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C Patrono

Publications and source records attributed to C Patrono.

At least 73 records · Page 4Linked to original sources
PubMed1995

Growth abnormalities in cultured mesangial cells from rats with spontaneous glomerulosclerosis.

Age-related glomerulosclerosis (GS) occurs in normotensive rats of the Milan strain (MNS), but not in genetically-matched hypertensive animals (MHS). Altered mesangial cell (MC) proliferation and matrix expansion are common features of the glomerular scarring process. We evaluated proliferation and matrix protein synthesis of cultured MC from MNS and MHS animals aged 1 and 8 months, that is, before and after the occurrence of GS. [3H]-thymidine (TdR) incorporation into DNA of MC from MNS rats stimulated by 10% FBS serum increased with donor aging from 115 +/- 6.0 to 176 +/- 15, P < 0.01 (% cpm/well over quiescent controls +/- SEM). Under the same experimental conditions, cell counts changed from 101 +/- 4.0 to 146 +/- 5.0, P < 0.01 (% cells/well over quiescent controls). Additionally, cytosolic Ca2+ concentration ([Ca2+]i) rised from 115 +/- 19 to 220 +/- 32 nM and from 112 +/- 24 to 734 +/- 136 nM when fura-2-loaded cells from young and old MNS rats, respectively, were stimulated with 1% FBS. The rate of collagen production also increased with donor age, as well as collagen IV and laminin B1 mRNA expression. In contrast, in MC from MHS rats both DNA synthesis and cell replication rate declined as function of donor age. No differences in the [Ca2+]i responses to FBS were observed, nor collagen production changed with MHS rat senescence. We conclude that the age-associated decline of proliferative activity in MC from MHS animals could actually reflect a normal process of cell aging, possibly protecting from the occurrence of GS.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1995

Long-term thromboxane-synthase inhibition prolongs survival in murine lupus nephritis.

Systemic lupus erythematosus (SLE) is an autoimmune disease, characterized by nephritis, in which mortality is largely influenced by the severity of renal involvement. As there are evidences that thromboxane (TX)A2 plays an important role in the pathogenesis of lupus nephritis, we decided to assess the effects of long-term suppression of TXA2 synthesis on the progression of the disease, by designing a study of TXA2-synthase inhibition having adequate size to detect an effect on mortality as the primary end-point. Thus, we randomized 362 NZBxNZW mice (11-week-old at entry) to one of the following treatments: a TXA2 synthase inhibitor, FCE 22178 (300 mg/kg daily), saline or cyclophosphamide (5 mg/mouse weekly x 4 weeks) used as reference treatment. The TXA2 synthase inhibitor suppressed TXA2 biosynthesis, as reflected by urinary TXB2 and 2,3-dinor-TXB2 excretion (by 78% and 90%, respectively) and significantly reduced mortality (death rate: 34% vs. 61% in controls, at 37 weeks, P < 0.01). A significant reduction in proteinuria (9 +/- 1.6 vs. 17.3 +/- 2.4 mg/24 hr in FCE 22178 vs. saline, P < 0.01) and glomerular lesions was observed up to 30 weeks but not at 37 weeks. In contrast, cyclophosphamide prevented the development of proteinuria and histologic lesions, and reduced mortality to 8% at 37 weeks. Renal plasma flow and glomerular filtration rate were lower (by 29% and 52%, respectively) in 37-week-old as compared to young NZBxNZW mice. These parameters were further depressed by cyclophosphamide (by 48% and 45% vs. age-matched controls, respectively, P < 0.01) but were not altered significantly by FCE 22178.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Effects of the novel anti-inflammatory compounds, N-[2-(cyclohexyloxy)-4-nitrophenyl] methanesulphonamide (NS-398) and 5-methanesulphonamido-6-(2,4-difluorothio-phenyl)-1-inda none (L-745,337), on the cyclo-oxygenase activity of human blood prostaglandin endoperoxide synthases.

1. We have evaluated the selectivity of ketoprofen and two novel nonsteroidal anti-inflammatory drugs, N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulphonamide (NS-398) and 5-methanesulphonamido-6-(2,4-difluorothiophenyl)-1-indano ne (L-745,337), in inhibiting the cyclo-oxygenase activity of prostaglandin endoperoxide synthase-2 (PGHS-2) vs PGHS-1 in human blood monocytes and platelets, respectively. 2. Heparinized whole blood samples were drawn from healthy volunteers pretreated with aspirin, 300 mg 48 h before sampling, to suppress the activity of platelet PGHS-1 and incubated at 37 degrees C for 24 h with increasing concentrations of the test compounds in the presence of lipopolysaccharide (LPS, 10 micrograms ml-1). Immunoreactive PGE2 levels were measured in plasma by a specific radioimmunoassay as an index of the cyclo-oxygenase activity of LPS-induced monocyte PGHS-2. 3. The effects of the same inhibitors on platelet PGHS-1 activity were assessed by allowing whole blood samples, drawn from the same subjects in aspirin-free periods, to clot at 37 degrees C for 1 h in the presence of the compounds and measuring immunoreactive thromboxane B2 (TXB2) levels in serum by a specific radioimmunoassay. 4. Under these experimental conditions, ketoprofen enantioselectively inhibited the cyclo-oxygenase activity of both PGHS-1 and PGHS-2 with equal potency (IC50 ratio: approx. 0.5 for both enantiomers), while L-745,337 and NS-398 achieved selective inhibition of monocyte PGHS-2 (IC50 ratio: > 150). L-745,337 and NS-398 did not affect LPS-induced monocyte PGHS-2 biosynthesis to any detectable extent. 5. We conclude that L-745,337 and NS-398 are selective inhibitors of the cyclo-oxygenase activity of human monocyte PGHS-2. These compounds may provide adequate tools to test the contribution of this novel pathway of arachidonate metabolism to human inflammatory disease.

Adult↗
PubMed1995

Inhibition of thromboxane biosynthesis and platelet function by simvastatin in type IIa hypercholesterolemia.

Thromboxane A2 (TXA2) biosynthesis is enhanced in the majority of patients with type IIa hypercholesterolemia. Because simvastatin (a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor) was previously shown to reduce platelet aggregation and TXB2 production ex vivo, we investigated TXA2 biosynthesis and platelet function in 24 patients with type IIa hypercholesterolemia randomized to receive in a double-blind fashion simvastatin (20 mg/d) or placebo for 3 months. The urinary excretion of 11-dehydro-TXB2, largely a reflection of platelet TXA2 production in vivo, was measured by a previously validated radioimmunoassay technique. Blood lipid levels and urinary 11-dehydro-TXB2 excretion were significantly (P < .001) reduced by simvastatin. In contrast, placebo-treated patients did not show any statistically significant changes in either blood lipids or 11-dehydro-TXB2 excretion. The reduction in 11-dehydro-TXB2 associated with simvastatin was correlated with the reduction in total cholesterol (r = .81, P < .0001), LDL cholesterol (r = .79, P < .0001), and apolipoprotein B (r = .76, P < .0001) levels. Platelets from patients with type IIa hypercholesterolemia required significantly (P < .01) more collagen and ADP to aggregate and synthesized less TXB2 in response to both agonists after simvastatin therapy. Bleeding time, platelet sensitivity to Iloprost, and blood lipoprotein(a) and HDL cholesterol levels were not significantly affected by either treatment. We conclude that enhanced TXA2 biosynthesis in type IIa hypercholesterolemia is, at least in part, dependent on abnormal cholesterol levels and/or other simvastatin-sensitive mechanisms affecting platelet function.

Adult↗
PubMed1995

Immunological characterization of urinary 8-epi-prostaglandin F2 alpha excretion in man.

F2-isoprostanes are prostaglandin (PG) F2-like compounds that are formed in vivo directly by free radical-catalyzed lipid peroxidation. One of the compounds that can be produced in abundance by such mechanism is 8-epi-PGF2 alpha, a potent vasoconstrictor. We have developed an enzyme immunoassay and a radioimmunoassay for measuring urinary concentrations of 8-epi-PGF2 alpha by raising antibodies against this compound. The antisera presented high titers (> 1/300,000) and provided highly sensitive assays (IC50, 8 and 24 pg/ml, for EIA and RIA, respectively); cross-reactivity with other PG was negligible. The interassay reproducibility of EIA was assessed by measuring the same urine stored frozen in aliquots after solid phase extraction and thin-layer chromatography (17%, n = 13). Measurements of urinary 8-epi-PGF2 alpha by immunoassays were validated using different antisera and by comparison with gas chromatography/mass spectrometry. Healthy volunteers excreted 25 +/- 12 ng of 8-epi-PGF2 alpha/mmol creatinine (n = 19), with no circadian variation over three consecutive 8-hr collection periods (n = 10); preliminary results showed that excretion increased as a function of age. Urinary excretion of 8-epi-PGF2 alpha was unchanged by treatment with two nonsteroidal antiinflammatory drugs, Ibuprofen at 1.2 g/day for 4 days (n = 4) or aspirin as a single administration of 1 g (n = 6). In contrast, the urinary excretion of 11-dehydro-thromboxane B2, a platelet cyclooxygenase-derived metabolite was reduced by more than 80% after aspirin administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

The effects of nonsteroidal anti-inflammatory drugs on human hypertensive vascular disease.

The co-administration of nonsteroidal anti-inflammatory drugs (NSAIDs) and antihypertensive agents often, but not always, results in blunting of the effect of antihypertensive therapy. Although NSAIDs have no detectable pressor effects in normal subjects or untreated hypertensive people, they seem to antagonize the action of the majority of antihypertensive agents, making it necessary to increase their dosage, and often preventing proper control of blood pressure, particularly in black and elderly patients. The mechanism of this pharmacodynamic interaction is not completely understood, but may involve inhibition of vascular and renal prostaglandin (PG) synthesis, with resulting vasoconstriction and impaired renal excretion of Na+ and water. Also, suppression of a possible intermediary action of PG in the antihypertensive action of agents such as angiotensin converting enzyme inhibitors has been proposed. Certain antihypertensive drugs, such as Ca(2+)-channel blockers, centrally acting alpha agonists, and diuretics seem less sensitive to antagonism by NSAIDs. Aspirin and sulindac seem to be devoid of pressor effects, and thus provide a safe alternative in patients at risk for this interaction. These compounds may, in selected circumstances, even potentiate the effects of antihypertensive medications. Studies are underway to evaluate the hemodynamic effects of more selective blockers of arachidonate metabolism, such as thromboxane synthase inhibitors and selective inhibitors of PGH synthase-2.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1995

Increased thromboxane biosynthesis in essential thrombocythemia.

In order to investigate the in vivo thromboxane (TX) biosynthesis in essential thrombocythemia (ET), we measured the urinary excretion of the major enzymatic metabolites of TXB2, 11-dehydro-TXB2 and 2,3-dinor-TXB2 in 40 ET patients as well as in 26 gender- and age-matched controls. Urinary 11-dehydro-TXB2 was significantly higher (p < 0.001) in thrombocythemic patients (4,063 +/- 3,408 pg/mg creatinine; mean +/- SD) than in controls (504 +/- 267 pg/mg creatinine), with 34 patients (85%) having 11-dehydro-TXB2 > 2 SD above the control mean. Patients with platelet number < 1,000 x 10 (9)/1 (n = 25) had significantly higher (p < 0.05) 11-dehydro-TXB2 excretion than patients with higher platelet count (4,765 +/- 3,870 pg/mg creatinine, n = 25, versus 2,279 +/- 1,874 pg/mg creatinine, n = 15). Average excretion values of patients aging > 55 was significantly higher than in the younger group (4,784 +/- 3,948 pg/mg creatinine, n = 24, versus 2,405 +/- 1,885 pg/mg creatinine, n = 16, p < 0.05). Low-dose aspirin (50 mg/d for 7 days) largely suppressed 11-dehydro-TXB2 excretion in 7 thrombocythemic patients, thus suggesting that platelets were the main source of enhanced TXA2 biosynthesis. The platelet count-corrected 11-dehydro-TXB2 excretion was positively correlated with age (r = 0.325, n = 40, p < 0.05) and inversely correlated with platelet count (r = -0.381, n = 40, p < 0.05). In addition 11 out of 13 (85%) patients having increased count-corrected 11-dehydro-TXB2 excretion, belonged to the subgroup with age > 55 and platelet count < 1,000 x 10(9)/1. We conclude that in essential thrombocythemia: 1) enhanced 11-dehydro-TXB2 excretion largely reflects platelet activation in vivo; 2) age as well as platelet count appear to influence the determinants of platelet activation in this setting, and can help in assessing the thrombotic risk and therapeutic strategy in individual patients.

Adult↗
PubMed1994

Evidence for episodic platelet activation in acute ischemic stroke.

BACKGROUND AND PURPOSE: Enhanced thromboxane biosynthesis has previously been reported in patients with acute ischemic stroke. In this study we examined the time course of thromboxane biosynthesis after the onset of symptoms in 13 patients with acute cerebral infarction. METHODS: We obtained five to eight consecutive 6-hour urine samples from each of these 13 patients within the first 48 hours after onset of symptoms to study the dynamics of platelet activation in this setting. The urinary excretion of the major enzymatic metabolite of thromboxane B2, 11-dehydro-thromboxane B2, was measured by a previously validated radioimmunoassay. The excretion rate was compared with that of 20 control patients with nonvascular neurological diseases. RESULTS: Eleven patients (85%) had at least one value exceeding 2 SD of the control mean (251 pmol/mmol creatinine). The proportion of samples with an elevated 11-dehydro-thromboxane B2 level was markedly similar in each of the eight 6-hour collection periods (mean, 52 +/- 8%; range, 40% to 67%). In 4 patients (31%) the excretion rate was elevated in all measurements obtained. In the 11 patients with enhanced thromboxane biosynthesis, no uniform pattern of changes over time in metabolite excretion emerged, with 3 patients having peak values at 0 to 12 hours, 3 at 12 to 24 hours, 3 at 24 to 36 hours, and 2 at 36 to 48 hours. The level and dynamics of 11-dehydro-thromboxane B2 excretion were related neither to the neurological symptoms nor to the type or site of the cerebral ischemia. CONCLUSIONS: We conclude that episodes of platelet activation occur repeatedly during the first 48 hours after the onset of symptoms of an acute ischemic stroke. Given its apparent dynamic nature, this ongoing process may be amenable to pharmacologic modulation.

Adult↗
PubMed1994

Effects of unfractionated and low molecular weight heparins on platelet thromboxane biosynthesis "in vivo".

Several "in vitro" and "in vivo" studies indicate that heparin administration may affect platelet function. In this study we investigated the effects of prophylactic heparin on thromboxane (Tx)A2 biosynthesis "in vivo", as assessed by the urinary excretion of major enzymatic metabolites 11-dehydro-TxB2 and 2,3-dinor-TxB2. Twenty-four patients who were candidates for cholecystectomy because of uncomplicated lithiasis were randomly assigned to receive placebo, unfractionated heparin, low molecular weight heparin or unfractionated heparin plus 100 mg aspirin. Measurements of daily excretion of Tx metabolites were performed before and during the treatment. In the groups assigned to placebo and to low molecular weight heparin there was no statistically significant modification of Tx metabolite excretion while patients receiving unfractionated heparin had a significant increase of both metabolites (11-dehydro-TxB2: 3844 +/- 1388 vs 2092 +/- 777, p < 0.05; 2,3-dinor-TxB2: 2737 +/- 808 vs 1535 +/- 771 pg/mg creatinine, p < 0.05). In patients randomized to receive low-dose aspirin plus unfractionated heparin the excretion of the two metabolites was largely suppressed thus suggesting that platelets are the primary source of enhanced thromboxane biosynthesis associated with heparin administration. These data indicate that unfractionated heparin causes platelet activation "in vivo" and suggest that the use of low molecular weight heparin may avoid this complication.

Adult↗
PubMed1994

A comparative evaluation of thromboxane receptor blockade, thromboxane synthase inhibition and both in animal models of arterial thrombosis.

The combination of thromboxane (TX) synthase inhibition and prostaglandin (PG) H2/TXA2 receptor antagonism yields enhanced antithrombotic effects as compared with either intervention alone. However, it is not known whether the enhancing effect of TX synthase inhibition is expressed also in the presence of complete blockade of PGH2/TXA2 receptors. Thus we evaluated the antithrombotic effects of increasing doses of the PGH2/TXA2 receptor antagonist L 670596 alone and in combination with a dose of the TX synthase inhibitor FCE 22178 causing > 95% inhibition of platelet TXB2 production. In the dog model of electrically induced coronary thrombosis, occlusion time in control animals (n = 14) averaged 72 +/- 29 min. L 670596 alone dose-dependently antagonized platelet PGH2/TXA2 receptors and prolonged occlusion time. The addition of FCE 22178 displaced the dose-occlusion time relation of L 670596 in a parallel fashion without modifying receptor occupancy. In the rabbit model of copper coil-induced carotid artery thrombosis, occlusion was very rapid (14 +/- 4 min) in control animals (n = 17) and was not modified by either aspirin or FCE 22178. L 670596 caused a dose-related receptor blockade and prolongation of occlusion time. The association with FCE 22178 enhanced significantly the antithrombotic effect of L 670596 at all doses. We conclude that the full therapeutic potential of PGH2/TXA2 receptor antagonism is expressed at > 90% platelet receptor occupancy. The additive effect of TX synthase inhibition suggests that conversion of PGH2 to platelet-inhibitor and vasodilator prostaglandins might be of therapeutic importance, irrespective of the extent of PGH2/TXA2 receptor blockade.

Animals↗
PubMed1993

Enzyme immunometric assay for endothelin using tandem monoclonal antibodies.

Seven distinct mouse monoclonal antibodies (mAbs) directed against human endothelin-1 (ET-1) have been obtained. On the basis of specificity studies performed with competitive immunoassays and of complementary binding studies, these mAbs were classified in two groups. mAbs of group A (Endo-4, -5, -6 and -10) were shown to be directed against the N terminal loop while those of group B (Endo-2, -8 and -18) recognized the C terminal part of the peptide. A pair of monoclonal antibodies with optimal properties for a two-site immunometric assay were selected and the test was performed in 96-well microtiter plates coated with one mAb (Endo-18), while another mAb (Endo-4) covalently labeled with enzyme acetylcholinesterase was used as tracer. Under optimal conditions, the assay appeared to be very sensitive since concentrations as low as 1 pg/ml could be significantly detected. The precision was also very good with a coefficient of variation below 10% from 3 to 250 pg/ml. The assay was specific for mature endothelin presenting no cross-reactivity with the precursor Big ET-1. On the other hand, strong cross-reactivity was observed with other ET-1-related peptides, including ET-2, ET-3, VIC peptide and sarafotoxin 6-b. The assay permitted specific determination of ET-1 in supernatants of cultured endothelial cells and the validity of the test was demonstrated by HPLC fractionation experiments. In addition, the assay also appeared to be suitable for direct determination of ET-1 in plasma. Studies performed with plasma from healthy subjects revealed that circulating levels of ET-1 are below or close to the detection limit of the method (< 8 pg/ml).

Amino Acid Sequence↗