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I Appleton

Publications and source records attributed to I Appleton.

At least 19 recordsLinked to original sources

The role of antioxidants in models of inflammation: emphasis on L-arginine and arachidonic acid metabolism.

Inflammatory processes are made up of a multitude of complex cascades. Under physiological conditions these processes aid in tissue repair. However, under pathophysiological environments, such as wound healing and hypoxia-ischaemia (HI), inflammatory mediators become imbalanced, resulting in tissue destruction. This review addresses the changes in reactive oxygen species (ROS), L-arginine and arachidonic acid metabolism in wound healing and HI and subsequent treatments with promising anti-oxidants. Even though these models may appear divergent, anti-oxidant treatments are nevertheless still having favourable effects. On the basis of recent findings, it is apparent that protection with anti-oxidants is not solely attributed to scavenging of ROS. In addition, the actions of anti-oxidants must be considered in light of the inflammatory process being assessed. To this end, there does not appear to be any universally applicable single mechanism to explain the actions of anti-oxidants.

Animals↗

Potential involvement of NOS and arginase in age-related behavioural impairments.

The present study investigated age-related changes in nitric oxide synthase (NOS) and arginase, which shares a substrate with NOS, in the hippocampus and parahippocampal region and the relationship between NOS/arginase and age-associated behavioural impairments. Aged rats (24 months old) displayed reduced exploratory activity, enhanced anxiety, poorer spatial learning and memory, and impaired object recognition memory relative to the young adults (4 months old). There were significant increases in total NOS activity in the aged hippocampus and perirhinal, postrhinal and temporal cortices and a dramatic decrease in endothelial NOS expression in the aged postrhinal cortex. Activity and protein expression of inducible NOS were not detected in any region from either group and a significant increase in total arginase activity was found in the aged perirhinal cortex. Multiple regression analysis revealed significant correlations between NOS/arginase and behavioural measures in both groups. The present findings provide further support for a contribution of nitric oxide to the normal aging process and suggest a potential involvement of arginase in aging and learning and memory.

Aging↗

Age-related changes in nitric oxide synthase and arginase in the rat prefrontal cortex.

Increasing evidence suggests that nitric oxide (NO), generated by nitric oxide synthase (NOS) from l-arginine, plays an important role in the ageing process. The present study, for the first time, investigates age-related changes in NOS and arginase, an enzyme that shares a common substrate with NOS, in the prefrontal cortex of rats assessed with and without prior behavioural testing. A significant increase in total NOS activity was found in the prefrontal cortex in aged (24-month-old) as compared with young (4-month-old) rats. Western blotting revealed that there were no significant differences between young and aged rats in neuronal NOS (nNOS) and endothelial NOS (eNOS) protein expression. Inducible isoform of NOS (iNOS), in terms of activity and protein expression, was not detected in either group. Total arginase activity and arginase I and II protein expression did not differ between the young and aged groups. The present findings support the contribution of NOS/NO to ageing but question the importance of iNOS in the normal ageing process.

Aging↗

Regional variations and age-related changes in nitric oxide synthase and arginase in the sub-regions of the hippocampus.

L-arginine can be metabolised by nitric oxide synthase (NOS) with the formation of L-citrulline and nitric oxide (NO), or arginase with the production of L-ornithine and urea. In contrast to studies showing a potential involvement of NOS/NO in the aging process, the role of arginase has not been well documented. The present study investigates for the first time the regional variations and age-related changes in both NOS and arginase in sub-regions of the hippocampus. In young adult rats, although the total NOS activity was not significantly different across the hippocampal CA1, CA2/3 and the dentate gyrus (DG) sub-regions, the total arginase activity showed a clear regional variation with the highest level in DG. Western blotting revealed that the highest levels of neuronal NOS (nNOS) and endothelial NOS (eNOS) proteins were located in CA1. Arginase I is expressed at a very low level in the brain (the whole hippocampus) as compared with the liver. By contrast, arginase II protein shows an extremely high expression in the brain with little or no expression in the liver. There was no regional variation in arginase I or arginase II protein expression across the sub-regions of the hippocampus. When a comparison was made between young (4-month-old) and aged (24-month-old) rats, a significant increase in total NOS activity was found in DG and significant decreases in arginase activity were observed in the CA1 and CA2/3 regions in the aged animals. Western blotting further revealed a dramatic decrease in eNOS protein expression in aged CA2/3 with no age-associated changes in nNOS, arginase I and II protein expression in any region examined. Interestingly, evidence of activity or protein expression of the inducible isoform of NOS (iNOS) was not detected in any tissue from either group. The present results, in conjunction with previous findings, support the contribution of NOS/NO to aging but question the involvement of iNOS in the normal aging process. Region-specific changes in arginase suggest that this enzyme may also contribute to aging.

Aging↗

Expression of cyclooxygenase isoforms in normal human skin and chronic venous ulcers.

Chronic venous ulcers are an example of abnormal wound healing showing chronic inflammation which together with the underlying vascular pathology results in delayed healing. Prostaglandins are among the most important mediators of inflammation. They have proinflammatory effects, predominantly by affecting the vasculature. Cyclooxygenase (COX) is the rate-limiting enzyme in prostanoid synthesis. It is present in two isoforms: COX-1 (constitutive cyclooxygenase) which is produced in the body to maintain normal haemostatic functions, and COX-2 (inducible cyclooxygenase), which is induced during inflammation in response to cytokines. Using immunoenzymatic labelling and western blot analysis, this study has shown that both COX-1 and COX-2 were up-regulated in chronic venous leg ulcers by comparison with normal human skin. De novo appearance of COX-2 in chronic venous ulcers was demonstrated, which is not seen in normal human skin. The main cellular sources of both COX isoforms are macrophages and endothelial cells. COX-2 is also produced by mast cells and fibroblasts. A COX radioimmunoassay showed up-regulation of COX activity in chronic venous ulcers compared with normal skin (p<0.05). Up-regulation of COX-1 in chronic venous leg ulcers could produce prostacyclin, which contributes to angiogenesis. Thus, inhibition of COX-1 by non-steroidal anti-inflammatory drugs (NSAIDs) could increase the local ischaemia and hypoxia associated with chronic venous ulcers. On the other hand, up-regulation of COX-2 is most likely responsible for the persistent inflammation in chronic venous leg ulcers. COX-2 selective inhibitors could therefore be effective in the treatment of chronic venous ulcers.

Blotting, Western↗

Damage to the vestibular inner ear causes long-term changes in neuronal nitric oxide synthase expression in the rat hippocampus.

The vestibular inner ear detects head acceleration and initiates compensatory eye movement and postural reflexes that help keep the visual image of the world stable on the retina, and maintain balance, during unexpected head movement. The most primitive vestibular systems are estimated to have evolved more than 500 million years ago and in mammalian and submammalian species the vestibular reflexes are mediated by basic brainstem pathways (see Wilson and Melvill Jones, 1979 for review). Although the contributions of the vestibular system to higher cognitive function have generally received less attention than its reflexive roles, vestibular sensory information is transmitted to higher centres in the brain and humans with vestibular damage are known to experience debilitating perceptual illusions (see Curthoys and Halmagyi, 1995; Berthoz, 1996 for reviews). Increasing behavioural and neurophysiological evidence suggests that the hippocampus uses information from the vestibular inner ear in order to build up maps of space that can be used in the development of spatial memory during learning tasks (McNaughton et al., 1991; Chapuis et al., 1992; Wiener and Berthoz, 1993; O'Mara et al., 1994; Wiener et al., 1995; Gavrilov et al., 1995; Stackman and Taube, 1996; Vitte et al., 1996; Taube et al., 1996; Save et al., 1998; Peruch et al., 1999; Cuthbert et al., 2000; Russell et al., 2000). However, to date, there has been no indication of the long-term neurochemical effects of the loss of vestibular input on hippocampal function. Since nitric oxide has been implicated in the mechanisms of hippocampal synaptic plasticity associated with the development of short-term memory (e.g. Schuman and Madison, 1991; Schuman et al., 1994; Arancio et al., 1996; Wu et al., 1997; Lu et al., 1999), we examined whether changes occur in the activity and expression of the enzymes responsible for nitric oxide production (nitric oxide synthases) in subregions of the rat hippocampus at different times following unilateral peripheral vestibular lesions, using western blotting and radioenzymatic assays. We found a decreased expression of neuronal nitric oxide synthase in the ipsilateral dentate gyrus at 2 weeks following the vestibular damage and not before, that may be related to the long-term effects of the loss of vestibular input on hippocampal function. These results support the hypothesis that head movement and position information derived from the vestibular inner ear may be important for the normal function of the hippocampus.

Afferent Pathways↗

Expression of nitric oxide synthase isoforms and arginase in normal human skin and chronic venous leg ulcers.

Chronic venous ulcers, an example of abnormal wound healing, show chronic inflammation with defective matrix deposition which together with the underlying vascular pathology, result in delayed healing. L-arginine is known to be metabolized by one of two pathways: nitric oxide synthase (NOS), producing nitric oxide (NO), or arginase, producing ornithine. NO is involved in many pathological conditions including vascular and inflammatory disorders. This study therefore investigated the distribution, level and activity of NOS and arginase in chronic venous ulcers in comparison with normal skin, using immunocytochemistry, western blotting, and enzyme assays. The results demonstrated an increased distribution of both NOS and arginase in chronic venous ulcer tissue compared with normal skin, with inflammatory cells and vascular endothelial cells as the main sources. These data were confirmed by western blot analysis, which showed increased levels of both enzymes in chronic venous ulcers. Moreover, there was significantly increased activity of both total NOS (p<0.04) and inducible NOS (p<0.05) in chronic venous ulcer tissue compared with normal skin, and significantly increased activity of arginase (p<0.01) in chronic venous ulcer tissue in comparison with normal skin. NO is known to combine with hydroxyl free radicals forming peroxynitrite, a potent free radical which causes tissue destruction. NO overexpression in chronic venous ulcers may be involved directly or indirectly (through production of peroxynitrite) in the pathogenesis and delayed healing of chronic venous ulcers, through its effects on vasculature, inflammation, and collagen deposition. Arginase is known to enhance matrix deposition. Thus, increased levels of arginase in chronic venous ulcers could contribute to the pathogenesis of lipodermatosclerosis associated with chronic venous insufficiency, predisposing to the formation of chronic venous ulcers and also to matrix cuff formation around blood vessels.

Arginase↗

The role of nitric oxide synthase isoforms and arginase in the pathogenesis of diabetic foot ulcers: possible modulatory effects by transforming growth factor beta 1.

AIMS/HYPOTHESIS: L-arginine, an amino acid involved in wound healing, is metabolised by one of two pathways; nitric oxide synthase and arginase. If metabolised by nitric oxide synthase, this can result in tissue destruction, or matrix deposition if metabolised by arginase. The aim therefore was to investigate the role of these enzymes in the pathogenesis of diabetic foot ulcers. METHODS: The activity, proteins by Western blot analysis and cellular distribution (using immunocytochemistry) of these enzymes were measured in diabetic foot ulcers, diabetic skin and normal skin. RESULTS: Total and inducible nitric oxide synthase (p < 0.001) and endothelial nitric oxide synthase were increased in diabetic ulcers compared with diabetic and normal skin and were associated with increased plasma nitrite concentrations in diabetic ulcers (p < 0.05). Inducible nitric oxide synthase was the major isoform, with the macrophage being the predominant cellular source. Similarly arginase activity was increased (p < 0.01) in diabetic ulcers. The protein levels corroborated with the activity data, with the fibroblast being the major cellular source. The spatial and cellular distribution of the two enzyme systems was distinct. Transforming growth factor-beta1 was decreased in diabetic ulcers in comparison with diabetic skin and normal skin. CONCLUSION/INTERPRETATION: Increased nitric oxide synthase activity in diabetic foot ulcers may be responsible for the impaired healing in this disease. Furthermore, the increased activity of arginase could account for the characteristic callus formation around these ulcers. In addition, the lower concentrations of transforming growth factor-betal in diabetic ulcers may explain the raised concentrations of nitric oxide in this condition.

Arginase↗

Vascular endothelial growth factor upregulates constitutive cyclooxygenase 1 in primary bovine and human endothelial cells.

The effect of the angiogenic cytokine vascular endothelial growth factor (VEGF) on nitric oxide synthase (NOS) and cyclooxygenase (COX) expression was examined in human (HUVEC) and bovine (BAE) endothelial cells. VEGF (10 ng/ml) induced constitutive COX-1 expression in both HUVEC and BAE, but not the cytokine-inducible isoform, COX-2, inducible NOS or endothelial NOS. In HUVEC, VEGF (10 ng/ml) increased COX activity, but COX inhibitors had no effect on the proliferative response of endothelial cells to this cytokine. In conclusion the induction of COX-1 by VEGF is not involved in the mitogenic response of endothelial cells, but may be an important regulatory mechanism in the maintenance of vascular integrity.

Animals↗

The role of vascular endothelial growth factor in a murine chronic granulomatous tissue air pouch model of angiogenesis.

Chronic granulomatous inflammation may be considered an angiogenic-dependent process. Recently it has been demonstrated that vascular endothelial growth factor (VEGF) or vascular permeability factor is essential for tumour angiogenesis. Its role in inflammation-mediated angiogenesis has yet to be determined. In this study, the murine chronic granulomatous air pouch model was used to investigate the role of VEGF in angiogenesis. Animals were treated twice weekly with 10 micrograms per animal of neutralizing antibody to rh VEGF and the vascularity and granuloma dry weight were assessed after 7 days. This resulted in significant suppression of both angiogenesis and granuloma dry weight. Western blot analysis demonstrated the presence of VEGF; the levels of protein paralleled the angiogenic response. These results demonstrate for the first time that VEGF may be an important regulator of angiogenesis in inflammation.

Air↗

Apoptosis, necrosis, and proliferation: possible implications in the etiology of keloids.

Keloids are collagenous lesions acquired as a result of abnormal wound heating. In this study we have assessed the potential role of proliferation, apoptosis, and necrosis in keloids. Samples were immunolabeled for proliferating cell nuclear antigen or DNA strand breaks or stained with acridine orange. Proliferating cells were observed in the basal layer of the epidermis and fibroblasts in the dermis, the numbers of the latter being increased in comparison with normal skin. No proliferating cells were observed in the central region of the keloid. In normal skin, apoptotic cells were restricted to the basal layer of the epidermis. In keloid samples, numerous apoptotic cells were observed in the epidermis and dermis; the number and distribution of positive cells decreased more distal to the keloid lesion. Apoptotic endothelial cells of a small proportion of blood vessels in the dermis were also observed. Evidence of necrosis was also seen in the dermis. These results suggest that, with maturity, progressive cell degeneration primarily by apoptosis results in clearance of certain cellular populations resulting in the typical keloid lesion. However, the persistence of fibroblast proliferation at the dermal/keloid interface propagates the fibrosis.

Acridine Orange↗

Distribution of cyclooxygenase isoforms in murine chronic granulomatous inflammation. Implications for future anti-inflammatory therapy.

Inhibition of the enzyme cyclooxygenase (COX) is the basis for the mechanism of action of non-steroidal anti-inflammatory drugs (NSAIDs). COX exists as a constitutive (COX-1) and a mitogen-inducible (COX-2) isoform. The relative contribution of COX-1 and COX-2 to inflammation is unknown. This study investigated COX activity and the distribution of COX-1 and COX-2 during the development of a murine air pouch model of chronic granulomatous inflammation. COX activity progressively rose and was maximal at day 14. Of the COX metabolites measured, PGE2 was the greatest > 6-keto PGF1a > TXB2 > PGF2a. By day 7, COX-2-labelled fibroblast- and macrophage-like cells were observed and their number and distribution increased with time. At all time points, endothelial cells of venules in the loose connective tissue of the dermis showed immunoreactivity for COX-2. After day 14, labelling of capillaries in the granuloma was also observed. This study is the first to show that COX-2 is the predominant COX isoform in all stages of the inflammatory response. These results suggest that selective inhibition of COX-2 may prove more beneficial, with fewer gastric and renal side-effects, than existing NSAID therapy for the treatment of chronic inflammatory diseases.

Animals↗

The pharmacological modulation of angiogenesis in chronic granulomatous inflammation.

Angiogenesis is required for the progression of chronic inflammation, and agents that alter it can affect the development of inflammation and the consequent tissue destruction. However, in vivo quantification of neovascularization and its modulation by angiostatic and angiogenic agents is difficult. Studies have relied on reported effects of drugs on embryonic and tumor vasculature to infer angiomodulatory actions. We have characterized a vascular casting method that incorporates carmine in gelatin. Vascularity expressed as micrograms dye/mg dry tissue (vascularity index, V.I.) was studied in the murine chronic granulomatous air pouch. Carmine was retained within the vasculature by gelatin, and its content increased before the granulomatous tissue, resulting in a V.I. peak at 5 days, regression and a second peak over 14 to 28 days. The modulation of prostaglandin synthesis, plasma exudation and vasomotor tone showed that the carmine V.I. remained unaffected, unlike Evans blue, illustrating independence from acute inflammatory processes such as vasomotor tone and plasma exudation. The angiogenic stimulus p.o. heparin increased the V.I., whereas a sub-anti-inflammatory dose of cortisone with 1000 U heparin reduced it. Higher doses of heparin overcame this. The potent angiostatic steroid tetrahydrocortisol significantly reduced the V.I. in the absence of heparin. Cortisone exhibited independence from heparin on topical administration in hyaluronan. Dexamethasone inhibited granulomatous tissue development with a resulting increase in V.I. These observations indicated the differential effects of angiostatic and anti-inflammatory steroid activity. The pharmacological modulation of angiogenesis in inflammation can therefore be quantified.

Animals↗

Inducible isoforms of cyclooxygenase and nitric-oxide synthase in inflammation.

Cyclooxygenase (COX) converts arachidonic acid to prostaglandin H2, which is further metabolized to prostanoids. Two isoforms of COX exist: a constitutive (COX-1) and an inducible (COX-2) enzyme. Nitric oxide is derived from L-arginine by isoforms of nitric-oxide synthase (NOS; EC 1.14.13.39): constitutive (cNOS; calcium-dependent) and inducible (iNOS; calcium-independent). Here we have investigated inducible isoforms of COX and NOS in the acute, chronic, and resolving stages of a murine air pouch model of granulomatous inflammation. COX and NOS activities were measured in skin samples in the acute phase, up to 24 h. Activities in granulomatous tissue were measured at 3, 5, 7, 14, and 21 days for the chronic and resolving stages of inflammation. COX-1 and COX-2 proteins were assessed by Western blot. COX activity in the skin increased over the first 24 h and continued to rise up to day 14. COX-2 protein rose progressively, also peaking at day 14. COX-1 protein remained unaltered throughout. The iNOS activity increased over the first 24 h in the skin, with a further major increase in the granulomatous tissue between days 3 and 7, followed by a decrease at day 14 and a further increase at day 21. The rise in COX and NOS activities in the skin during the acute phase reinforces the proinflammatory role for prostanoids and suggests one also for nitric oxide. However, in the chronic and resolving stages, a dissociation of COX and NOS activity occurred. Thus, there may be differential regulation of these enzymes, perhaps due to the changing pattern of cytokines during the inflammatory response.

Amino Acid Oxidoreductases↗

Cyclo-oxygenase and nitric oxide synthase isoforms in rat carrageenin-induced pleurisy.

1. The profiles of cyclo-oxygenase (COX) and nitric oxide synthase (NOS) isoforms were determined in the rat carrageenin-induced pleurisy model of acute inflammation. 2. The enzymes were assessed in peripheral blood leucocyte (PBL) cell pellets taken from untreated animals and at 2, 6 and 24 h after injection of the irritant in pleural exudate cell pellets and lung homogenates. 3. COX activity was assessed by the generation of prostacyclin (PGI2, measured as the stable metabolite, 6-keto prostaglandin F1 alpha) and prostaglandin E2 (PGE2). Western blot analysis and immunohistochemistry were also carried out. 4. NOS activity was based on the conversion of [3H]-L-arginine to [3H]-L-citrulline in the presence (total NOS activity) or absence of Ca2+ (inducible NOS; iNOS). 5. Peripheral blood leucocyte samples contained low levels of COX activity. In pleural exudate cell pellets, COX activity peaked at 2 to 6 h after injection of the carrageenin. At 24 h, COX activity was significantly reduced. 6. Western blot analysis demonstrated that the inducible isoform of COX (COX-2), was the predominant enzyme at all time points. Low levels of COX-2 were seen in PBLs. In pleural exudate cell pellets maximal COX-2 protein levels were seen at 2 h. 7. Immunohistochemistry confirmed the findings of Western blot studies. Approximately 10% of polymorphonuclear neutrophils (PMNs) in PBLs from untreated animals were immunopositive for COX-2. In cell pellet smears from carrageenin-induced pleurisy taken 2 h after injection of the irritant, PMNs were also the major source of COX-2 immunoreactivity. A small proportion of macrophages and mesothelial cells were also immunolabelled for COX-2.8. Low levels of NOS activity were seen in PBLs. In pleural exudates NOS activity was maximum at 6 h and greatly reduced by 24 h. This activity was solely attributable to iNOS.9. The present results illustrated a similar profile of COX and NOS activity in the carrageenin-induced pleurisy model of acute inflammation. It was demonstrated that COX-2 and iNOS were the predominant isoforms of their respective enzymes.

6-Ketoprostaglandin F1 alpha↗