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Biomedical subjects

Andrea Semplicini

Publications and source records attributed to Andrea Semplicini.

At least 37 records · Page 2Linked to original sources
PubMed2005

Angiotensin II-induced over-activation of p47phox in fibroblasts from hypertensives: which role in the enhanced ERK1/2 responsiveness to angiotensin II?

BACKGROUND: Fibroblasts are involved in the remodeling of the heart and of the vasculature associated to arterial hypertension, and an abnormal extracellular signal-regulated kinase 1/2 (ERK1/2) activation by angiotensin II (Ang II) plays a pivotal role in this process. However, the intracellular pathways leading to cell hypertrophy and hyperplasia, as well as to collagen production, are still incompletely known. OBJECTIVE: To investigate the role of superoxide anion (O2) and of nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase in Ang II-stimulated ERK1/2 over-activation in fibroblasts from hypertensive patients. METHODS: O2 production was measured in skin fibroblasts from hypertensives (HT, n = 11) and from normotensive controls (NT, n = 10) by electron spin resonance technique. ERK1/2 phosphorylation and p47phox NAD(P)H oxidase subunit translocation were measured by western blot. RESULTS: Ang II (1 micromol/l) induced a larger p47phox subunit translocation and increased intracellular O2 production to a larger extent in HT in comparison to NT and this effect was blocked by apocynin, an inhibitor of the NAD(P)H oxidase. Ang II increased ERK1/2 phosphorylation more in HT than in NT. The Ang II-induced ERK1/2 phosphorylation was inhibited by apocynin in a dose-dependent manner in NT, but not in HT. CONCLUSIONS: The chain of cellular events leading to increased ERK1/2 responsiveness to Ang II in hypertension include an exaggerated response of p47phox, NAD(P)H oxidase and O2, but it is partially resistant to apocynin. Therefore, NAD(P)H-dependent reactive oxygen species (ROS) production is not the only determinant of the exaggerated ERK1/2 responsiveness in fibroblasts of hypertensives (HT).

Acetophenones↗
PubMed2005

Metformin prevents glucose-induced protein kinase C-beta2 activation in human umbilical vein endothelial cells through an antioxidant mechanism.

Hyperglycemia determines the vascular complications of diabetes through different mechanisms: one of these is excessive activation of the isoform beta2 of protein kinase C (PKC-beta2). Metformin, a widely used antidiabetic agent, is associated with decreased cardiovascular mortality in obese type 2 diabetic patients. Therefore, we assessed the role of metformin in glucose-induced activation of PKC-beta2 and determined the mechanism of its effect in human umbilical venous endothelial cells grown to either normo- (5 mmol/l) or hyperglycemia (10 mmol/l) and moderately and acutely exposed to 25 mmol/l glucose. We studied PKC-beta2 activation by developing adenovirally expressed chimeras encoding fusion protein between green fluorescent protein (GFP) and conventional beta2 isoform (PKC-beta2-GFP). Glucose (25 mmol/l) induced the translocation of PKC-beta2-GFP from the cytosol to the membrane in cells grown to hyperglycemia but not in those grown in normal glucose medium. Metformin (20 micromol/l) prevented hyperglycemia-induced PKC-beta2-GFP translocation. We also assessed oxidative stress under the same conditions with a 4-((9-acridine-carbonyl)amino)-2,2,6,6-tetramethylpiperidin-oxyl,free radical (TEMPO-9-AC) fluorescent probe. We observed significantly increased radical oxygen species production in cells grown in hyperglycemia medium, and this effect was abolished by metformin. We show that in endothelial cells, metformin inhibits hyperglycemia-induced PKC-beta2 translocation because of a direct antioxidant effect. Our data substantiate the findings of previous large intervention studies on the beneficial effect of this drug in type 2 diabetic patients.

Cells, Cultured↗
PubMed2004

Callipeltin A: sodium ionophore effect and tension development in vascular smooth muscle.

Callipeltin A is a cyclic depsidecapeptide isolated from the marine sponges Callipelta sp. and Latrunculia sp. that has been previously shown to increase the force of contraction of guinea-pig atria through the inhibition of Na+/Ca2+ exchanger (NCX). We investigated the effect of callipeltin A on guinea-pig aortic rings contracted by procedures that activate NCX in "calcium entry mode". Callipeltin A did not inhibit these contractions. Resting aorta responded to callipeltin A with a remarkable contraction that was concentration-dependent (EC50 0.44microM). This contraction was not inhibited by the calcium channel blocker verapamil and was not mediated by the activation of alpha-adrenergic or endothelin-1 receptors. Pre-incubation of aortic rings with 0.5mM amiloride, an inhibitor of NCX, completely prevented callipeltin A-induced contraction. Furthermore, callipeltin A (EC50 0.51microM) increased Na+ efflux of Na-loaded erythrocytes. 1H and 13C NMR resonances of callipeltin A revealed small but significant changes in the titration with K+ and Na+ salts. It is suggested that the effect of callipeltin A on cardiac and vascular preparations is linked to a Na-ionophore action.

Animals↗
PubMed2004

Effects of angiotensin II and insulin on ERK1/2 activation in fibroblasts from hypertensive patients.

BACKGROUND: Insulin resistance, a frequent finding in hypertensive patients, leads to accelerated cardiovascular damage. It has been suggested that a crosstalk between angiotensin II and insulin signaling pathways may provoke insulin resistance, and may contribute to the development of cardiovascular damage. To identify a common pathophysiologic pathway between metabolic disorders and cardiovascular remodeling, we investigated the effect of angiotensin II and insulin on extracellular signal regulated kinases 1 and 2 (ERK1/2), isoforms of mitogen-activated protein kinases (MAPK) involved in cellular proliferation and extracellular matrix deposition. METHODS: Skin fibroblasts from normotensive subjects, insulin sensitive hypertensive subjects, and insulin resistant hypertensive subjects were cultured and used after four passages. The ERK1/2 expression and phosphorylation were measured by Western blot using specific antibodies, respectively anti-ERK1/2 and anti-pERK1/2. Expression of AT1 receptor for angiotensin II was determined by reverse transcriptase-polymerase chain reaction in real time. RESULTS: The ERK1/2 were similarly expressed in skin fibroblasts from all groups; ERK1/2 phosporylation evoked by angiotensin II was significantly higher in fibroblasts from hypertensive patients in comparison to normotensive subjects, but the increase was observed only in insulin resistant hypertensive subjects. The effect of insulin on ERK1/2 phosphorylation was not significantly different in the three groups. Treatment with the combination of insulin and angiotensin II increased ERK1/2 phosphorylation to a greater extent in comparison to the single agonists in normotensive subjects and in insulin sensitive but not in insulin resistant hypertensive subjects. CONCLUSIONS: Angiotensin II stimulated ERK1/2 activation is increased in insulin resistant hypertensive subjects, and it may play a role in the pathogenesis of insulin resistance and accelerated cardiovascular damage.

Adult↗
PubMed2004

Abnormal regulation of G protein alpha(i2) subunit in skin fibroblasts from insulin-resistant hypertensive individuals.

BACKGROUND: Studies in experimental animals and human cells have demonstrated increased intracellular calcium (Ca(i2) signalling and Galphai signal transduction associated with hypertension. We have recently shown that angiotensin II-induced mobilization of Ca(i2) is enhanced in fibroblasts from hypertensive individuals in comparison with that in normotensive individuals and that it is blunted by insulin and pertussis toxin in insulin-sensitive, but not in insulin-resistant, patients. This suggests that G(i)-mediated signal transduction is reduced in insulin-resistant hypertension. OBJECTIVE: To investigate the expression and regulation of Galpha(i2) subunit in insulin-sensitive and insulin-resistant hypertensive individuals. METHODS: G protein alpha(i2) subunit mRNA was measured in cultured skin fibroblasts from patients with insulin-sensitive and insulin-resistant hypertension, by real-time reverse transcriptase polymerase chain reaction. We also investigated the effects of short-term exposure to fetal calf serum, angiotensin II and insulin, alone and in combination, on the expression of Galpha(i2) in vitro. Spectrofluorophotometric measurement of free Cai was performed in monolayers of 24 h serum-deprived cells in basal conditions and after exposure to angiotensin II, with and without pre-incubation with insulin. RESULTS: Expression of Galpha(i2) was significantly greater in fibroblasts from hypertensive individuals than in normotensive individuals and the increase was unrelated to age and body mass. The difference was largely accounted for by greater values in insulin-sensitive than in insulin-resistant hypertensive individuals. In fibroblasts from those with insulin-sensitive hypertension, angiotensin II and insulin were additive to fetal calf serum in increasing the expression of Galpha(i2). In these patients, insulin blunted the angiotensin-II induced Cai transient. In contrast, in those with insulin-resistant hypertension, Galpha(i2) was lower and unresponsive to angiotensin II and insulin. Finally, in fibroblasts from insulin-resistant patients, insulin was unable to reduce the angiotensin II-induced Cai peak. CONCLUSIONS: A subnormal Galpha(i2)-mediated signal transduction may be involved in the pathogenesis of cellular insulin resistance in hypertension. This novel Galpha(i2)-mediated signal transduction associated with insulin sensitivity in fibroblasts may help to control excessive angiotensin II signalling.

Adult↗
PubMed2004

Rho kinase and PAI-1 in Bartter's/Gitelman's syndromes: relationship to angiotensin II signaling.

OBJECTIVE: Angiotensin II (Ang II)-mediated activation of Rho kinase (ROK) is involved in the pathophysiology of hypertension and cardiovascular remodeling. ROK also controls plasminogen activator inhibitor-1 (PAI-1) which promotes vascular fibrosis contributing to atherogenesis. Bartter's and Gitelman's syndromes (BS/GS) are useful models to investigate abnormalities of vascular tone regulation, due to their reduced short- and long-term signaling pathways of Ang II. This study evaluated, using BS/GS as a model, ROK and PAI-1 gene and protein expression and the effect of Ang II co-incubation on ROK and PAI-1 gene and protein expression. DESIGN, METHODS AND RESULTS: We measured ROK and PAI-1 gene and protein expression [reverse transcription-polymerase chain reaction (RT-PCR) and Western blot] in mononuclear cells (PBM) from one BS and eight GS patients. The effect of Ang II on ROK and PAI-1 gene and protein expression was also evaluated and compared with 10 controls. ROK gene and protein expression was reduced in BS/GS [0.47 +/- 0.11 densitometric units (d.u.) versus 0.70 +/- 0.04 d.u., P = 0.0038 and 0.39 +/- 0.07 d.u. versus 0.55 +/- 0.07 d.u., P = 0.0026, respectively]. The basal level of PAI-1 gene and protein expression did not differ (0.40 +/- 0.03 d.u. versus 0.39 +/- 0.02 d.u. and 0.81 +/- 0.02 d.u. versus 0.83 +/- 0.02 d.u., respectively). Ang II increased ROK and PAI-1 gene and protein expression only in controls: from 0.70 +/- 0.04 to 0.90 +/- 0.06 d.u., P = 0.007 (ROK mRNA); from 0.55 +/- 0.07 to 0.86 +/- 0.07 d.u., P = 0.0005 (ROK protein); from 0.40 +/- 0.02 to 0.63 +/- 0.03 d.u., P = 0.001 (PAI-1 mRNA); and from 0.83 +/- 0.02 to 1.34 +/- 0.16 d.u., P = 0.0023 (PAI-1 protein). CONCLUSIONS: This study confirms BS/GS as a human model to investigate interrelated systems involved in the pathophysiology of hypertension and throws more light on the cellular mechanisms of BS/GS reduced Ang II short- and long-term signaling pathways.

Adult↗
PubMed2004

Effect of manidipine on gene expression and protein level of oxidative stress-related proteins: p22phox and HO-1: relevance for antihypertensive and anti-remodeling effects.

Oxidative stress (OxSt) is a major damaging factor in arterial hypertension and its long-term complications. This is why considerable attention is paid to the possible effects of antihypertensive drugs on OxSt. Manidipine is a dihydropiridine calcium channel blocker with reported nephroprotective activities, but no information is available on its effect on OxSt and related mechanisms. This study assessed the effect of manidipine on normal subjects' monocyte gene and protein expression of OxSt-related proteins such as p22(phox), a NAD(P)H oxidase system subunit, critical in generating O2-, and heme oxygenase-1 (HO-1), induced by and protective from OxSt, and compared manidipine with the ACE inhibitor captopril and the calcium channel blocker nifedipine, in the presence and absence of sodium arsenite (NaAsO2) as an inducer of OxSt.Co-incubation of manidipine with NaAsO2 dose-dependently decreased p22(phox) mRNA production from basal: 0.87 +/- 0.1 d.u., 0.69 +/- 0.06 and 0.66 +/- 0.09 at 100, 300 and 500 nM respectively versus 0.99 +/- 0.2, P < 0.04, while HO-1 mRNA production was increased by the same concentrations of the drug: 0.87 +/- 0.1 d.u., 0.92 +/- 0.1, 0.98 +/- 0.1 respectively versus 0.63 +/- 0.07; P < 0.03. Monocyte p22(phox) mRNA production was reduced both by manidipine and captopril: 0.48 +/- 0.04 d.u. and 0.43 +/- 0.08, respectively versus 0.58 +/- 0.07, P < 0.006, while no changes were induced by nifedipine (0.61 +/- 0.07, P = ns). Manidipine increased monocyte HO-1 mRNA production (1.6 +/- 0.4 versus 1.2 +/- 0.4, P < 0.008), while nifedipine and captopril showed no effect (1.2 +/- 0.3 and 1.1 +/- 0.3, respectively). The effects of M on p22(phox) and HO-1 gene expression in the presence of OxSt were also paralleled by the same effects at protein level. In conclusion, manidipine decreases p22(phox) and increases HO-1 mRNA production and protein level. The manidipine-induced increase of HO-1 gene and protein expression seems to be a peculiar effect of this drug since it is not observed with captopril and nifedipine. This effect, together with the reduction of p22(phox) mRNA production, could play a role in its protective mechanism against OxSt.

Adult↗
PubMed2004

Increased expression of regulator of G protein signaling-2 (RGS-2) in Bartter's/Gitelman's syndrome. A role in the control of vascular tone and implication for hypertension.

Regulator of G protein signaling-2 (RGS-2) plays a key role in the G protein-coupled receptor (GPCR) angiotensin II (Ang II) signaling. NO and cGMP exert a vasodilating action also through activation and binding to RGS-2 of cGMP dependent protein kinase 1-alpha, which phosphorylates RGS-2 and dephosphorylates myosin light chain. In Bartter's/Gitelman's patients (BS/GS) Ang II related signaling and vasomotor tone are blunted. Experiments were planned to explore whether RGS-2 may play a role in BS/GS vascular hyporeactivity. NO metabolites and cGMP urinary excretion were also measured. Mononuclear cells (PBM) from six BS/GS patients and six healthy controls were used. PBM RGS-2 mRNA and RGS-2 protein were increased in BS/GS: 0.47 +/- 0.06 d.u. vs 0.32 +/- 0.04, (p < 0.006) (RGS-2 mRNA), and 0.692 +/- 0.02 vs 0.363 +/- 0.06 (p < 0.0001) (RGS2 protein). Incubation of PBM with Ang II increased RGS-2 protein in controls (from 0.363 +/- 0.06 d.u. to 0.602 +/- 0.05; p < 0.0001) but not in BS/GS (from 0.692 +/- 0.02 to 0.711 +/- 0.02). NO(2)(-)/NO(3)(-) and cGMP urinary excretion were increased in BS/GS (0.46 +/- 0.13 vs 0.26 +/- 0.05 micromol/micromol of urinary creatinine, p < 0.005, and 0.060 +/- 0.030 vs 0.020 +/- 0.01 p < 0.009, respectively). These results demonstrate that RGS-2 is increased and maximally stimulated in BS/GS and human RGS-2 system reacts as predicted by knockout mice experiments. This is the first report of RGS-2 level in a human clinical condition characterized by altered vascular tone, underlines the importance of RGS-2 as a key regulator element for Ang II signaling and provides insight into the links between BS/GS genetic abnormalities and abnormal vascular tone regulation.

Adult↗
PubMed2004

Insulin generates free radicals by an NAD(P)H, phosphatidylinositol 3'-kinase-dependent mechanism in human skin fibroblasts ex vivo.

Oxidative stress may be involved in the development of vascular complications associated with diabetes; however, the molecular mechanism responsible for increased production of free radicals in diabetes remains uncertain. Therefore, we examined whether acute hyperinsulinemia increases the production of free radicals and whether this condition affects proliferative extracellular signal-regulated kinase (ERK-1 and -2) signaling in human fibroblasts in vitro. Insulin treatment significantly increased intracellular superoxide anion (O(2)(-)) production, an effect completely abolished by Tiron, a cell-permeable superoxide dismutase (SOD) mimetic and by polyethylene glycol (PEG)-SOD, but not by PEG catalase. Furthermore, insulin-induced O(2)(-) production was attenuated by the NAD(P)H inhibitor apocynin, but not by rotenone or oxypurinol. Inhibition of the phosphatidylinositol 3'-kinase (PI 3'-kinase) pathway with LY294002 blocked insulin-stimulated O(2)(-) production, suggesting a direct involvement of PI 3'-kinase in the activation of NAD(P)H oxidase. The insulin-induced free radical production led to membranous translocation of p47phox and markedly enhanced ERK-1 and -2 activation in human fibroblasts. In conclusion, these findings provided direct evidence that elevated insulin levels generate O(2)(-) by an NAD(P)H-dependent mechanism that involves the activation of PI 3'-kinase and stimulates ERK-1- and ERK-2-dependent pathways. This effect of insulin may contribute to the pathogenesis and progression of cardiovascular disease in the insulin resistance syndrome.

Biological Transport↗
PubMed2003

G-protein beta3-subunit gene 825T allele and hypertension: a longitudinal study in young grade I hypertensives.

The 825T allele of the GNB3 gene has been associated with essential hypertension and obesity in cross-sectional studies. We have therefore planned a longitudinal cohort study to assess whether the GNB3 825T allele is predictive of blood pressure increase in young subjects with grade I hypertension. We genotyped at the GNB3 825 locus 461 participants of the Hypertension and Ambulatory Recording Venetia Study (HARVEST) study (age, 18 to 45 years) at low cardiovascular risk, according to 1999 ISH/WHO criteria. The study end point was eligibility for antihypertensive medication, that is, progression to grade II hypertension during the first year of observation or office systolic blood pressure > or =150 mm Hg and/or office diastolic blood pressure > or =95 mm Hg in two later consecutive visits during follow-up. At baseline, there was no statistically significant difference among genotypes with respect to body mass index, blood pressure, and heart rate. During follow-up (mean, 4.7 years), 113 (51.1%) patients with CC genotype and 145 (60.4%) patients with TT/TC genotype reached the end point. According to survival analysis, the patients carrying the 825T allele had an increased risk of reaching the blood pressure end point (CI, 1.108 to 1.843; P=0.006). In young patients with grade I hypertension, the 825T allele is associated with increased risk of progression to more severe hypertension requiring antihypertensive therapy. The GNB3 825T allele may be considered a genetic marker of predisposition for hypertension.

Adolescent↗
PubMed2003

The Li+/Na+ exchange in hypertension.

The red cell membrane Li+/Na+exchange is a heteroexchange that operates in either direction across the cell membrane. It binds either Li+ or Na+ on one side of the membrane and it exchanges the transported species for either Li+ or Na+ on the opposite side in a stoichiometric ratio of 1:1. In the population, Li+/Na+exchange is unimodally distributed but skewed to the right. Such distribution results from superimposition of two normal distributions. Many laboratories have shown that red-cell Li+/Na+ exchange is increased in patients with essential hypertension, compared with normotensive controls. Among the various alterations of cell membrane cation transport reported in hypertension, the increase of red-cell Li+/Na+ exchange has been most widely investigated and confirmed. Moreover, increased Li+/Na+ exchange has been found in some clinical conditions related to hypertension, such as overweight and diabetes. Among diabetic patients, Li+/Na+ exchange is particularly high in patients with nephropathy, hypertension, and microalbuminuria, leading to the hypothesis that it can be considered a cellular marker of the risk of developing diabetic nephropathy. Furthermore, it is associated with severe and drug-resistant hypertension, insulin resistance, vascular and cardiac hypertrophy, hyperlipidemia, obesity, family history of hypertension, and of major cardiovascular accidents suggesting that high Li+/Na+ exchange could be a biochemical marker for increased cardiovascular risk. Regardless of its the pathophysiological significance, its measurement could be of clinical use as an intermediate phenotype of increased cardiovascular risk.

Antiporters↗
PubMed2003

Hypertension in acute ischemic stroke: a compensatory mechanism or an additional damaging factor?

BACKGROUND: In acute ischemic stroke, a transient blood pressure (BP) elevation is common, but the best management is still unknown. Therefore, we investigated retrospectively the relationship between BP after ischemic stroke and neurological outcome (evaluated by means of the National Institutes of Health Stroke Scale score at day 7). METHODS: The medical records of 92 consecutive patients with acute ischemic stroke, aged 47 to 96 years, were examined. Blood pressure was measured on admission, 4 times during the first 24 hours, 3 times daily for the first 4 days, and twice daily on day 7 (or at discharge). Antihypertensive treatment was given according to American Heart Association guidelines. RESULTS: The region damaged by the stroke was total anterior in 16 patients (17%), partial anterior in 30 (33%), lacunar in 34 (37%), and posterior circulation in 12 (13%). Stroke pathogenesis was cardioembolic in 28 (30%), atherothrombotic in 29 (32%), and lacunar in 34 (37%). The systolic BP range was 140 to 220 mm Hg; diastolic BP, 70 to 110 mm Hg. Initial BP was higher in the group with lacunar infarction than in the other groups (P<.05). The patients with the best outcome had the highest BP during the first 24 hours. The neurological outcome was strongly influenced by baseline stroke severity (NIH Scale score) and admission BP. Better initial neurological conditions and higher initial BP resulted in better neurological outcomes. CONCLUSIONS: The outcome of stroke is influenced by the type of stroke and initial BP. Lacunar stroke and the highest BP on admission carry the best prognosis, whereas the reverse is true for posterior circulation infarction and low BP. We found no evidence that, within the present BP range, hypertension is harmful and that its lowering is beneficial.

Aged↗