Inhibition of platelet aggregation ex vivo is repressed in apolipoprotein E deficient mice.

In the present study, we assessed whether the endogenous platelet inhibitory mechanisms are altered in the early to moderate stages of the atherosclerotic process. Apolipoprotein E deficient mice (ApoE-/-), a mouse model of atherosclerosis, and their wild-type (WT) counterparts were used to assess agonist-stimulated synthesis of prostacyclin (PGI2), inhibition of platelet aggregation ex vivo, and intra-platelet cAMP levels. Basal U46619 and ADP -induced platelet aggregation in vitro were increased in ApoE-/- mice at 18-20 weeks in comparison with 8-10 weeks of age. Systemically administered endothelin-1 (ET-1) or bradykinin (BK) inhibited platelet aggregation in a similar fashion in 8- to 10-week-old ApoE-/- and WT mice, but not in the ApoE-/- mice at 18-20 weeks of age, although both peptides maintained their capacity to increase plasma levels of the PGI2. Intravenous infusion of PGI2 also failed to inhibit platelet aggregation ex vivo in 18- to 20-week-old ApoE-/- mice. Interestingly, both BK and PGI2 retained their ability to increase intraplatelet cAMP in WT and ApoE-/- mice. Our results suggest that a loss of activity of endogenous inhibitorymechanisms could contribute to the increased platelet reactivity in ApoE-/- mice, and that this phenomenon occurs early in the intermediate stage of the atherosclerotic process.

Pharmacological assessment of ibuprofen arginate on platelet aggregation and colon cancer cell killing.

Nonsteroidal anti-inflammatory drugs (NSAIDs), including ibuprofen, are amongst the most commonly used medications and produce their anti-inflammatory and analgesic benefits by blocking cyclooxygenase (COX)-2. These drugs also have the potential to prevent and treat cancer and some members of the class including ibuprofen can produce anti-platelet effects. Despite their utility, all NSAIDs are associated with increased risk of cardiovascular side effects which our recent work suggests could be mediated by increased levels of the endogenous NO synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) leading to reduced endothelial NOS activity and associated endothelial cell dysfunction. ADMA is a cardiotoxic hormone and biomarker of cardiovascular risk whose effects can be prevented by l-arginine. The ibuprofen salt, ibuprofen arginate (Spididol®) was created to increase drug solubility but we have previously established that it not only effectively blocks COX-2 but also provides an arginine source able to reverse the effects of ADMA in vitro and in vivo. Here we have gone on to explore whether the formulation of ibuprofen with arginine influences the potency and efficacy of the parent molecule using a range of simple in vitro assays designed to test the effects of NSAIDs on (i) platelet aggregation and (iii) colon cancer cell killing. Our findings demonstrate that ibuprofen arginate retains these key functional effects of NSAIDs with similar or increased potency compared to ibuprofen sodium, further illustrating the potential of ibuprofen arginate as an efficacious drug with the possibility of improved cardiovascular safety.

Aspirin has little additional anti-platelet effect in healthy volunteers receiving prasugrel.

BACKGROUND: Strong P2Y(12) blockade, as can be achieved with novel anti-platelet agents such as prasugrel, has been shown in vitro to inhibit both ADP and thromboxane A(2) -mediated pathways of platelet aggregation, calling into question the need for the concomitant use of aspirin. OBJECTIVE: The present study investigated the hypothesis that aspirin provides little additional anti-aggregatory effect in a group of healthy volunteers taking prasugrel. STUDY PARTICIPANTS/METHODS: In all, 9 males, aged 18 to 40 years, enrolled into the 21-day study. Prasugrel was loaded at 60 mg on day 1 and maintained at 10 mg until day 21. At day 8, aspirin 75 mg was introduced and the dose increased to 300 mg on day 15. On days 0, 7, 14 and 21, platelet function was assessed by aggregometry, response to treatments was determined by VerifyNow and urine samples were collected for quantification of prostanoid metabolites. RESULTS: At day 7, aggregation responses to a range of platelet agonists were reduced and there was only a small further inhibition of aggregation to TRAP-6, collagen and epinephrine at days 14 and 21, when aspirin was included with prasugrel. Urinary prostanoid metabolites were unaffected by prasugrel, and were reduced by the addition of aspirin, independent of dose. CONCLUSIONS: In healthy volunteers, prasugrel produces a strong anti-aggregatory effect, which is little enhanced by the addition of aspirin. The addition of aspirin as a dual-therapy with potent P2Y(12) receptor inhibitors warrants further investigation.

4-Methylnitrosamino-1-3-pyridyl-1-butanone (NNK) promotes lung cancer cell survival by stimulating thromboxane A2 and its receptor.

The role of thromboxane A(2) (TxA(2)) in smoking-associated lung cancer is poorly understood. This study was conducted to study the role of TxA(2) in smoking carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-promoted cell survival and growth in human lung cancer cells. We found that NNK increased TxA(2) synthase (TxAS) expression and thromboxane B(2) (TxB(2)) generation in cultured lung cancer cells, the result of which was supported by the increased level of TxAS in lung cancer tissues of smokers. Both TxAS-specific inhibitor furegrelate and TxA(2) receptor antagonist SQ29548 completely blocked NNK-mediated cell survival and growth via inducting apoptosis. TxA(2) receptor agonist U46619 reconstituted a near-full survival and growth response to NNK when TxAS was inhibited, affirming the role of TxA(2) receptor in NNK-mediated cell survival and growth. Suppression of cyclic adenosine monophosphate response element binding protein (CREB) activity by its small interference RNA blocked the effect of NNK. Phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) also had a positive role. Altogether, our results have revealed that NNK stimulates TxA(2) synthesis and activates its receptor in lung cancer cells. The increased TxA(2) may then activate CREB through PI3K/Akt and extracellular ERK pathways, thereby contributing to the NNK-promoted survival and growth of lung cancer cells.

In the presence of strong P2Y12 receptor blockade, aspirin provides little additional inhibition of platelet aggregation.

BACKGROUND: Aspirin and antagonists of platelet ADP P2Y(12) receptors are often coprescribed for protection against thrombotic events. However, blockade of platelet P2Y(12) receptors can inhibit thromboxane A(2) (TXA(2))-dependent pathways of platelet activation independently of aspirin. OBJECTIVES: To assess in vitro whether aspirin adds additional antiaggregatory effects to strong P2Y(12) receptor blockade. METHODS: With the use of platelet-rich plasma from healthy volunteers, determinations were made in 96-well plates of platelet aggregation, TXA(2) production and ADP/ATP release caused by ADP, arachidonic acid, collagen, epinephrine, TRAP-6 amide and U46619 (six concentrations of each) in the presence of prasugrel active metabolite (PAM; 0.1-10 µmol L(-1)), aspirin (30 µmol L(-1)), PAM + aspirin or vehicle. results: PAM concentration-dependently inhibited aggregation; for example, aggregation in response to all concentrations of ADP and U46619 was inhibited by ≥ 95% by PAM at > 3 µmol L(-1) . In further tests of PAM (3 µmol L(-1)), aspirin (30 µmol L(-1)) and PAM + aspirin, aspirin generally failed to produce more inhibition than PAM or additional inhibition to that caused by PAM. The antiaggregatory effects of PAM were associated with reductions in the platelet release of both TXA(2) and ATP + ADP. Similar effects were found when either citrate or lepirudin were used as anticoagulants, and when traditional light transmission aggregometry was conducted at low stirring speeds. CONCLUSIONS: P2Y(12) receptors are critical to the generation of irreversible aggregation through the TXA(2) -dependent pathway. As a result, strong P2Y(12) receptor blockade alone causes inhibition of platelet aggregation that is little enhanced by aspirin. The clinical relevance of these observations remains to be determined.

18F-labeled resin microspheres as surrogates for 90Y resin microspheres used in the treatment of hepatic tumors: a radiolabeling and PET validation study.

(90)Y-labeled resin microspheres (SIR-Spheres) are currently used to treat patients with primary and metastatic solid liver tumors. This treatment is typically palliative since patients have exhausted all other standard treatment options. Improving the quality of life and extending patient survival are typical benchmarks for tracking patient response. However, the current method for predicting microsphere biodistributions with (99m)Tc-labeled macroaggregated albumin (MAA) does not correlate well with patient response. This work presents the development of a new (18)F-labeled resin microsphere to serve as a surrogate for the treatment microsphere and to employ the superior resolution and sensitivity of positron emission tomography (PET). The (18)F microsphere biodistributions were determined in a rabbit using PET imaging and histological review. The PET-based uptake ratio was shown to agree with the histological findings to better than 3%. In addition, the radiolabeling process was shown to be rapid, efficient and relatively stable in vivo.

A clinical and genetic study of SPG5A linked autosomal recessive hereditary spastic paraplegia.

The authors performed a clinical and genetic study of a large consanguineous English family with uncomplicated autosomal recessive hereditary spastic paraplegia (ARHSP). Linkage to the previously described SPG5A locus was established with maximum multipoint lod score of 4.84. The locus was refined to a 23.6 cM interval between markers D8S1833 and D8S285. No evidence of oxidative phosphorylation defects was found in muscle biopsies from two affected individuals.

Screening of patients with hereditary spastic paraplegia reveals seven novel mutations in the SPG4 (Spastin) gene.

Hereditary spastic paraplegia (HSP) is a heterogeneous condition characterised in its pure form by progressive lower limb spasticity. Mutations in SPG4 (encoding spastin) may be responsible for up to 40% of autosomal dominant (AD) cases. A cohort of 41 mostly pure HSP patients from Britain and Austria, 30 of whom displayed AD inheritance, was screened for mutations in SPG4 by single strand conformation polymorphism (SSCP) analysis followed by sequencing of samples with mobility shifts. We identified eight SPG4 mutations in pure AD HSP patients, seven of which were novel: one missense mutation within the AAA cassette (1633G>T), two splice site mutations (1130-1G>T, 1853+2T>A) and four frameshift mutations (190_208dup19, 1259_1260delGT, 1702_1705delGAAG, 1845delG). A novel duplication in intron 11 (1538+42_45dupTATA) was also detected. We report the HUGO-approved nomenclature of these mutations as well. Furthermore, we detected a silent change (1004G>A; P293P), previously reported as a mutation, which was also present in controls. The frequency of SPG4 mutations detected in pure AD HSP was 33.3%, suggesting that screening of such patients for SPG4 mutations is worthwhile. Most patients will have unique mutations. Screening of SPG4 in apparently isolated cases of HSP may be of less value.

Immunization by particle-mediated transfer of the granulocyte-macrophage colony-stimulating factor gene into autologous tumor cells in melanoma or sarcoma patients: report of a phase I/IB study.

The primary objective of this phase I study was to determine the safety of an autologous tumor vaccine given by intradermal injection of lethally irradiated granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transfected autologous melanoma and sarcoma cells. Secondary objectives included validation of the gene delivery technology (particle-mediated gene transfer), determining the host immune response to the tumor after vaccination, and monitoring patients for evidence of antitumor response. Sixteen patients were treated with either of two different doses of GM-CSF-treated tumor cells. One patient received treatment with both doses of tumor cells. No treatment-related local or systemic toxicity was noted in any patient. Patients administered 100% treated cells (i.e., with a preparation of tumor cells that had all been exposed to GM-CSF DNA transfection) had a more extensive lymphocytic infiltrate at the vaccine site than did patients given 10% treated cells (a preparation of tumor cells in which 10% had been exposed to GM-CSF transfection) or nontreated tumor. The generation of a systemic immune response to autologous tumor by a delayed-type hypersensitivity response to the intradermal placement of nontransfected tumor cells was noted in one patient. One patient had a transient partial response of metastatic tumor sites. The entire procedure, from tumor removal to vaccine placement, was accomplished in less than 6 hr in all patients. Four of 17 patient tumor preparations produced greater than 3.0 ng of GM-CSF per 10(6) cells per 24 hr in vitro. The one patient with greater than 30 ng of GM-CSF per 10(6) cells per 24 hr in vitro had positive DTH, a significant histologic inflammatory response, and clinically stable disease. This technique of gene transfer was safe and feasible, but resulted in clinically relevant levels of gene expression in only a minority of patients.

Coronary artery brachytherapy.

Coronary artery disease is the leading cause of mortality in the West with over 1.2 million angioplasties performed annually. Despite the introduction of stents, restenosis occurs in 30-40% of vessels, which until recently has only been treated effectively by coronary artery bypass surgery. Coronary artery brachytherapy appears to provide an alternative, less invasive remedy. The mechanisms of restenosis and how these are inhibited by radiation are described here. The practicalities of radiation delivery and the history of the development of intravascular radiation as an effective clinical tool are outlined. Finally, the pitfalls of the current technology and the areas in which future research must be targeted for the field to develop are discussed.

Modulation of colony stimulating factor release and apoptosis in human colon cancer cells by anticancer drugs.

Modulation of the immune response against tumour cells is emerging as a valuable approach for cancer treatment. Some experimental studies have shown that secretion of colony stimulating factors by cancer cells reduces their tumorigenicity and increases their immunogenicity probably by promoting the cytolitic and antigen presenting activities of leukocytes. We have observed that human colon cancer cells (HT-29) are able to secrete granulocyte-macrophage-colony stimulating factor, granulocyte-colony stimulating factor and macrophage-colony stimulating factor when stimulated with cytokines (IL-1beta and TNF-alpha). In this study we assessed, for the first time, the effects of several anticancer drugs on colony stimulating factor release or apoptosis in HT-29 cells. Cytokine-induced release of granulocyte-macrophage-colony stimulating factor, granulocyte-colony stimulating factor and macrophage-colony stimulating factor was significantly increased by cisplatin and 6-mercaptopurine. Taxol only increased macrophage-colony stimulating factor release while reduced that of granulocyte-colony stimulating factor. No changes in colony stimulating factor secretion were observed after treatment with methotrexate. Only cisplatin and taxol induced apoptosis in these cells. Secretion of colony stimulating factors by colon cancer cells may contribute to the immune host response against them. Anticancer drugs such as cisplatin and 6-mercaptopurine increase colony stimulating factor secretion by cytokine stimulated cancer cells probably through mechanisms different to those leading to cell apoptosis, an effect that may contribute to their anti-neoplasic action.

A large family with hereditary spastic paraparesis due to a frame shift mutation of the spastin (SPG4) gene: association with multiple sclerosis in two affected siblings and epilepsy in other affected family members.

Hereditary spastic paraparesis (HSP) is a clinically and genetically heterogeneous neurodegenerative disorder characterised by progressive lower limb spasticity and weakness. Some forms have been associated with white matter lesions and complex phenotypes. This study was prompted by the diagnosis of multiple sclerosis (MS) in two sisters from a large pedigree with hereditary spastic paraparesis. Twelve affected members of the extended family were examined (MRI and EEG were carried out and evoked potentials measured in five), and historical information was obtained from six affected deceased persons. The inherited disease phenotype was confirmed as autosomal dominant hereditary spastic paraparesis associated with epilepsy in four affected persons. None of the extended family had evidence of MS. Genetic analysis of the family has shown linkage to chromosome 2p and sequencing of the spastin gene has identified a 1406delT frameshift mutation in exon 10. This kindred demonstrates the clinical heterogeneity of HSP associated with spastin mutations. The possible relevance of the concurrence of HSP and MS in the sib pair is discussed.

Relationship between endogenous colony stimulating factors and apoptosis in human colon cancer cells: role of cyclo-oxygenase inhibitors.

1. Nonsteroidal anti-inflammatory drug (NSAID) usage is associated with gastrointestinal inflammatory damage and aggravation of gut inflammatory conditions. NSAIDs also exert a preventive effect against colon cancer that seems to be due to increased colon cell apoptosis. NSAIDs have been shown to modulate the release of colony stimulating factors (CSFs) in some cells. In the present study we analysed the effect of these drugs on secretion of CSFs and apoptosis in human colon epithelial cells (HT-29). 2. HT-29 cells secreted bioactive levels of GM-CSF, G-CSF and M-CSF when stimulated with IL-1ss and TNF-alpha, and diclofenac (10(-7)-10(-4) M), indomethacin (10(-7)-10(-4) M) and sodium salicylate (10(-5)-10(-2) M) induced concentration-dependent increases in GM-CSF secretion. 3. Reduced secretion of G-CSF and M-CSF and increased cell apoptosis were observed with the highest concentrations of these non-selective NSAIDs. 4. No changes in any CSF release or HT-29 cell apoptosis were detected in the presence of the COX-2 selective inhibitor DFP (10(-7)-10(-4) M). 5. Neither the exogenous addition of CSFs nor the blockade of secreted CSFs modified apoptosis in HT-29 cells stimulated with cytokines and/or NSAIDs. 6. These results suggest that colon epithelial cells can contribute to local inflammatory responses by releasing CSFs and thus extend the life span of local leukocytes. Modulation of CSF levels by non-selective NSAIDs may be involved in the pro-inflammatory effects of these agents in the gut.

Evaluation of poly-L-lactic acid and polyglycolic acid resorbable stents for repair of tracheomalacia in a porcine model.

Poly-L-lactic acid and polyglycolic acid (PLPG) resorbable stents may offer a potential solution to the problem of tracheomalacia. Advantages of this material include its strength, its versatile shaping characteristics, and its resorbability, which would preclude surgical removal and allow for airway growth. The purpose of this pilot study was to examine the usefulness of PLPG stents for temporary external airway stenting of tracheomalacia in a porcine model. A severe tracheomalacia was created in 6 pigs by submucosal resection of segments of tracheal cartilage from 6 consecutive rings. The PLPG stent was then shaped to recreate the tracheal contour and sutured to the underlying airway. Endoscopic photodocumentation during spontaneous ventilation was obtained before and after reconstruction. After creation of the malacic tracheal segment, all animals developed stridor, retractions, and cyanosis during spontaneous ventilation. After repair, all animals were extubated without complication. All animals survived the follow-up period of 9 to 12 weeks without evidence of respiratory distress and with rapid weight gain. Repeat bronchoscopy showed no evidence of airway collapse during spontaneous ventilation. Tracheal measurements revealed growth of the stented segment with a mild narrowing within the repaired region. Histologic examination showed preservation of respiratory epithelium. These preliminary findings suggest that PLPG stents may serve a useful role in the surgical management of tracheomalacia.

Cyclooxygenase selectivity of non-steroid anti-inflammatory drugs in humans: ex vivo evaluation.

We have recently described a novel assay to assess ex vivo the activity and selectivity on cyclooxygenase-1 and -2 (EC 1.14.99.1) of non-steroid anti-inflammatory drugs (NSAID) administered to rats [Br. J. Pharmacol. 126 (1999) 1824.]. Here, we have extended these studies to humans. Healthy male volunteers were given orally one of the following drugs (mg) for 5 days: etodolac (200 or 400 b.i.d.), meloxicam (7.5 or 15 q.d.), nimesulide (100 or 200 b.i.d.), nabumetone (500 or 1000 b.i.d.) or naproxen (500 b.i.d.). Blood samples were withdrawn from the volunteers before and up to 24 h after the last dose. Plasma obtained from the blood was tested for its ability to inhibit prostanoid formation in interleukin-1beta-treated A549 cells (cyclooxygenase-2 system) and human washed platelets (cyclooxygenase-1 system). Plasma from etodolac-treated subjects demonstrated a slight selectivity towards the inhibition of cyclooxygenase-2. This effect was more prominent in plasma from subjects receiving meloxicam or nimesulide. Plasma from nabumetone-treated subjects showed no or little selectivity towards cyclooxygenase-1 depending on the dose of drug administered, while plasma taken from subjects receiving naproxen was more active at inhibiting cyclooxygenase-1 than cyclooxygenase-2. In conclusion, we have demonstrated that this assay can be used to assess ex vivo the relative activity against cyclooxygenase-1 and cyclooxygenase-2 of NSAIDs consumed by human volunteers. It is to be hoped that data from such systems will aid in our understanding of the relationships between the differential inhibition of cyclooxygenase-1 and cyclooxygenase-2 by NSAIDs and their reported efficacies and (gastrointestinal) toxicities.

Total chemical synthesis of human activin beta(A)[12-116] and related large-loop polypeptides.

We report here the synthesis, purification, and characterization of several large polypeptides related to the human activin beta(A) subunit and their cyclic counterparts. In particular, we describe for the first time the total chemical synthesis of a 105-mer polypeptide, des[1-11] activin beta(A), and related large-loop polypeptide, by an optimized solid phase synthetic protocol based on 9-flouroenylmethyoxycarbonyl (Fmoc) chemistry. These studies show that automated chemical synthesis utilizing Fmoc-based solid phase synthetic strategies provides a practical alternative to recombinant DNA technology for the production of activin-related subunits, with the opportunity to rapidly provide different analogues and structural variants for subsequent structure-function and associated biophysical investigations.

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.