Biomedical subjects

J A Mitchell

Publications and source records attributed to J A Mitchell.

At least 37 records · Page 2Linked to original sources

PubMed2001

Effects of prostaglandin E2 and cAMP elevating drugs on GM-CSF release by cultured human airway smooth muscle cells. Relevance to asthma therapy.

Human airway smooth muscle (HASM) cells release granulocyte macrophage-colony stimulating factor (GM-CSF) and express cyclooxygenase (COX)-2 (resulting in the release of prostaglandin [PG] E2) after stimulation with cytokines. Because COX-2 activity can regulate a number of inflammatory processes, we have assessed its effects, as well as those of agents that modulate cyclic adenosine monophosphate (cAMP), on GM-CSF release by HASM cells. Cells stimulated with a combination of proinflammatory cytokines (interleukin-1beta and tumor necrosis factor-alpha each at 10 ng/ml) for 24 h released significant amounts of PGE2 (measured by radioimmunoassay) and GM-CSF (measured by enzyme-linked immunosorbent assay). Indomethacin and other COX-1/COX-2 inhibitors caused concentration-dependent inhibitions of PGE2 concomitantly with increases in GM-CSF formation. Addition of exogenous PGE2 or the beta2-agonist fenoterol, which increase cAMP, to cytokine-treated HASM cells had no effect on GM-CSF release unless COX activity was first blocked with indomethacin. The type 4 phosphodiesterase inhibitors rolipram and SB 207499 both caused concentration-dependent reductions in GM-CSF production. Thus, when HASM cells are activated with cytokines they release PGE2, which acts as a "braking mechanism" to limit the coproduction of GM-CSF. Moreover, agents that elevate cAMP also reduce GM-CSF formation by these cells.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2001

Endogenously released endothelin-1 from human pulmonary artery smooth muscle promotes cellular proliferation: relevance to pathogenesis of pulmonary hypertension and vascular remodeling.

Endothelin (ET)-1 is a potent vasoconstrictor and comitogen/ proliferation factor for vascular smooth muscle (VSM). As such, it has been implicated in the vascular wall remodeling observed in pulmonary hypertension (PH). Although the endothelium is considered the main source of ET-1, it can be released by other cells including VSM and may mediate proliferation in an autocrine manner. We investigated this possibility using human pulmonary artery smooth-muscle (HPASM) cells. Serum stimulated the release of ET-1 from HPASM cells in a concentration-dependent fashion and caused proliferation as determined by [(3)H]thymidine uptake and increase in cell number. Addition of an ET-A receptor antagonist (BQ123) or an inhibitor of ET-1 synthesis (phosphoramidon) reduced the proliferation induced by serum, confirming an autocrine role for ET-1. In addition, treatment of HPASM cells with two drug types used in the management of PH-cicaprost, a stable prostacyclin-mimetic; or diltiazem, a calcium-channel blocker-reduced ET-1 release from these cells. We conclude that ET-1 released from HPASM cells has an autocrine function in serum-induced proliferation, with important implications for the pathogenesis of human vascular remodeling. Drugs used in the treatment of PH may act, at least in part, by inhibiting this autocrine loop.

Base Sequence↗

PubMed2001

Parathyroid hormone-induced up-regulation of connexin-43 messenger ribonucleic acid (mRNA) is mediated by sequences within both the promoter and the 3'untranslated region of the mRNA.

The gap junction protein connexin 43 (Cx43) mediates communication between osteoblasts and is important for maximal PTH responsiveness. We examined the role of the Cx43 promoter and messenger RNA 3' untranslated region (UTR) in conferring responsiveness to PTH in the rat osteosarcoma cell line UMR-106. PTH induced a 4-fold increase in luciferase activity of a reporter construct containing 1.6 kb 5' of the transcription start site. Deletion analysis of the promoter localized responsive sequences to between -31 to +1 bp. PTH treatment of transgenic mice containing the 1.6 kb promoter luciferase construct induced increases in luciferase and Cx43 immunoreactivity in bone cells underlying the tibial growth plate. The full Cx43 3'UTR conferred a 3-fold response to PTH when placed 3' of a CMV-luciferase construct. Deletion analysis localized responsive sequences to between 2510 and 3132 of the 3'UTR. Cloning of this segment 5' of the CMV promoter disrupted the PTH response, indicating this sequence does not function as an enhancer. Sequences within the promoter conferred responsiveness to forskolin, whereas the 3'UTR responded to both TPA and forskolin. These data indicate that PTH responsive sequences are present in the Cx43 promoter and 3'UTR, suggesting that transcriptional and posttranscriptional pathways operate to regulate PTH-induced Cx43 expression in osteoblast cells.

3' Untranslated Regions↗

PubMed2001

Regulation of connexin43 expression by c-fos and c-jun in myometrial cells.

Labor is associated with a dramatic increase in the myometrial expression of connexin43 (Cx43) which is thought to mediate myocyte contractile coupling. The transcription factor c-fos is also dramatically increased prior to the onset of labor and is therefore a potential regulator of Cx43. The promoter region of Cx43 contains a conserved activator protein-1 (AP-1) site, which binds dimers of Fos and Jun proteins. We constructed expression vectors for c-Fos and c-Jun to investigate the role of these transcription factors in the regulation of Cx43 expression. These expression vectors were then co-transfected into SHM (syrian hamster myocyte) cells with a Cx43 promoter-Luciferase vector. The combinations of c-Fos and c-Jun proteins activated the Cx43 promoter while c-Jun alone had no effect on Cx43 promoter activity. Mutation of the AP-1 site was found to reduce this responsiveness. These data indicate that the transcription factors c-Fos and c-Jun are important in the regulation of Cx43 expression.

Animals↗

PubMed2001

Sodium salicylate inhibits prostaglandin formation without affecting the induction of cyclooxygenase-2 by bacterial lipopolysaccharide in vivo.

The mechanisms underlying the anti-inflammatory properties of salicylate are not well understood. In particular, while salicylate inhibits prostaglandin production in vivo it only weakly inhibits cyclooxygenase (COX)-1 or -2 activity in vitro. Thus, it has often been suggested that in vivo salicylate may inhibit the expression rather than the activity of COX, particularly COX-2. Using a model of acute COX-2 expression in the rat, we show that salicylate inhibits COX-2 activity in vivo without affecting COX-2 expression. In anesthetized rats LPS (6 mg kg(-1), i.p.) increased the expression of COX-2 as evidenced by increased circulating levels of 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha), a stable breakdown product of PGI(2)), greatly exaggerated formation of 6-keto-PGF(1alpha) following arachidonic acid (AA) challenge (3 mg kg(-1), i.v.), and increased expression of COX-2, but not COX-1, protein. Diclofenac (3 mg kg(-1), i.p.) or the COX-2 selective agent diisopropyl fluorophosphate (10 mg kg(-1), i.p.) decreased the LPS-induced increase in circulating 6-keto-PGF(1alpha) and the exaggerated 6-keto-PGF(1alpha) production following AA challenge. Sodium salicylate (20 or 120 mg kg(-1), i.p.) (administered either 1 h prior, or once per day for 3 days prior, to LPS injection) reduced only the LPS-induced increase in circulating 6-keto-PGF(1alpha), but not the exaggerated 6-keto-PGF(1alpha) production following AA challenge or the expression of COX-2. Thus, salicylate inhibits LPS-induced COX-2 activity in a manner that is overcome by provision of excess substrate and independent of effects on COX-2 expression. In conclusion, our results exclude mechanisms other than direct enzyme inhibition as responsible for the anti-COX effects of salicylate.

6-Ketoprostaglandin F1 alpha↗

PubMed2000

Effects of nitric oxide-modulating amino acids on coronary vessels: relevance to sepsis.

Excessive nitric oxide (NO) production in septic shock is thought to contribute to the associated profound hypotension. Here we show that despite induction of NO synthase (NOS) in the hearts of endotoxin-treated rats, coronary vascular responses to the contractile peptide endothelin-1, were not modified. This was not due to any change in the expression of endothelin receptors. However, when the substrate for NOS, L-arginine, was added to the perfusate, increases in coronary perfusion pressure stimulated by endothelin were reduced in hearts from endotoxin-treated animals compared to those from controls. In addition, L-glutamine, which blocks the generation of L-arginine from intracellular stores, enhanced the increase in perfusion pressure stimulated by endothelin-1. These data suggest that L-arginine becomes rate limiting for the production of NO in the coronary vessels during septic shock. Moreover, it suggests that vascular reactivity may be modulated positively or negatively by supplementation with the relevant amino acids.

Animals↗

PubMed2000

The pathogenesis of lung injury following pulmonary resection.

Postpneumonectomy pulmonary oedema (PPO) develops in approximately 5% of patients undergoing pneumonectomy or lobectomy, and has a high associated mortality (>50%). In its extreme form, PPO follows a clinical and histopathological course indistinguishable from acute respiratory distress syndrome. Perioperative fluid overload, impaired lymphatic drainage following node dissection and trauma caused by surgical manipulation have been implicated in the pathogenesis of PPO. However, PPO more probably represents the pulmonary manifestation of a panendothelial injury consequent upon inflammatory processes induced by the surgical procedure, which involves collapse and re-expansion of the operative lung to permit hilar dissection and pulmonary resection. High inspired oxygen concentrations are required to overcome the effects of shunt. Animal studies have shown that pulmonary ischaemia/reperfusion can result in oedema formation, possibly due to the generation of pro-oxidant forces. Moreover, plasma taken from patients undergoing lobectomy or pneumonectomy (but not lesser resections) shows evidence of oxidative damage. Such evidence suggests either that the high inspired oxygen concentrations associated with one-lung ventilation, or ischaemia/reperfusion injury, may modulate post-pneumonectomy pulmonary oedema. Mechanisms by which redox imbalance may result in tissue damage and postpneumonectomy pulmonary oedema are discussed.

Female↗

PubMed2000

Release of GM-CSF and G-CSF by human arterial and venous smooth muscle cells: differential regulation by COX-2.

In addition to their traditional contractile function, vascular smooth muscle cells can be stimulated under inflammatory conditions to release a range of potent biological mediators. Indeed, we and others have shown that human vascular smooth muscle release the colony stimulating factors (CSF) granulocyte macrophage-CSF (GM-CSF) and granulocyte-CSF (G-CSF) as well as large amounts of prostaglandins following the induction of cyclo-oxygenase-2 (COX-2), when stimulated with cytokines. Here we demonstrate, for the first time, that co-induced COX-2 activity simultaneously suppresses GM-CSF release and potentiates G-CSF release by human vascular cells. Moreover, the differential regulation of GM-CSF and G-CSF release by COX-2 was mimicked by the prostacyclin (PGI(2)) mimetic, cicaprost. These observations suggest that PGI(2), released following the induction of COX-2, differentially regulates the release of GM-CSF (suppresses) and G-CSF (potentiates) from human vascular cells.

Arteries↗

PubMed2000

A brief review of the effects of chronic hydrocephalus on the gonadotropin releasing hormone system: implications for amenorrhea and precocious puberty.

Precocious puberty and amenorrhea have been associated with hydrocephalus, but the pathogenesis has not been determined. Approximately 22 cases of amenorrhea, and a few cases of precocious puberty, have been reported in hydrocephalic patients. Shunt treatment leads to initiation and maintenance of normal reproductive cycles in most cases. An underlying mechanism responsible for reproductive dysfunction may involve the role of gonadotrophin releasing hormone (GnRH). The exact pathway by which hydrocephalus disrupts the hypothalamic GnRH system is unknown. However, compressive forces, ischemia, and impairment of neurotransmitter feedback loops are likely candidates.

Amenorrhea↗

PubMed2000

Cyclic AMP regulates cytokine stimulation of endothelin-1 release in human vascular smooth muscle cells.

Exposure of human vascular smooth muscle cells (HVSMCs) to cytokines markedly elevates endothelin-1 (ET-1) mRNA expression and peptide production. Cyclic AMP is a key regulator of many physiological processes. The aim of this study was to determine whether an elevation of intracellular cAMP may affect cytokine-induced production of ET-1 in HVSMCs. Cicaprost, a prostacyclin analogue, forskolin, an activator of adenylate cyclase, and Ro-20-1724, a phosphodiesterase type IV inhibitor, inhibited cytokine-stimulated ET-1 release in a concentration-dependent manner. Ro-20-1724 also inhibited cytokine-induced upregulation of preproendothelin-1 mRNA expression in saphenous vein (SV) vascular smooth muscle cells (VSMCs). These findings demonstrate that ET-1 mRNA expression and peptide production is modulated by elevations in cAMP levels. Further studies are necessary to elucidate which other pathways may be involved in the regulation of cytokine-stimulated ET-1 release from HVSMCs.

4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗

PubMed2000

Signal transduction pathways involved in cytokine stimulation of endothelin-1 release from human vascular smooth muscle cells.

Endothelin-1 (ET-1) mRNA expression and peptide production in human vascular smooth muscle cells (HVSMCs) are markedly increased by exposure to cytokines. As the transcription factor nuclear factor-kappaB (NF-kappaB) often mediates the effects of cytokines in target cells the aim of this study was to determine whether the production of ET-1 following exposure of HVSMCs to cytokines depends upon activation of NF-kappaB. BAY 11-7082, an inhibitor of cytokine-induced inhibitor protein (IkappaB) phosphorylation, inhibited cytokine-stimulated upregulation of preproendothelin-1 mRNA expression and ET-1 peptide production. In addition, immunoblotting showed that cytokine stimulation of ET-1 release in VSMCs involves nuclear translocation of NF-kappaB. In conclusion, NF-kappaB activation is involved in the stimulation by cytokines of ET-1 release from HVSMCs. As upregulated production of ET-1 within VSMCs may underlie the causative role of ET-1 in a number of disease states this finding indicates that NF-kappaB within HVSMCs could be central to a number of vascular pathologies.

Cells, Cultured↗

PubMed2000

The prostacyclin-mimetic cicaprost inhibits endogenous endothelin-1 release from human pulmonary artery smooth muscle cells.

There is increasing evidence supporting a role for endothelin-1 (ET-1) in human pulmonary hypertension. The aim of this study was to determine the relative roles of human pulmonary microvascular endothelial cells (HPMVE) and human pulmonary artery smooth muscle (HPASM) cells to produce ET-1 under inflammatory conditions and to investigate further possible control mechanisms of ET-1 production by HPASM. Although HPMVE cells produced more ET-1 than HPASM when cultured with fetal calf serum (FCS) alone and after treatment with cytokines; HPASM produced significant amounts of ET-1 after stimulation with cytokines. Cytokine-stimulated increase in ET-1 production by HPASM was inhibited by cicaprost, a prostacyclin analogue, and other agents that are known to increase intracellular cyclic AMP. Cicaprost also inhibited proliferation of HPASM in response to FCS lending support to the theory that part of the clinical benefit seen in long-term treatment with prostacyclin in pulmonary hypertension may be a result of inhibition of ET-1 production in these cells.

Cell Division↗

PubMed2000

Cyclooxygenase-2 regulates granulocyte-macrophage colony-stimulating factor, but not interleukin-8, production by human vascular cells: role of cAMP.

Vascular smooth muscle is now recognized as an important site of mediator generation under inflammatory conditions. Indeed, the release of leukocyte activators, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-8, by human arterial smooth muscle cells has recently been demonstrated. However, the potential for venous cells to release GM-CSF has not been addressed. We have shown that human vascular smooth muscle cells express the "inflammatory" form of cyclooxygenase (COX), cyclooxygenase-2 (COX-2), when stimulated with cytokines. In some nonvascular cell types, the COX activity has been shown to regulate the release of GM-CSF and IL-8, although the nature of the isoform responsible was not addressed. We show that human venous smooth muscle cells, like their arterial counterparts, release GM-CSF after stimulation with IL-1beta. Similarly, both cell types released IL-8. Under the same conditions, we found that COX-2 activity suppressed GM-CSF, but not IL-8, release by both types of human vascular cells. Moreover, the prostacyclin mimetic, cicaprost, and the cAMP analogue, dibutyryl cAMP, inhibited GM-CSF release from these cells. These observations suggest that COX-2 activity suppresses GM-CSF release via a cAMP-dependent pathway in human vascular cells and illustrates a novel mechanism by which this enzyme can modulate immune and inflammatory events.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1999

Potent, orally active GPIIb/IIIa antagonists containing a nipecotic acid subunit. Structure-activity studies leading to the discovery of RWJ-53308.

Although intravenously administered antiplatelet fibrinogen receptor (GPIIb/IIIa) antagonists have become established in the acute-care clinical setting for the prevention of thrombosis, orally administered drugs for chronic use are still under development. Herein, we present details from our exploration of structure-activity surrounding the prototype fibrinogen receptor antagonist RWJ-50042 (racemate of 1), which was derived from a unique approach involving the gamma-chain of fibrinogen (Hoekstra et al. J. Med. Chem. 1995, 38, 1582). Our analogue studies culminated in the discovery of RWJ-53308 (2), a potent, orally active GPIIb/IIIa antagonist. To progress from RWJ-50042 to a suitable candidate for clinical development, we conducted a series of optimization cycles that employed solid-phase parallel synthesis for the rapid, efficient preparation of nearly 250 analogues, which were assayed for fibrinogen receptor affinity and inhibition of platelet aggregation induced by four different activators. This strategy produced several promising analogues for advanced study, including 3-(3,4-methylenedioxybenzene)-beta-amino acid analogue 3 (significant improved in vivo potency) and 3-(3-pyridyl)-beta-amino acid 2 (significantly improved potency, oral absorption, and duration of action). In dogs, 2 displayed significant ex vivo antiplatelet activity on oral administration at 1.0 mg/kg, 16% systemic oral bioavailability, minimal metabolic transformation, and an excellent safety profile. Additionally, 2 was found to be efficacious in three in vivo thrombosis models: canine arteriovenous (AV) shunt (0.01-0.1 mg/kg, iv), guinea pig photoactivation-induced injury (0.3-3 mg/kg, iv), and guinea pig ferric chloride-induced injury (0.3-1 mg/kg, iv). On the basis of its noteworthy preclinical data, RWJ-53308 (2) was selected for clinical evaluation.

Administration, Oral↗

PubMed1999

Nonsteroid drug selectivities for cyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated with human gastrointestinal toxicity: a full in vitro analysis.

The beneficial actions of nonsteroid anti-inflammatory drugs (NSAID) can be associated with inhibition of cyclo-oxygenase (COX)-2 whereas their harmful side effects are associated with inhibition of COX-1. Here we report data from two related assay systems, the human whole blood assay and a modified human whole blood assay (using human A549 cells as a source of COX-2). This assay we refer to as the William Harvey Modified Assay. Our aim was to make meaningful comparisons of both classical NSAIDs and newer COX-2-selective compounds. These comparisons of the actions of >40 NSAIDs and novel COX-2-selective agents, including celecoxib, rofecoxib and diisopropyl fluorophosphate, demonstrate a distribution of compound selectivities toward COX-1 that aligns with the risk of serious gastrointestinal complications. In conclusion, this full in vitro analysis of COX-1/2 selectivities in human tissues clearly supports the theory that inhibition of COX-1 underlies the gastrointestinal toxicity of NSAIDs in man.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1999

Kaolin-induced hydrocephalus in the hamster: temporal sequence of changes in intracranial pressure, ventriculomegaly and whole-brain specific gravity.

The development of kaolin-induced hydrocephalus in adult hamsters was monitored by measuring changes in intracranial pressure (ICP), ventriculomegaly (VG) and whole-brain specific gravity (SG). Controls were intact or sham operated animals. Relative to controls, ICP of experimental animals increased at 24 h post intracisternal kaolin injection (by approximately 7-fold), reached a maximum on day 6 (by approximately 12-fold) and remained markedly elevated through day 15 (by approximately 5-fold). Ventricles differed in time of onset of distension (third: day 1, lateral: day 2, fourth: day 4) and in time of maximum ventriculomegaly (fourth: day 6; third: day 7; and lateral: day 9). Ventricular distension resulted in alterations in the ependyma; cilia were lost and apical cell surfaces were distorted. The ependyma was ruptured and the subjacent neuropil was exposed to the cerebrospinal fluid in some regions. Whole-brain SG remained constant in controls but declined in hydrocephalic hamsters after day 3 post-kaolin injection and reached its nadir on day 9 when whole-brain water content was 18% greater than in controls. Consistent with the fact that causal relationships exist between increased ICP, ventricular distension and brain edema, the alterations in each parameter occurred sequentially rather than simultaneously, and the time-course of each manifestation of hydrocephalus differed. The data suggest that the pathophysiology of kaolin-induced hydrocephalus in the hamster is tri-phasic: an initial period of rapid change, a brief interval of maximum alteration, and a subsequent period of compensation.

Age Factors↗

PubMed1999

Cyclo-oxygenase-2: pharmacology, physiology, biochemistry and relevance to NSAID therapy.

Cyclo-oxygenase is expressed in cells in two distinct isoforms. Cyclo-oxygenase-1 is present constitutively whilst cyclo-oxygenase-2 is expressed primarily after inflammatory insult. The activity of cyclo-oxygenase-1 and -2 results in the production of a variety of potent biological mediators (the prostaglandins) that regulate homeostatic and disease processes. Inhibitors of cyclo-oxygenase include the nonsteroidal anti-inflammatory drugs (NSAIDs) aspirin, ibuprofen and diclofenac. NSAIDs inhibit cyclo-oxygenase-2 at the site of inflammation, to produce their therapeutic benefits, as well as cyclo-oxygenase-1 in the gastric mucosa, which produces gastric damage. Most recently selective inhibitors of cyclo-oxygenase-2 have been developed and introduced to man for the treatment of arthritis. Moreover, recent epidemiological evidence suggests that cyclo-oxygenase inhibitors may have important therapeutic relevance in the prevention of some cancers or even Alzheimer's disease. This review will discuss how the new advancements in NSAIDs research has led to the development of a new class of NSAIDs that has far reaching implications for the treatment of disease.

Alzheimer Disease↗

PubMed1999

Selective COX-2 inhibitors and human inflammatory bowel disease.

BACKGROUND: Much recent effort has been made to produce selective inhibitors of cyclo-oxygenase-2 (COX-2) in the belief that these will lack the gastrointestinal damaging effects of traditional non-steroidal anti-inflammatory drugs (NSAIDs). Inflammatory bowel disease is associated with increased local production of prostanoids. These prostanoids, particularly PGE2 and PGI2, may well be protective as inflammatory bowel disease is aggravated by NSAID use. AIM: To examine the effects of a traditional NSAID and a highly selective COX-2 inhibitor on the production of these prostanoids in human inflammatory bowel disease. METHODS: Colonic mucosal biopsies were obtained from patients undergoing routine colonoscopy and biopsy for diagnostic or surveillance purposes. Biopsies were incubated in culture medium containing 10% foetal calf serum and antibiotics, plus test drugs or vehicle for 24 h, after which time the medium was removed and the content of PGE2, PGI2 (measured as 6 keto-PGF1alpha) and thromboxane (Tx) A2 (measured as TxB2) determined. RESULTS: Biopsies obtained from diseased colonic mucosa produced significantly more PGE2, PGI2 and thromboxane A2 than did controls (for example, PGE2: ulcerative colitis, 4.17+/-1.06; Crohn's disease, 3.97+/-1.66; control, 0.12 +/-0.13 ng/mL, n = 8-12). These increases were inhibited to a similar extent by either a highly selective COX-2 inhibitor (L-745,337) or a traditional non-selective NSAID (indomethacin). CONCLUSIONS: Until selective COX-2 inhibitors have been assessed adequately in human inflammatory bowel disease, these compounds should not be assumed to be safe for the gastrointestinal tract in inflammatory bowel disease.

Anti-Inflammatory Agents, Non-Steroidal↗