/- mice, as demonstrated by significant reductions in lung weight index, lung volume index, and volume of respiratory region. Also, pneumonectomy-induced increases in alveolar surface density and cell proliferation were prevented in eNOS-/- mice, indicating that eNOS plays a role in alveolar hyperplasia. Compensatory lung growth was also impaired in wild-type mice treated with the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester. Together, these results indicate that eNOS is critical for compensatory lung growth.     

Pharmacological induction of vascular extracellular superoxide dismutase expression in vivo

Pentaerythritol tetranitrate (PETN) treatment reduces progression of atherosclerosis and endothelial dysfunction and decreases oxidation of low-density lipoprotein (LDL) in rabbits. These effects are associated with decreased vascular superoxide production, but the underlying molecular mechanisms remain unknown. Previous studies demonstrated that endogenous nitric oxide could regulate the expression of extracellular superoxide dismutase (ecSOD) in conductance vessels in vivo . We investigated the effect of PETN and overexpression of endothelial nitric oxide synthase (eNOS⁺⁺) on the expression and activity of ecSOD. C57BL/6 mice were randomized to receive placebo or increasing doses of PETN for 4 weeks and eNOS⁺⁺ mice with a several fold higher endothelial-specific eNOS expression were generated. The expression of ecSOD was determined in the lung and aortic tissue by real-time PCR and Western blot. The ecSOD activity was measured using inhibition of cytochrome C reduction. There was no effect of PETN treatment or eNOS overexpression on ecSOD mRNA in the lung tissue, whereas ecSOD protein expression increased from 2.5-fold to 3.6-fold ( P < 0.05) by 6 mg PETN/kg body weight (BW)/day and 60 mg PETN/kg BW/day, respectively. A similar increase was found in aortic homogenates. eNOS⁺⁺ lung cytosols showed an increase of ecSOD protein level of 142 ± 10.5% as compared with transgene-negative littermates ( P < 0.05), which was abolished by N^ω-nitro-L-arginine treatment. In each animal group, the increase of ecSOD expression was paralleled by an increase of ecSOD activity. Increased expression and activity of microvascular ecSOD are likely induced by increased bioavailability of vascular nitric oxide. Up-regulation of vascular ecSOD may contribute to the reported antioxidative and anti-atherosclerotic effects of PETN.     

Multiple deficiencies in antioxidant enzymes in mice result in a compound increase in sensitivity to oxidative stress

To examine the effect of compound deficiencies in antioxidant defense, we have generated mice (Sod2^+/−/Gpx1^−/−) that are deficient in Mn superoxide dismutase (MnSOD) and glutathione peroxidase 1 (Gpx1) by breeding Sod2^+/− and Gpx1^−/− mice together. Although Sod2^+/−/Gpx1^−/− mice showed a 50% reduction in MnSOD and no detectable Gpx1 activity in either mitochondria or cytosol in all tissues, they were viable and appeared normal. Fibroblasts isolated from Sod2^+/−/Gpx1^−/− mice were more sensitive (4- to 6-fold) to oxidative stress (t-butyl hydroperoxide or γ irradiation) than fibroblasts from wild-type mice, and were twice as sensitive as cells from Sod2^+/− or Gpx1^−/− mice. Whole-animal studies demonstrated that survival of the Sod2^+/−/Gpx1^−/− mice in response to whole body γ irradiation or paraquat administration was also reduced compared with that of wild-type, Sod2^+/−, or Gpx1^−/− mice. Similarly, endogenous oxidative stress induced by cardiac ischemia/reperfusion injury led to greater apoptosis in heart tissue from the Sod2^+/−/Gpx1^−/− mice than in that from mice deficient in either MnSOD or Gpx1 alone. These data show that Sod2^+/−/Gpx1^−/− mice, deficient in two mitochondrial antioxidant enzymes, have significantly enhanced sensitivity to oxidative stress induced by exogenous insults and to endogenous oxidative stress compared with either wild-type mice or mice deficient in either MnSOD or Gpx1 alone.     

Vasodilator mechanisms in the coronary circulation of endothelial nitric oxide synthase-deficient mice

Previous studies have demonstrated that responses to endothelium-dependent vasodilators are absent in the aortas from mice deficient in expression of endothelial nitric oxide synthase (eNOS -/- mice), whereas responses in the cerebral microcirculation are preserved. We tested the hypothesis that in the absence of eNOS, other vasodilator pathways compensate to preserve endothelium-dependent relaxation in the coronary circulation. Diameters of isolated, pressurized coronary arteries from eNOS -/-, eNOS heterozygous (+/-), and wild-type mice (eNOS +/+ and C57BL/6J) were measured by video microscopy. ACh (an endothelium-dependent agonist) produced vasodilation in wild-type mice. This response was normal in eNOS +/- mice and was largely preserved in eNOS -/- mice. Responses to nitroprusside were also similar in arteries from eNOS +/+, eNOS +/-, and eNOS -/- mice. Dilation to ACh was inhibited by N(G)-nitro-L-arginine, an inhibitor of NOS in control and eNOS -/- mice. In contrast, trifluoromethylphenylimidazole, an inhibitor of neuronal NOS (nNOS), decreased ACh-induced dilation in arteries from eNOS-deficient mice but had no effect on responses in wild-type mice. Indomethacin, an inhibitor of cyclooxygenase, decreased vasodilation to ACh in eNOS-deficient, but not wild-type, mice. Thus, in the absence of eNOS, dilation of coronary arteries to ACh is preserved by other vasodilator mechanisms.     

5'-AMP-activated protein kinase activation prevents postischemic leukocyte-endothelial cell adhesive interactions

Preconditioning (PC) with nitric oxide (NO) donors or agents that increase endothelial NO synthase (eNOS) activity 24 h before ischemia-reperfusion (I/R) prevents postischemic leukocyte rolling (LR) and stationary leukocyte adhesion (LA). Since 5'-AMP-activated protein kinase (AMPK) phosphorylates eNOS at Ser1177, resulting in activation, we postulated that AMPK activation may trigger the development of a preconditioned anti-inflammatory phenotype similar to that induced by NO donors. Wild-type (WT) C57BL/6J and eNOS(-/-) mice were treated with the AMPK agonist 5-aminoimidazole-4-carboxamide 1-beta-d-furanoside (AICAR) 30 min (early AICAR PC) or 24 h (late AICAR PC) before I/R; LR and LA were quantified in single postcapillary venules in the jejunum using intravital microscopy. I/R induced comparable marked increases in LR and LA in WT and eNOS(-/-) mice relative to sham-operated (no ischemia) animals. Late AICAR PC prevented postischemic LR and LA, whereas early AICAR PC prevented LA in WT mice. Late AICAR PC was ineffective in preventing I/R-induced LR but not LA in the eNOS(-/-) mice, and the same pattern was seen in WT animals treated with the NOS inhibitor N(omega)-nitro-l-arginine. Early AICAR PC remained effective in preventing LA in eNOS(-/-) mice. Our results indicate that both early and late PC with an AMPK agonist produces an anti-inflammatory phenotype in postcapillary venules. Since the protection afforded by late AICAR PC on postischemic LR was prevented by NOS inhibition in WT mice and absent in eNOS-deficient mice, it appears that eNOS triggers this protective effect. In stark contrast, antecedent AMPK activation prevented I/R-induced LA by an eNOS-independent mechanism.     

Reduced sensitivity of inducible nitric oxide synthase-deficient mice to chronic colitis.

BACKGROUND: Overproduction of nitric oxide by the inducible form of nitric oxide synthase (iNOS) has been implicated in colitis. Different authors have postulated both toxic and protective effects of nitric oxide (NO) in the pathophysiology of active inflammation. The objective of this study was to examine the role of iNOS in experimental chronic colitis using iNOS-deficient mice. METHODS: For induction of colitis, mice received three cycles of 2% of dextran sodium sulfate (DSS) (M.W. 40,000) treatment in drinking water. The degree of colonic inflammation, leukocyte infiltration, and the expression of cell adhesion molecules were determined. INOS expression and nitrotyrosine were also determined by immunohistochemistry. RESULTS: After DSS treatment, a moderate colitis with marked cell infiltration was observed. Intense expression of iNOS was observed on infiltrating cells as well as on the colonic mucosal epithelium in these animals. In the iNOS-deficient mice, tissue damage was significantly diminished. No iNOS or nitrotyrosine staining was found in iNOS-deficient mice. The number of infiltrating cells and the expression of mucosal adressin cell adhesion molecule-1 were significantly attenuated in the DSS-treated colon of iNOS-deficient mice. CONCLUSION: Induction of iNOS seems to act as a critical toxic effector molecule in the pathogenesis of chronic colonic inflammation.     

Increased arginase activity and endothelial dysfunction in human inflammatory bowel disease

Nitric oxide (.NO) generation from conversion of l-arginine to citrulline by nitric oxide synthase isoforms plays a critical role in vascular homeostasis. Loss of .NO is linked to vascular pathophysiology and is decreased in chronically inflamed gut blood vessels in inflammatory bowel disease (IBD; Crohn's disease and ulcerative colitis). Mechanisms underlying decreased .NO production in IBD gut microvessels are not fully characterized. Loss of .NO generation may result from increased arginase (AR) activity, which enzymatically competes with nitric oxide synthase for the common substrate l-arginine. We characterized AR expression in IBD microvessels and endothelial cells and its contribution to decreased .NO production. AR expression was assessed in resected gut tissues and human intestinal microvascular endothelial cells (HIMEC). AR expression significantly increased in both ulcerative colitis and Crohn's disease microvessels and submucosal tissues compared with normal. TNF-alpha/lipopolysaccharide increased AR activity, mRNA and protein expression in HIMEC in a time-dependent fashion. RhoA/ROCK pathway, a negative regulator of .NO generation in endothelial cells, was examined. The RhoA inhibitor C3 exoenzyme and the ROCK inhibitor Y-27632 both attenuated TNF-alpha/lipopolysaccharide-induced MAPK activation and blocked AR expression in HIMEC. A significantly higher AR activity and increased RhoA activity were observed in IBD submucosal tissues surrounding microvessels compared with normal control gut tissue. Functionally, inhibition of AR activity decreased leukocyte binding to HIMEC in an adhesion assay. Loss of .NO production in IBD microvessels is linked to enhanced levels of AR in intestinal endothelial cells exposed to chronic inflammation in vivo.     

Critical role of endothelial cell-derived nitric oxide synthase in sickle cell disease-induced microvascular dysfunction

Superoxide, which can limit nitric oxide bioavailability, has been implicated in blood cell-vessel wall interactions observed in sickle cell transgenic (beta(S)) mice. Here we report that nonselective chemical inhibition of nitric oxide synthase isoforms dramatically reduces the enhanced leukocyte and platelet adhesion normally observed in cerebral venules of beta(S) mice. Although genetic deficiency of vascular wall inducible nitric oxide synthase does not alter adhesion responses in beta(S) mice, a significant attenuation is noted in beta(S) mice with vascular wall endothelial nitric oxide synthase (eNOS) deficiency, while the adhesion responses are exacerbated when eNOS is overexpressed in microvessels. The eNOS-mediated enhancement of blood cell adhesion is reversible by pretreatment with sepiapterin (which generates the eNOS cofactor tetrahydrobiopterin) or polyethyleneglycol-superoxide dismutase, implicating a role for eNOS-dependent superoxide production. These findings suggest that an imbalance between eNOS-derived nitric oxide and superoxide, both generated by the vessel wall, is critical to the proinflammatory and prothrombogenic phenotype that is assumed by the microvasculature in sickle cell disease.     

Aged endothelial nitric oxide synthase knockout mice exhibit higher mortality concomitant with impaired open-field habituation and alterations in forebrain neurotransmitter levels

Endothelial nitric oxide synthase (eNOS) has been implicated in various brain and peripheral pathologies such as renal failure, heart failure or stroke. Consequently, the mortality rate of aged eNOS knockout mice (eNOS^–/–) was higher than that of age-matched (18–22 months old) controls. Only seven of the original 14 eNOS^–/– animals that participated in the study reached the age of 18 months or older, whereas no control mice died during this life span. In order to assess the behavioral and neurochemical consequences of chronic eNOS deficiency we examined whether the surviving aged eNOS^–/– mice showed changes in terms of motor, emotional, exploratory and neurochemical parameters. Aged eNOS^–/– mice showed reduced exploratory activity in the open-field with no habituation observable neither within sessions nor after repeated exposures. Pole test performance of eNOS^–/– mice was comparable to controls. In the elevated plus-maze eNOS^–/– mice did not differ from controls in terms of time spent in and entries into arms, but showed less locomotion on the open arms. The most prominent neurochemical alterations in the forebrains of aged eNOS^–/– mice were: (a) increased acetylcholine levels in the neostriatum; (b) decreased noradrenaline concentrations in the ventral striatum; and (c) lower serotonin levels in the frontal cortex and ventral striatum. The present findings suggest that mice which survived chronic eNOS-deficiency into old age, show some behavioral and neurochemical phenotypes distinct from adult eNOS^–/– mice.     

Mechanisms of H2O2-induced oxidative stress in endothelial cells

Hydrogen peroxide, produced by inflammatory and vascular cells, induces oxidative stress that may contribute to endothelial dysfunction. In smooth muscle cells, H₂O₂ induces production of O₂⁻ by activating NADPH oxidase. However, the mechanisms whereby H₂O₂ induces oxidative stress in endothelial cells are poorly understood. We examined the effects of H₂O₂ on O₂⁻ levels on porcine aortic endothelial cells (PAEC). Treatment with 60 μmol/L H₂O₂ markedly increased intracellular O₂⁻ levels (determined by conversion of dihydroethidium to hydroxyethidium) and produced cytotoxicity (determined by propidium iodide staining) in PAEC. Overexpression of human manganese superoxide dismutase in PAEC reduced O₂⁻ levels and attenuated cytotoxicity resulting from treatment with H₂O₂. L-NAME, an inhibitor of nitric oxide synthase (NOS), and apocynin, an inhibitor of NADPH oxidase, reduced O₂⁻ levels in PAEC treated with H₂O₂, suggesting that both NOS and NADPH oxidase contribute to H₂O₂-induced O₂⁻ in PAEC. Inhibition of NADPH oxidase using apocynin and NOS rescue with L-sepiapterin together reduced O₂⁻ levels in PAEC treated with H₂O₂ to control levels. This suggests interaction-distinct NOS and NADPH oxidase pathways to superoxide. We conclude that H₂O₂ produces oxidative stress in endothelial cells by increasing intracellular O₂⁻ levels through NOS and NADPH oxidase. These findings suggest a complex interaction between H₂O₂ and oxidant-generating enzymes that may contribute to endothelial dysfunction.     

Mild hypoxia impairs alveolarization in the endothelial nitric oxide synthase-deficient mouse

In addition to its vasodilator properties, nitric oxide (NO) promotes angiogenesis in the systemic circulation and tumors. However, the role of NO in promoting normal lung vascular growth and its impact on alveolarization during development or in response to perinatal stress is unknown. We hypothesized that NO modulates lung vascular and alveolar growth and that decreased NO production impairs distal lung growth in response to mild hypoxia. Litters of 1-day-old mouse pups from parents that were heterozygous for endothelial nitric oxide synthase (eNOS) deficiency were placed in a hypobaric chamber at a simulated altitude of 12,300 ft (Fi(O(2)) = 0.16). After 10 days, the mice were killed, and lungs were fixed for morphometric and molecular analysis. Compared with wild-type controls, mean linear intercept (MLI), which is inversely proportional to alveolar surface area, was increased in the eNOS-deficient (eNOS -/-) mice [51 +/- 2 micro m (eNOS -/-) vs. 41 +/- 1 micro m (wild type); P < 0.01]. MLI was also increased in the eNOS heterozygote (+/-) mice (44 +/- 1 micro m; P < 0.03 vs. wild type). Vascular volume density was decreased in the eNOS -/- mice compared with wild-type controls (P < 0.03). Lung vascular endothelial growth factor (VEGF) protein and VEGF receptor-1 (VEGFR-1) protein content were not different between the study groups. In contrast, lung VEGFR-2 protein content was decreased from control values by 63 and 34% in the eNOS -/- and eNOS +/- mice, respectively (P < 0.03). We conclude that exposure to mild hypoxia during a critical period of lung development impairs alveolarization and reduces vessel density in the eNOS-deficient mouse. We speculate that NO preserves normal distal lung growth during hypoxic stress, perhaps through preservation of VEGFR-2 signaling.     

GPR65 (TDAG8) inhibits intestinal inflammation and colitis-associated colorectal cancer development in experimental mouse models

GPR65 (TDAG8) is a proton-sensing G protein-coupled receptor predominantly expressed in immune cells. Genome-wide association studies (GWAS) have identified GPR65 gene polymorphisms as an emerging risk factor for the development of inflammatory bowel disease (IBD). Patients with IBD have an elevated risk of developing colorectal cancer when compared to the general population. To study the role of GPR65 in intestinal inflammation and colitis-associated colorectal cancer (CAC), colitis and CAC were induced in GPR65 knockout (KO) and wild-type (WT) mice using dextran sulfate sodium (DSS) and azoxymethane (AOM)/DSS, respectively. Disease severity parameters such as fecal score, colon shortening, histopathology, and mesenteric lymph node enlargement were aggravated in GPR65 KO mice compared to WT mice treated with DSS. Elevated leukocyte infiltration and fibrosis were observed in the inflamed colon of GPR65 KO when compared to WT mice which may represent a cellular mechanism for the observed exacerbation of intestinal inflammation. In line with high expression of GPR65 in infiltrated leukocytes, GPR65 gene expression was increased in inflamed intestinal tissue samples of IBD patients compared to normal intestinal tissues. Moreover, colitis-associated colorectal cancer development was higher in GPR65 KO mice than WT mice when treated with AOM/DSS. Altogether, our data demonstrate that GPR65 suppresses intestinal inflammation and colitis-associated tumor development in murine colitis and CAC models, suggesting potentiation of GPR65 with agonists may have an anti-inflammatory therapeutic effect in IBD and reduce the risk of developing colitis-associated colorectal cancer.     

Micronucleated erythrocyte frequency in control and azidothymidine-treated Tk+/+, Tk+/- and Tk-/- mice

The first step in the activation of the anti-retroviral nucleoside analogue azidothymidine (AZT) involves its conversion to a 5′-monophosphate. In this study, we have evaluated the role of cytosolic thymidine kinase (Tk), the major enzyme involved in phosphorylating thymidine and its analogues, in the nuclear DNA damage produced by AZT in neonatal mice. Tk^+/+, Tk^+/− and Tk^−/− mice were treated intraperitoneally with 200 mg/kg/day of AZT on postnatal days 1 through 8, and micronuclei were measured in peripheral blood 24 h after the last dose. AZT treatment increased the micronucleus (MN) frequencies to similar extents in both the reticulocytes (RETs) and normochromatic erythrocytes (NCEs) of Tk^+/+ and Tk^+/− mice; AZT did not increase the frequency of micronucleated RETs (MN-RETs) or micronucleated NCEs (MN-NCEs) in Tk^−/− mice. Unexpectedly, neonatal Tk^−/− mice treated with the vehicle had significantly elevated MN frequencies for both RETs and NCEs relative to Tk^+/+ and Tk^+/− mice (e.g., 3.4% MN-RETs and 4.8% MN-NCEs in Tk^−/− mice versus 0.7 and 0.6% MN-RETs and MN-NCEs in neonatal Tk^+/+ mice). Additional assays performed on untreated Tk^−/− mice showed that elevated spontaneous MN frequencies persisted until at least 20 weeks of age, which approaches the average lifespan of Tk^−/− mice. These results indicate that metabolism by Tk is necessary for the genotoxicity of AZT in neonatal mice; however, the genotoxicity of AZT is not altered by reducing the Tk gene dose by half. The elevated spontaneous MN frequencies in Tk^−/− mice suggest the presence of an endogenous genotoxic activity in these mice.