Signals ensuing from trimeric G-protein-coupled receptors synergize to induce platelet activation... more Signals ensuing from trimeric G-protein-coupled receptors synergize to induce platelet activation. At low doses, the thromboxane A2 analogue U46619 does not activate integrin alphaIIbbeta3 or trigger platelet aggregation, but it induces shape changes. In the present study, we addressed whether low doses of U46619 trigger tyrosine phosphorylation independently of integrin alphaIIbbeta3 activation and ADP secretion, and synergize with adrenaline (epinephrine) to induce aggregation in acetylsalicylic acid (aspirin)-treated platelets. Low doses of U46619 triggered tyrosine phosphorylation of different proteins, including FAK (focal adhesion kinase), Src and Syk, independently of signals ensuing from integrin alphaIIbbeta3 or ADP receptors engaged by secreted ADP. The G(12/13)-mediated Rho/Rho-kinase pathway was also increased by low doses of U46619; however, this pathway was not upstream of tyrosine phosphorylation, because this occurred in the presence of the Rho-kinase inhibitor Y-276...
MDs (mitochondrial diseases) are a clinically heterogeneous group of disorders characterized by i... more MDs (mitochondrial diseases) are a clinically heterogeneous group of disorders characterized by impairment of the respiratory chain function with altered oxidative phosphorylation. We tested the hypothesis that the function of vascular endothelium is affected by increased oxidative stress in MDs. A total of 12 patients with MDs and pair-matched controls were studied. Endothelial function was assessed by measuring FMD (flow-mediated vasodilation) of brachial and common femoral arteries. The test was repeated after vitamin C (500 mg, twice a day) and E (400 mg, once a day) supplementation for 30 days and 90 days after vitamin withdrawal. FMD was reduced in patients compared with controls [AUC/τ (time-averaged area under the curve) for the brachial artery, 1.05±0.24 compared with 4.19±0.59% respectively, P<0.001; AUC/τ for the femoral artery, 0.98±0.19 compared with 2.36±0.29% respectively, P=0.001; values are means±S.E.M.] and correlated (brachial artery) with plasma lactate (r=-0.63, P<0.01). Urinary 8-iso-PGF2α (8-iso-prostaglandin F2α) was higher in patients than controls (505.6±85.9 compared with 302.5±38.7 pg/mg of creatinine; P<0.05) and correlated with plasma lactate (r=0.70, P<0.05). Immunohistochemical analysis showed 8-iso-PGF2α staining in MD-affected striated muscle cells and in blood vessels in muscle biopsies of patients. Antioxidant vitamins transiently restored FMD in patients [ΔAUC/τ (change in AUC/τ) for the brachial artery, +1.38±0.49%, P<0.05; ΔAUC/τ for the femoral artery, +0.98±0.24%, P<0.01] but had no effect on FMD in controls (brachial artery, -1.3±0.63%; and common femoral artery, -0.58±0.30%), thus abolishing the differences between patients and controls. The results of the present study indicate that oxidative stress is increased and is, at least partly, responsible for endothelial dysfunction in MDs.
It has been suggested that angiotensin-converting enzyme (ACE) inhibitors may also act through th... more It has been suggested that angiotensin-converting enzyme (ACE) inhibitors may also act through the renal prostaglandin (PG) system; moreover, it has been shown that nonsteroidal antiinflammatory drugs (NSAIDs) are capable of reducing the antihypertensive activity of ACE inhibitors. Sixteen essential hypertensive patients (WHO stages, I-II, eight on a low-sodium diet and eight on a high-sodium diet) were treated with enalapril, 20 mg/day per os, for 4 days. On days 3 and 4, ibuprofen, 1,200 mg/day per os, was also given. Enalapril reduced blood pressure, particularly in the group on the low-sodium diet. Urinary 6-keto-PGF1 alpha, an indicator of renal PGI2 production (determined by HPLC-RIA), increased in the first few hours after enalapril in the low-sodium group. Ibuprofen did not reduce the antihypertensive effect of enalapril, nor did it affect plasma renin activity or plasma aldosterone. Only a slight reduction in 6-keto-PGF1 alpha excretion was observed after enalapril plus ibu...
F 2-isoprostanes are prostaglandin (PG) isomers produced in vivo through free radical- catalyzed ... more F 2-isoprostanes are prostaglandin (PG) isomers produced in vivo through free radical- catalyzed peroxidation of arachidonic acid, which may affect platelet function. The current study investigated the effects of 8-epiprostaglandin F 2a (8-epi-PGF2a) on critical events of platelet activation. A dose-dependent increase in platelet adhesion to fibrinogen- and plasma-coated microwells by 8-epi-PGF2a (1 to 1000 nmol/L) was observed when resting
The aim of the study was to evaluate the effects of acute hyperhomocysteinemia on distensibility ... more The aim of the study was to evaluate the effects of acute hyperhomocysteinemia on distensibility and compliance of large peripheral arteries. Isoprostanes generation and antioxidant vitamins were used to assess the role of oxidative stress. A cross-over, double-blind study on distensibility (DC: distensibility coefficient) and compliance (CC: cross-sectional compliance) of common femoral and brachial arteries was performed in 12 healthy young male volunteers by means of a wall track system before and 4 h after a single oral methionine (100 mg/kg) or placebo administration. The effects of methionine load were investigated also after oral administration of vitamin C (1g/day) and vitamin E (800 mg/day) for 8 consecutive days. Oral methionine induced a significant increase in plasmatic levels of homocysteine. Distensibility and compliance of brachial and femoral arteries were significantly reduced after methionine load in comparison to placebo. This acute impairment of arterial wall mechanical properties was associated to endothelial dysfunction, since altered flow-dependent vasodilatation (P < 0.05 versus placebo) was observed in the same arterial districts. A significant increase in urinary 8-iso-prostaglandin F2alpha was observed after methionine. Pretreatment with vitamins C and E prevented the effects of methionine on femoral and brachial arteries as well as on urinary 8-iso-prostaglandin F2alpha excretion. Hyperhomocysteinemia seems responsible for altered arterial wall elasticity and for endothelial dysfunction. A pivotal role can be attributed to oxidative stress.
The effect of indomethacin (2 X 50 mg daily) and carprofen (2 X 150 mg daily) on gastric secretio... more The effect of indomethacin (2 X 50 mg daily) and carprofen (2 X 150 mg daily) on gastric secretion and the generation of prostaglandins PGE2 and PGF2 alpha in gastric juice, was investigated in a single blind cross-over study in eight healthy volunteers lasting one week. We observed no statistically significant change in basal and pentagastrin-stimulated gastric secretory parameters (outputs of gastric acid, N-acetyl-neuraminic acid and pepsin) before and after treatment with indomethacin and carprofen. However, an inhibitory effect was found on the output of PGE2 and PGF2 alpha after pentagastrin stimulation. While both drugs diminished the output of PGF2 alpha to a similar extent, carprofen exerted a markedly weaker inhibitory effect on the output of PGE2 than did indomethacin. It is suggested that the gastric tolerability of non-steroidal anti-inflammatory drugs (NSAIDs) is related to their inhibitory potency on PGE2 formation, in the sense that weak inhibitors of PGE2 cause less...
Oxidative stress may be one of the important complex pathogenetic mechanisms that lead to damage ... more Oxidative stress may be one of the important complex pathogenetic mechanisms that lead to damage in scleroderma; free radicals may provoke endothelial injury, fibroblast proliferation and fragmentation of autoantigens favouring induction of autoantibodies. The present study investigates the oxidant status in scleroderma patients by measuring the urinary concentration of 8-isoprostaglandin-F2alpha, an F2-isoprostane, and a product of free radical-mediated peroxidation of arachidonic acid. Forty-three scleroderma patients (42 women and 1 man, mean age 54.1 yr, mean disease duration 9.0 yr) underwent clinical evaluation and instrumental investigations in order to assess skin, vascular, lung and heart involvement. Von Willebrand factor was evaluated as marker of vascular dysfunction in 36 out of the 43 cases. The urinary level of 8-isoprostaglandin-F2alpha was measured in all scleroderma patients and in the 43 age- and sex-matched healthy controls. Urinary levels of 8-isoprostaglandin-F2alpha were higher in scleroderma patients than in the healthy control group (341.7 vs 147.6 pg/mg creatinine; P < 0.001). Values of 8-isoprostaglandin-F2alpha were strongly correlated with the nailfold videocapillaroscopy pattern and lung involvement (P = 0.002 and 0.003, respectively), showing increasing levels with the progression of pulmonary severity. Correlation between 8-isoprostaglandin-F2alpha level and von Willebrand factor narrowly failed to reach statistical significance (P = 0.05). There was no correlation between 8-isoprostaglandin-F2alpha concentration and disease activity, vascular, skin and heart involvement, disease pattern or autoantibody profile. Our study further supports the role of oxidant stress in the pathogenesis of scleroderma, showing a strong correlation between a marker of free radical damage with both the severity of lung involvement and the videocapillaroscopic patterns.
The antiplatelet activity of two new nitrocompounds, chemically related to acetylsalicylic acid (... more The antiplatelet activity of two new nitrocompounds, chemically related to acetylsalicylic acid (NCX 4215 and NCX 4016), was studied in vitro to verify the hypothetical dual action of these drugs. Both drugs, in a dose-dependent way, inhibited arachidonic acid-induced platelet aggregation and thromboxane A2 production, measured as thromboxane B2 concentration in whole blood. These effects are likely to be related to cyclo-oxygenase inhibition. NCX 4215 and NCX 4016 in a dose-dependent way inhibited also thrombin-induced aggregation of platelets pretreated with acetylsalicylic acid. These inhibitory effects are related to nitric oxide release and cGMP increase and significantly reversed by oxyhaemoglobin and methylene blue. Either as a cyclo-oxygenase inhibitor or as a nitric oxide donor, NCX 4016 proved to be significantly more potent than NCX 4215.
Plasma from 37 essential hypertensive patients, from 11 subjects with primary aldosteronism and f... more Plasma from 37 essential hypertensive patients, from 11 subjects with primary aldosteronism and from 23 normotensive subjects was tested for ouabain-like activity. Despite a very substantial overlap, hypertensive patients (both essential and secondary) showed significantly higher levels of a ouabain-like plasma factor compared to normotensive controls. No substantial differences could be detected, however, between the two forms of hypertension; in particular, no significant changes were observed in the low-PRA subgroup. Our results are hardly compatible with the hypothesis that this substance may be of crucial importance in the development either of essential hypertension or of primary aldosteronism.
Signals ensuing from trimeric G-protein-coupled receptors synergize to induce platelet activation... more Signals ensuing from trimeric G-protein-coupled receptors synergize to induce platelet activation. At low doses, the thromboxane A2 analogue U46619 does not activate integrin alphaIIbbeta3 or trigger platelet aggregation, but it induces shape changes. In the present study, we addressed whether low doses of U46619 trigger tyrosine phosphorylation independently of integrin alphaIIbbeta3 activation and ADP secretion, and synergize with adrenaline (epinephrine) to induce aggregation in acetylsalicylic acid (aspirin)-treated platelets. Low doses of U46619 triggered tyrosine phosphorylation of different proteins, including FAK (focal adhesion kinase), Src and Syk, independently of signals ensuing from integrin alphaIIbbeta3 or ADP receptors engaged by secreted ADP. The G(12/13)-mediated Rho/Rho-kinase pathway was also increased by low doses of U46619; however, this pathway was not upstream of tyrosine phosphorylation, because this occurred in the presence of the Rho-kinase inhibitor Y-276...
MDs (mitochondrial diseases) are a clinically heterogeneous group of disorders characterized by i... more MDs (mitochondrial diseases) are a clinically heterogeneous group of disorders characterized by impairment of the respiratory chain function with altered oxidative phosphorylation. We tested the hypothesis that the function of vascular endothelium is affected by increased oxidative stress in MDs. A total of 12 patients with MDs and pair-matched controls were studied. Endothelial function was assessed by measuring FMD (flow-mediated vasodilation) of brachial and common femoral arteries. The test was repeated after vitamin C (500 mg, twice a day) and E (400 mg, once a day) supplementation for 30 days and 90 days after vitamin withdrawal. FMD was reduced in patients compared with controls [AUC/τ (time-averaged area under the curve) for the brachial artery, 1.05±0.24 compared with 4.19±0.59% respectively, P<0.001; AUC/τ for the femoral artery, 0.98±0.19 compared with 2.36±0.29% respectively, P=0.001; values are means±S.E.M.] and correlated (brachial artery) with plasma lactate (r=-0.63, P<0.01). Urinary 8-iso-PGF2α (8-iso-prostaglandin F2α) was higher in patients than controls (505.6±85.9 compared with 302.5±38.7 pg/mg of creatinine; P<0.05) and correlated with plasma lactate (r=0.70, P<0.05). Immunohistochemical analysis showed 8-iso-PGF2α staining in MD-affected striated muscle cells and in blood vessels in muscle biopsies of patients. Antioxidant vitamins transiently restored FMD in patients [ΔAUC/τ (change in AUC/τ) for the brachial artery, +1.38±0.49%, P<0.05; ΔAUC/τ for the femoral artery, +0.98±0.24%, P<0.01] but had no effect on FMD in controls (brachial artery, -1.3±0.63%; and common femoral artery, -0.58±0.30%), thus abolishing the differences between patients and controls. The results of the present study indicate that oxidative stress is increased and is, at least partly, responsible for endothelial dysfunction in MDs.
It has been suggested that angiotensin-converting enzyme (ACE) inhibitors may also act through th... more It has been suggested that angiotensin-converting enzyme (ACE) inhibitors may also act through the renal prostaglandin (PG) system; moreover, it has been shown that nonsteroidal antiinflammatory drugs (NSAIDs) are capable of reducing the antihypertensive activity of ACE inhibitors. Sixteen essential hypertensive patients (WHO stages, I-II, eight on a low-sodium diet and eight on a high-sodium diet) were treated with enalapril, 20 mg/day per os, for 4 days. On days 3 and 4, ibuprofen, 1,200 mg/day per os, was also given. Enalapril reduced blood pressure, particularly in the group on the low-sodium diet. Urinary 6-keto-PGF1 alpha, an indicator of renal PGI2 production (determined by HPLC-RIA), increased in the first few hours after enalapril in the low-sodium group. Ibuprofen did not reduce the antihypertensive effect of enalapril, nor did it affect plasma renin activity or plasma aldosterone. Only a slight reduction in 6-keto-PGF1 alpha excretion was observed after enalapril plus ibu...
F 2-isoprostanes are prostaglandin (PG) isomers produced in vivo through free radical- catalyzed ... more F 2-isoprostanes are prostaglandin (PG) isomers produced in vivo through free radical- catalyzed peroxidation of arachidonic acid, which may affect platelet function. The current study investigated the effects of 8-epiprostaglandin F 2a (8-epi-PGF2a) on critical events of platelet activation. A dose-dependent increase in platelet adhesion to fibrinogen- and plasma-coated microwells by 8-epi-PGF2a (1 to 1000 nmol/L) was observed when resting
The aim of the study was to evaluate the effects of acute hyperhomocysteinemia on distensibility ... more The aim of the study was to evaluate the effects of acute hyperhomocysteinemia on distensibility and compliance of large peripheral arteries. Isoprostanes generation and antioxidant vitamins were used to assess the role of oxidative stress. A cross-over, double-blind study on distensibility (DC: distensibility coefficient) and compliance (CC: cross-sectional compliance) of common femoral and brachial arteries was performed in 12 healthy young male volunteers by means of a wall track system before and 4 h after a single oral methionine (100 mg/kg) or placebo administration. The effects of methionine load were investigated also after oral administration of vitamin C (1g/day) and vitamin E (800 mg/day) for 8 consecutive days. Oral methionine induced a significant increase in plasmatic levels of homocysteine. Distensibility and compliance of brachial and femoral arteries were significantly reduced after methionine load in comparison to placebo. This acute impairment of arterial wall mechanical properties was associated to endothelial dysfunction, since altered flow-dependent vasodilatation (P < 0.05 versus placebo) was observed in the same arterial districts. A significant increase in urinary 8-iso-prostaglandin F2alpha was observed after methionine. Pretreatment with vitamins C and E prevented the effects of methionine on femoral and brachial arteries as well as on urinary 8-iso-prostaglandin F2alpha excretion. Hyperhomocysteinemia seems responsible for altered arterial wall elasticity and for endothelial dysfunction. A pivotal role can be attributed to oxidative stress.
The effect of indomethacin (2 X 50 mg daily) and carprofen (2 X 150 mg daily) on gastric secretio... more The effect of indomethacin (2 X 50 mg daily) and carprofen (2 X 150 mg daily) on gastric secretion and the generation of prostaglandins PGE2 and PGF2 alpha in gastric juice, was investigated in a single blind cross-over study in eight healthy volunteers lasting one week. We observed no statistically significant change in basal and pentagastrin-stimulated gastric secretory parameters (outputs of gastric acid, N-acetyl-neuraminic acid and pepsin) before and after treatment with indomethacin and carprofen. However, an inhibitory effect was found on the output of PGE2 and PGF2 alpha after pentagastrin stimulation. While both drugs diminished the output of PGF2 alpha to a similar extent, carprofen exerted a markedly weaker inhibitory effect on the output of PGE2 than did indomethacin. It is suggested that the gastric tolerability of non-steroidal anti-inflammatory drugs (NSAIDs) is related to their inhibitory potency on PGE2 formation, in the sense that weak inhibitors of PGE2 cause less...
Oxidative stress may be one of the important complex pathogenetic mechanisms that lead to damage ... more Oxidative stress may be one of the important complex pathogenetic mechanisms that lead to damage in scleroderma; free radicals may provoke endothelial injury, fibroblast proliferation and fragmentation of autoantigens favouring induction of autoantibodies. The present study investigates the oxidant status in scleroderma patients by measuring the urinary concentration of 8-isoprostaglandin-F2alpha, an F2-isoprostane, and a product of free radical-mediated peroxidation of arachidonic acid. Forty-three scleroderma patients (42 women and 1 man, mean age 54.1 yr, mean disease duration 9.0 yr) underwent clinical evaluation and instrumental investigations in order to assess skin, vascular, lung and heart involvement. Von Willebrand factor was evaluated as marker of vascular dysfunction in 36 out of the 43 cases. The urinary level of 8-isoprostaglandin-F2alpha was measured in all scleroderma patients and in the 43 age- and sex-matched healthy controls. Urinary levels of 8-isoprostaglandin-F2alpha were higher in scleroderma patients than in the healthy control group (341.7 vs 147.6 pg/mg creatinine; P < 0.001). Values of 8-isoprostaglandin-F2alpha were strongly correlated with the nailfold videocapillaroscopy pattern and lung involvement (P = 0.002 and 0.003, respectively), showing increasing levels with the progression of pulmonary severity. Correlation between 8-isoprostaglandin-F2alpha level and von Willebrand factor narrowly failed to reach statistical significance (P = 0.05). There was no correlation between 8-isoprostaglandin-F2alpha concentration and disease activity, vascular, skin and heart involvement, disease pattern or autoantibody profile. Our study further supports the role of oxidant stress in the pathogenesis of scleroderma, showing a strong correlation between a marker of free radical damage with both the severity of lung involvement and the videocapillaroscopic patterns.
The antiplatelet activity of two new nitrocompounds, chemically related to acetylsalicylic acid (... more The antiplatelet activity of two new nitrocompounds, chemically related to acetylsalicylic acid (NCX 4215 and NCX 4016), was studied in vitro to verify the hypothetical dual action of these drugs. Both drugs, in a dose-dependent way, inhibited arachidonic acid-induced platelet aggregation and thromboxane A2 production, measured as thromboxane B2 concentration in whole blood. These effects are likely to be related to cyclo-oxygenase inhibition. NCX 4215 and NCX 4016 in a dose-dependent way inhibited also thrombin-induced aggregation of platelets pretreated with acetylsalicylic acid. These inhibitory effects are related to nitric oxide release and cGMP increase and significantly reversed by oxyhaemoglobin and methylene blue. Either as a cyclo-oxygenase inhibitor or as a nitric oxide donor, NCX 4016 proved to be significantly more potent than NCX 4215.
Plasma from 37 essential hypertensive patients, from 11 subjects with primary aldosteronism and f... more Plasma from 37 essential hypertensive patients, from 11 subjects with primary aldosteronism and from 23 normotensive subjects was tested for ouabain-like activity. Despite a very substantial overlap, hypertensive patients (both essential and secondary) showed significantly higher levels of a ouabain-like plasma factor compared to normotensive controls. No substantial differences could be detected, however, between the two forms of hypertension; in particular, no significant changes were observed in the low-PRA subgroup. Our results are hardly compatible with the hypothesis that this substance may be of crucial importance in the development either of essential hypertension or of primary aldosteronism.
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Papers by M. Degan