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Biomedical subjects

A G Stewart

Publications and source records attributed to A G Stewart.

At least 19 recordsLinked to original sources
PubMed2006

Collagen impairs glucocorticoid actions in airway smooth muscle through integrin signalling.

BACKGROUND AND PURPOSE: Airway wall remodelling in asthma is characterised by a number of structural changes, including an increase in the volume of airway smooth muscle (ASM), and the abundance of the extracellular matrix (ECM) protein, collagen, is increased. We have investigated the mechanism of collagen-induced glucocorticoid resistance of proliferation, and migration of ASM. EXPERIMENTAL APPROACH: ASM cultured from human airways has been seeded on to either type I monomeric collagen or a laminin pentapeptide, YIGSR. The role of alpha2beta1 integrin in the collagen-induced glucocorticoid resistance was investigated using a function blocking monoclonal antibody. KEY RESULTS: Culture of ASM on collagen I, but not laminin, led to a greater proliferative response that was insensitive to regulation by dexamethasone (100 nM). The anti-migratory effects of the glucocorticoid, fluticasone propionate (1 nM) were also impaired by contact of ASM with collagen. The impaired anti-mitogenic action of dexamethasone was associated with a failure to reduce the levels of the rate-limiting cell cycle regulatory protein, cyclin D1. When signalling through the alpha2beta1 integrin was reduced, dexamethasone-mediated reductions in proliferation and cyclin D1 levels were restored. CONCLUSIONS AND IMPLICATIONS: In the collagen-rich microenvironment of the inflamed and fibrotic asthmatic airway, integrin/ECM interactions may contribute to glucocorticoid resistance.

Androstadienes↗
PubMed2005

The PPARgamma ligand, rosiglitazone, reduces airways hyperresponsiveness in a murine model of allergen-induced inflammation.

There is considerable interest in the role of peroxisome proliferator activated receptors (PPARs) as ligand-activated transcription factors in the airways. This study examines the effects of a potent synthetic PPARgamma ligand, rosiglitazone (RG), in a murine model of allergen-induced inflammation, to explore its potential regulation of airways inflammation, structure and function. C57BL/6 mice were sensitised with ovalbumin (OVA, 50 microg i.p., days 0, 12) and challenged with aerosolized OVA (1% w v(-1), 30 min day(-1)) for 7 days (days 20-26). Mice were treated with RG (5 mg kg(-1) i.p.) or vehicle during the challenge period. The OVA challenge induced increases in leukocyte number and MMP-2 activity in bronchoalveolar lavage fluid and in goblet cell number in lung tissue obtained on Day 27. RG failed to inhibit inflammatory cell infiltration, MMP-2 activity or goblet cell hyperplasia. Respiratory resistance in response to methacholine (MCh i.v.) was greater in OVA-challenged mice than saline-challenged mice and this airways hyperresponsiveness (AHR) was reduced by RG. However, RG did not affect MCh-induced contraction in isolated guinea-pig tracheal rings, nor did it influence the airway obstruction induced by MCh in saline-challenged mice, so a direct effect on airway obstruction is unlikely. These data suggest that RG modulates AHR in this model, by a mechanism that is also potentially independent of an anti-inflammatory action.

Allergens↗
PubMed2005

Osteomalacia in Hazara District, Pakistan.

Osteomalacia is most commonly seen in the remoter northern regions (Kohistan District) of Hazara District, Pakistan. Low serum calcium is common, as is tetany, but not universal. A 2% prevalence was found retrospectively in all obstetric patients from 1978--1985. Overall, there was a 12% caesarean section rate (61/annum), of which 37% (22) exhibited cranio-pelvic disproportion, nearly half of which (n=83, 46%) were thought clinically to be due to osteomalacia. Osteomalacia was found prospectively in 3.6% of all female outpatients (3600/100,000). Purdah did not appear to influence the incidence of osteomalacia, although sunlight exposure varied significantly due to place of abode (0.05 > P> 0.025); those living in the deeper, darker valleys suffered more from osteomalacia and its side effects, such as cranio-pelvic disproportion and the resulting need for caesarean sections. Diet is an important factor, showing little variety in the affected region; it lacks animal protein and is low in calories.The estimated intake of vitamin D is approximately 1 microg per day, seriously short of the daily requirement of 2.5 microg. The other main factor is higher parity in the women with osteomalacia (15/18 affected women had more than three pregnancies compared with 9/18 controls; odds ratio 13, 0.05 > P> 0.025). These all indicate that in a marginal situation added metabolic stress can precipitate the condition. While supplementation of the diet is essential in such communities it will be difficult to initiate and maintain. We therefore also recommend that strategies for prevention be focused on the men to encourage them to help improve the diet and lifestyle of their womenfolk.

Adult↗
PubMed2003

The illusion of environmental iodine deficiency.

The distribution of endemic goitre in England and Wales was compared with the distribution of environmental iodine (atmospheric deposition, soil, surface water). Despite a very clear goitre belt through the west of England and Wales there was no patterning in the environmental iodine distribution. A clear seasonal variation in depositional iodine exists, with an unusually high concentration of iodine in March 1997. The temporal variation in iodine concentration is determined at the monthly and not the annual level. The presence of endemic goitre is no indicator of how iodine is distributed in the environment or vice versa!

England↗
PubMed2002

Antigen-induced airway inflammation in the Brown Norway rat results in airway smooth muscle hyperplasia.

Asthma is characterized by chronic airways inflammation, airway wall remodeling, and airway hyperresponsiveness (AHR). An increase in airway smooth muscle has been proposed to explain a major part of AHR in asthma. We have used unbiased stereological methods to determine whether airway smooth muscle hyperplasia and AHR occurred in sensitized, antigen-challenged Brown Norway (BN) rats. Ovalbumin (OA)-sensitized BN rats chronically exposed to OA aerosol displayed airway inflammation and a modest level of AHR to intravenously administered ACh 24 h after the last antigen challenge. However, these animals did not show an increase in smooth muscle cell (SMC) number in the left main bronchus, suggesting that short-lived inflammatory mechanisms caused the acute AHR. In contrast, 7 days after the last aerosol challenge, there was a modest increase in SMC number, but no AHR to ACh. Addition of FCS to the chronic OA challenge protocol had no effect on the degree of inflammation but resulted in a marked increase in both SMC number and a persistent (7-day) AHR. These results raise the possibility that increases in airway SMC number rather than, or in addition to, chronic inflammation contribute to the persistent AHR detected in this model.

Acetylcholine↗
PubMed2001

Effects of the endothelin receptor antagonist Bosentan on ischaemia/reperfusion injury in rat skeletal muscle.

We examined the role of endothelin in ischaemia/reperfusion injury in skeletal muscle, using the endothelin receptor antagonist Bosentan. In the rat hindlimb tourniquet ischaemia model, one hindlimb was rendered ischaemic for 2 h at 36 degrees C, then blood flow was re-established for either 24 h to assess muscle survival or 1.5 h for a study of capillary perfusion. In the first set of rats, the gastrocnemius muscle was removed from the postischaemic limb and assessed for viability histochemically using the nitro blue tetrazolium stain. Tissue water content (a measure of oedema) and myeloperoxidase activity (a measure of neutrophil accumulation) were also assessed in the ischaemic muscle, the contralateral non-ischaemic muscle and the lungs. In the second set of rats, the hind limb was infused with India ink after 2-h ischaemia and 1.5-h reperfusion and the muscle was harvested, fixed and cleared. In control rats, muscle viability was 17+/-2% (S.E.M.). In rats treated with Bosentan (10 mg/kg, i.p.) 30 min before release of the tourniquet, muscle viability (48+/-7%) was significantly increased compared to the control group (P<0.01). Bosentan treatment had no significant effect on tissue water content or myeloperoxidase activity in the ischaemic muscle, the contralateral non-ischaemic muscle or the lung. Immunoreactive endothelin levels in serum increased to a peak at 90 min of reperfusion and returned to control levels by 24-h reperfusion. India ink studies demonstrated a significantly increased functional capillary density in postischaemic Bosentan-treated muscles compared with postischaemic control muscles (P<0.05). These results suggest that endothelin plays an important role in the necrosis which results from a period of ischaemia and reperfusion in skeletal muscle, by mediating a decrease in postischaemic microvascular perfusion.

Animals↗
PubMed2001

Lipopolysaccharide-induced cell cycle arrest in macrophages occurs independently of nitric oxide synthase II induction.

Lipopolysaccharide (LPS, a Gram-negative bacterium cell wall component) is a potent macrophage activator that inhibits macrophage proliferation and stimulates production of nitric oxide (NO) via NO synthase II (NOSII). We investigated whether NO mediates the LPS-stimulated cell cycle arrest in mouse bone marrow-derived macrophages (BMM). The addition of the NO donor DETA NONOate (200 microM) inhibited BMM proliferation by approx. 80%. However, despite NO being an antimitogen, LPS was as potent at inhibiting proliferation in BMM derived from NOSII-/- mice as from wild-type mice. Consistent with these findings, LPS-induced cell cycle arrest in normal BMM was not reversed by the addition of the NOSII inhibitor S-methylisothiourea. Moreover, in both normal and NOSII-/- BMM, LPS inhibited the expression of cyclin D1, a protein that is essential for proliferation in many cell types. Despite inhibiting proliferation DETA NONOate had no effect on cyclin D1 expression. Our data indicate that while both LPS and NO inhibit BMM proliferation, LPS inhibition of BMM proliferation can occur independently of NOSII induction.

Animals↗
PubMed2001

Nitric oxide synthase II gene disruption: implications for tumor growth and vascular endothelial growth factor production.

The expression of a primary initiator of tumor angiogenic responses, vascular endothelial growth factor (VEGF), may be induced by nitric oxide (NO) in carcinoma cells. However, the net impact of NO on carcinogenesis remains unclear, because manipulation of NO levels has been shown to either stimulate or inhibit tumor growth. We have investigated the relationship between inducible NO synthase (NOS II), VEGF expression, and growth of B16-F1 melanoma over 14 days in wild-type (NOS II+/+) mice and in those in which the gene for NOS II has been deleted (NOS II-/-). B16-F1 tumor growth was measured as wet weight of the excised tissue. Tumor NOS II and VEGF localization were evaluated by immunohistochemistry, and VEGF mRNA levels were measured by Northern blot analysis. In NOS II+/+ mice inoculated with B16-F1 melanoma cells, macroscopic tumors were always observed at 14 days; however, 22% of NOS II-/- mice had no detectable tumor mass. Immunoreactive NOS II was detected in tumor cells of tumors grown in NOS II+/+ but not in NOS II-/- mice. Although immunoreactive VEGF was detected in the granules of tumor-associated mast cells from both NOS II+/+ and NOS II-/- mice, VEGF mRNA expression in tumors from NOS II-/- was half that in NOS II+/+ mice. Neither NOS II inhibition, exogenous NO, nor peroxynitrite influenced DNA synthesis in culture B16-F1 melanoma cells. The NO donor did not alter either VEGF mRNA levels or degranulation in cultures of the mast cell line RBL-2H3, but peroxynitrite increased both VEGF mRNA expression and degranulation. We conclude that host expression of NOS II contributes to induction of NOS II in the tumor and to melanoma growth in vivo, possibly by regulating the amount and availability of VEGF.

Animals↗
PubMed2001

Targeted disruption of the nitric oxide synthase 2 gene protects against ischaemia/reperfusion injury to skeletal muscle.

To provide definitive insight into the complicated roles of the nitric oxide synthase (NOS) enzymes in ischaemia/reperfusion (I/R) injury of skeletal muscle, experiments were undertaken in mice with targeted disruption of the inducible NOS (NOS-2 KO) isoform, compared with the wild-type mouse strain. The degree of I/R injury in the NOS-2 KO mice was attenuated relative to that in the wild-type strain. After 70 min of ischaemia (24 h reperfusion), nitroblue tetrazolium (NBT) staining of skeletal muscle showed significant necrosis (40%) in wild-type mice, whilst in NOS-2 KO mice, ischaemia could be prolonged to 90 min before significant necrosis (38%) was apparent. Specific enzyme activities of the mitochondrial respiratory chain enzymes, measured in skeletal muscle homogenates, suggested that direct inhibition of the enzymes is not causal in the I/R injury. Immunohistological examination of skeletal muscle for NOS-2 showed its induction selectively in mast cells. In vitro experiments using bone marrow-derived mast cells showed that NOS-2 induction was associated with increased degranulation of mast cells. These findings suggest that NO generated by induction of NOS-2 has a deleterious effect in I/R injury of skeletal muscle and that NO exerts its damaging effect through factors released by degranulation of mast cells.

Animals↗
PubMed2001

Airway wall remodelling and hyperresponsiveness: modelling remodelling in vitro and in vivo.

Airway wall remodelling contributes to the airway hyperresponsiveness that characterizes asthma. An increase in airway smooth muscle (ASM) volume is quantitatively important in the overall remodelling response and may be considered as a target for new therapeutic approaches to chronic asthma. ASM volume increases result from both hypertrophic as well as hyperplastic growth, the latter having been more extensively investigated. There are relatively few in vivo models available for analysis of the underlying mechanism(s) or their regulation by drugs. Human ASM in culture has been used to investigate potential stimuli for ASM proliferation and the signal transduction pathways that subserve these responses. The mitogen-activated protein kinase (MAPK) family members, ERK 1/2 and the phosphoinositol-3-kinase (PI3K) pathways each contribute to the signalling of G1 progression/S-phase entry in ASM. Glucocorticoids and beta(2)-adrenoceptor agonists attenuate, but do not prevent proliferative responses of ASM. Thus, there is scope for improved pharmacological control of this chronic and progressive aspect of asthma pathogenesis.

Adrenergic beta-Agonists↗
PubMed2001

Inducible nitric oxide synthase (iNOS) activity promotes ischaemic skin flap survival.

We have examined the role of nitric oxide (NO) in a model of functional angiogenesis in which survival of a skin flap depends entirely on angiogenesis to provide an arterial blood supply to maintain tissue viability. The different effects of nitric oxide synthase (NOS) inhibitors on rat skin flap survival appeared to be explained on the basis of their NOS isoform selectivity. Skin flap survival was decreased by iNOS-selective (inducible NOS) inhibitors, S-methyl-isothiourea, aminoguanidine and aminoethylthiorea; unaffected by the non-selective inhibitor nitro-imino-L-ornithine; and enhanced by the cNOS (constitutive NOS, that is endothelial NOS (eNOS) and neuronal NOS (nNOS)) inhibitor, nitro-L-arginine methyl ester. Skin flap survival was reduced in mice with targeted disruption of the iNOS gene (iNOS knockout mice), and the administration of nitro-L-arginine methyl ester significantly increased flap survival in iNOS knockout mice (P<0.05). iNOS immunoreactivity was identified in mast cells in the angiogenic region. Immunoreactive vascular endothelial growth factor (VEGF) and basic fibroblast growth factor were also localized to mast cells. The combination of interferon-gamma and tumour necrosis factor-alpha induced NO production and increased VEGF levels in mast cells cultured from bone marrow of wild-type, but not iNOS KO mice. The increased tissue survival associated with the capacity for iNOS expression may be related to iNOS-dependent enhancement of VEGF levels and an ensuing angiogenic response. Our results provide both pharmacological and genetic evidence that iNOS activity promotes survival of ischaemic tissue.

Animals↗
PubMed2001

Anti-remodelling drugs for the treatment of asthma: requirement for animal models of airway wall remodelling.

1. Airway wall remodelling (AWR), the structural change induced by acute and chronic inflammation in the airways, may be one of the most significant and difficult to reverse components of progressive asthma. 2. The mechanisms underlying the development of AWR are not known. Studies of only the most superficial wall structures of large airways can be conducted in living humans because of the degree of invasiveness required to measure airway structural changes. These studies reveal that currently available agents do not fully prevent or reverse AWR. Thus, animal models of asthma pathology may be used to assess the contribution of particular mediators and cells to the development of remodelling and may also prove to be useful in the initial screening of potential anti-remodelling agents. 3. Airway hyperresponsiveness and AWR stimulated by chronic antigen challenge in previously disease-free animals is the most popular of the currently used models of remodelling. Other animal models include the use of specially bred strains with intrinsic airway hyperresponsiveness or animals that have a naturally occurring asthma-like disease, such as cats with feline asthma or horses with heaves. The further development of animal models of AWR will facilitate the development of novel anti-asthma therapies.

Airway Obstruction↗
PubMed2000

Prior heat stress improves survival of ischemic-reperfused skeletal muscle in vivo.

The ability of heat stress to improve the survival of ischemic-reperfused skeletal muscle in vivo was investigated. Ischemia-reperfusion was applied using the rat hindlimb tourniquet model. The viability of ischemic-reperfused muscle (11 +/- 1%) was increased by prior mild heat stress (86 +/- 2%). To investigate whether heat shock protein 70 (Hsp 70) expression in the muscle of the heated limb was responsible for this protection, the survival of Hsp 70-expressing transduced myoblasts and myocytes was measured after exposure to mediators of ischemia-reperfusion injury. Survival was improved in Hsp 70-positive myoblasts but not in myocytes, suggesting that the mechanism of protection conferred by heat stress in vivo cannot be explained by the expression of Hsp 70 in myocytes and may involve a more complex mechanism. In conclusion, prior heat stress is effective in protecting mature skeletal muscle in vivo against necrosis after ischemia-reperfusion and has potential for use in microsurgical procedures requiring tourniquet applications.

Animals↗
PubMed2000

Thrombin-stimulated DNA synthesis in human cultured airway smooth muscle occurs independently of products of cyclo-oxygenase or 5-lipoxygenase.

Arachidonic acid (AA) liberation and metabolism via cyclo-oxygenase or lipoxygenases may be an important regulatory pathway for mitogenic signalling in human cultured airway smooth muscle (ASM) cells. In cytokine-treated cells, thrombin markedly enhances production of the anti-mitogenic arachidonic acid metabolite, PGE(2). In this study, in the absence of cytokines, we examined the role of endogenous AA metabolism in thrombin-stimulated ASM DNA synthesis. Selective inhibitors of cyclo-oxygenase of 5-lipoxygenase metabolism had no significant effect on 0.3 U/ml thrombin-stimulated DNA synthesis. However, the non-selective, redox-active lipoxygenase inhibitors NDGA and BWA4C inhibited thrombin-stimulated DNA synthesis. Under basal conditions, and following stimulation by thrombin, the levels of the AA metabolites PGE(2), TxA(2), and LTC(4), remained below assay detection limits. Exogenous addition of AA, LTD(4), or 5-, 12-, and 15-HETE and HpETE metabolites had no consistent or substantial stimulatory effect on either basal or thrombin-stimulated DNA synthesis. These data suggest that the non-selective lipoxygenase inhibitors influence DNA synthesis via effects unrelated to lipoxygenase inhibition. The lack of detection of AA metabolites, the lack of influence of selective antagonists/inhibitors of the AA pathway, and the failure of selected AA metabolites to either enhance or directly stimulate DNA synthesis suggest that in the absence of cytokines, cyclo-oxygenase and lipoxygenase metabolism has little role in signalling of human ASM DNA synthesis by thrombin.

Arachidonate 5-Lipoxygenase↗
PubMed2000

Localization of inducible nitric oxide synthase to mast cells during ischemia/reperfusion injury of skeletal muscle.

Nitric oxide contributes to tissue necrosis after ischemia-reperfusion (IR). A biochemical and immunohistochemical study was made of the amounts and localization of both Ca++-independent nitric oxide synthase (NOS) II and Ca++-dependent (NOS I and NOS III) in rat skeletal muscle after ischemia and 0.5, 2, 8, 16, and 24 hours reperfusion. NOS II was not detectable in control muscle or during ischemia, was first detected after 2 hours reperfusion, increased further by 8 hours, and remained elevated at 24 hours. Both NOS II and nitrotyrosine, a marker of peroxynitrite formation, were localized exclusively to mast cells except after 24 hours reperfusion when some macrophages and neutrophils also showed positive immunoreactivity. Mast cells underwent extensive degranulation during reperfusion. NOS I was not detected in injured or control muscle. The level of NOS III, which was localized to the endothelium of blood vessels of all sizes in control muscle, decreased progressively during ischemia and reperfusion to reach undetectable levels after 16 hours reperfusion. These findings indicate that most of the nitric oxide formed during IR injury is generated by NOS II located almost exclusively in mast cells.

Animals↗
PubMed2000

Neutrophil-independent protective effect of r-metHuG-CSF in ischaemia-reperfusion injury in rat skeletal muscle.

The aim of this study was to investigate the effect of the cytokine, r-metHuG-CSF, in a rat model of ischaemia-reperfusion (IR) injury and the pathophysiological mechanism involved. The administration of r-metHuG-CSF (20 (g/kg, s.c.) 4 h prior to either 100 min or 2 h of tourniquet ischaemia to the upper thigh significantly improved the viability of skeletal muscle after 24 h reperfusion compared with saline-treated rats (P < 0.05). Administration of r-metHuG-CSF earlier (24 h before ischaemia) or later (immediately before ischaemia) had no protective effect. At the dose used, r-metHuG-CSF caused a three-fold increase in the level of circulating blood neutrophils and a modest but significant increase in the neutrophil content of ischaemic muscle after 24 h reperfusion. Reduction of neutrophils to 1.4% of normal levels by cyclophosphamide (150 mg/kg, i.p.) prior to injury had no significant effect on the survival of muscle subjected to 2 h ischaemia and 24 h reperfusion or on the protective effect of r-metHuG-CSF. IR injury to skeletal muscle was accompanied by a time-dependent increase in plasma TNFalpha levels during the first 8 h of reperfusion and the increase was reduced significantly by pretreatment with r-metHuG-CSF. However, a similar time-dependent increase in plasma nitrite/nitrate levels was unaffected by pretreatment with r-metHuG-CSF. These findings suggest that the protective effect of r-metHuG-CSF may be mediated by the attenuated release of TNFalpha and indicate that the level of neutrophils in either blood or injured tissue does not influence significantly the viability of rat muscle after IR injury.

Animals↗
PubMed1999

Nitric oxide synthase-independent generation of nitric oxide in rat skeletal muscle ischemia-reperfusion injury.

We have used electron paramagnetic resonance to investigate the time course of nitric oxide (NO) generation and its susceptibility to inhibitors of nitric oxide synthase (NOS) in ischemia-reperfusion (IR) injury to rat skeletal muscle in vivo. Significant levels of muscle nitroso-heme complexes were detected 24 h postreperfusion, but not after at 0.05, 3, and 8 h of reperfusion. The levels of muscle nitroso-heme complexes were not decreased by the NOS inhibitor N-nitro-L-arginine methyl ester as a single dose (30 mg/kg) prior to reperfusion or as multiple doses continued throughout the reperfusion (total administered, 120 mg/kg) or by the potent NOS inhibitor S-methylisothiourea (3 mg/kg). In contrast, nitroso-heme levels were reduced by the glucocorticoid dexamethasone (2.5 mg/kg). Muscle necrosis in vitro did not result in the formation of nitroso-heme complexes. The finding that reperfusion after ischemia is necessary for NO formation suggests that an inflammatory pathway is responsible for NOS-independent NO formation in IR injury to skeletal muscle.

Animals↗