Claudio Ferri

Angiotensin II Stimulates Intercellular Adhesion Molecule-1 (ICAM-1) Expression by Human Vascular Endothelial Cells and Increases Soluble ICAM-1 Release In Vivo

BACKGROUNDnWe evaluated whether angiotensin II (Ang II) influenced intercellular adhesion molecule (ICAM)-1 expression by human vascular endothelial cells derived from umbilical cord veins (HUVECs) and plasma soluble ICAM-1 levels in vivo.nnnMETHODS AND RESULTSnCultured HUVECs were incubated with Ang II (from 10(-9) to 10(-6) mol/L) with or without candesartan and PD12319 (inhibitors of Ang II AT(1) and AT(2) receptors, respectively) for various times up to 4 hours. Total RNA was then extracted from HUVECs, and Northern blots were probed with a 1.9-kb ICAM-1 cDNA fragment. HUVEC supernatants were used to assess soluble ICAM-1 release by ELISA. Northern blot analysis detected a strong increase of ICAM-1 mRNA after 2-hour incubation with Ang II. The response was inhibited by candesartan. Soluble ICAM-1 release by HUVECs also increased (P<0. 002) after 2-hour Ang II stimulation. In vivo, Ang II (at an initial rate of 1.0 ng. kg(-1). min(-1), to be increased each 30 minutes by 2.0 ng. kg(-1). min(-1) to the final rate of 7.0 ng. kg(-1). min(-1)) was infused in 8 normotensive and 12 essential hypertensive individuals. In the latter, Ang II was reinfused after 4 weeks on either placebo (n=3), losartan (50 mg UID, n=5), or atenolol (50 mg UID, n=4) treatment. Plasma soluble ICAM-1 levels increased after Ang II infusion in hypertensives and normotensives (P<0.0001 after 90 minutes). Losartan reduced baseline soluble ICAM-1 levels (P<0.05) and Ang II-related ICAM-1 increments.nnnCONCLUSIONSnAng II upregulates ICAM-1 expression by HUVECs and stimulates in vitro and in vivo soluble ICAM-1 release. AT(1) receptor blockade inhibits such endothelial effects of Ang II.

Effects of Naloxone on Myocardial Ischemic Preconditioning in Humans

OBJECTIVESnWe attempted to establish whether naloxone, an opioid receptor antagonist, abolishes the adaptation to ischemia observed in humans during coronary angioplasty after repeated balloon inflations.nnnBACKGROUNDnExperimental studies indicate that myocardial opioid receptors are involved in ischemic preconditioning.nnnMETHODSnTwenty patients undergoing angioplasty for an isolated stenosis of a major epicardial coronary artery were randomized to receive intravenous infusion of naloxone or placebo during the procedure. Intracoronary electrocardiogram and cardiac pain (using a 100-mm visual analog scale) were determined at the end of the first two balloon inflations. Average peak velocity in the contralateral coronary artery during balloon occlusion, an index of collateral recruitment, was also assessed by using a Doppler guide wire in the six patients of each group with a stenosis on the left anterior descending coronary artery.nnnRESULTSnIn naloxone-treated patients, ST-segment changes and cardiac pain severity during the second inflation were similar to those observed during the first inflation (12+/-6 vs. 11+/-7 mm, p = 0.3, and 58+/-13 vs. 56+/-12 mm, p = 0.3, respectively), whereas in placebo-treated patients, they were significantly less (6+/-3 vs. 13+/-6 mm, p = 0.002 and 31+/-21 vs. 55+/-22 mm, p = 0.008, respectively). In both naloxone- and placebo-treated patients, average peak velocity significantly increased from baseline to the end of the first inflation (p = 0.04 and p = 0.02, respectively), but it did not show any further increase during the second inflation.nnnCONCLUSIONSnThe adaptation to ischemia observed in humans after two sequential coronary balloon inflations is abolished by naloxone and is independent of collateral recruitment. Thus, it is due to ischemic preconditioning and is, at least partially, mediated by opioid receptors, suggesting their presence in the human heart.

Plasma endothelin-1 levels, pulmonary hypertension, and lung fibrosis in patients with systemic sclerosis

PURPOSEnTo investigate the behavior of circulating endothelin (ET)-1 concentrations in patients affected by systemic sclerosis, and to elucidate the possible relationships existing in this disease among plasma peptide levels, pulmonary hypertension, and lung fibrosis.nnnPATIENTS AND METHODSnCirculating ET-1 levels were determined by reverse-phase, high-pressure liquid chromatography followed by sensitive radioimmunoassay in 20 patients affected by systemic sclerosis (18 women and 2 men, mean age 48.1 +/- 13.7 years) with or without pulmonary hypertension as evaluated by Doppler echocardiography, or lung fibrosis as measured by a score method based on lung examination by high-resolution computed tomography (HRCT). A group of 18 normal volunteers served as controls (15 women and 3 men, mean age 45.0 +/- 10.1 years).nnnRESULTSnPlasma ET-1 levels were significantly higher (P < 0.001) in patients with systemic sclerosis (1.72 +/- 0.28 pg/mL) than in control subjects (0.63 +/- 0.06 pg/mL). Pulmonary artery systolic hypertension was detected in 10 patients (50%) with systemic sclerosis (56.2 +/- 18.0 mm Hg, range 37 to 97) versus none of the control subjects (30.2 +/- 2.2 mm Hg, P < 0.0001). Lung fibrosis was present in 12 patients (60%), with an HRCT overall score of 9.0 +/- 4.6. There were no significant differences in plasma ET-1 levels between patients with pulmonary hypertension (1.58 +/- 0.20 pg/mL) or without it (1.76 +/- 0.39 pg/mL, P = 0.188, not significant [NS]); or between patients with lung fibrosis (1.65 +/- 0.14 pg/mL) or without fibrosis (1.78 +/- 0.37 pg/mL, P = 0.290, NS). In particular, 6 patients had neither pulmonary hypertension nor lung fibrosis. In these patients, plasma ET-1 levels were similar compared with the others (1.85 +/- 0.49 versus 1.66 +/- 0.13, respectively; P = 0.180, NS). No correlations were observed between ET-1 levels and either pulmonary pressure levels or HRCT overall scores.nnnCONCLUSIONSnThe use of a sensitive assay, highly selective for ET-1, showed higher levels of circulating peptide in patients affected by systemic sclerosis than in control subjects. Neither pulmonary hypertension nor lung fibrosis was accompanied by a further rise in plasma ET-1 concentrations.

Reduction of oxidative stress by oral N-acetyl-L-cysteine treatment decreases plasma soluble vascular cell adhesion molecule-1 concentrations in non-obese, non-dyslipidaemic, normotensive, patients with non-insulin-dependent diabetes.

Summary To assess in vivo effects of antioxidants on vascular cell adhesion molecule (VCAM)-1 expression, circulating soluble VCAM-1 and intraerythrocytic reduced glutathione (GSH) and GSH disulphide (GSSG) concentrations were evaluated in non-insulin-dependent diabetic patients without complications (9 men, 6 women, 48 ± 6 years old) before and after 1 month of either oral N-acetyl-L-cysteine (1.200 mg/day) or placebo treatments, given in randomized, cross-over, double-blind fashion. Ten healthy subjects (7 men, 3 women, 52 ± 4 years old) served as control subjects. Baseline plasma VCAM-1 concentrations were higher (p = 0.007) in non-insulin-dependent diabetic patients (707.9 ± 52.5 ng/ml) than in control subjects (627.3 ± 84.6 ng/ml). Intraerythrocytic GSSG content was higher (non-insulin dependent diabetic patients: 0.618 ± 0.185 μmol/g Hb; control subjects: 0.352 ± 0.04 μmol/g Hb, p = 0.0002), whereas intraerythrocytic GSH concentrations were lower (p = 0.001) in non-insulin dependent diabetic patients (6.0 ± 0.7 μmol/g Hb) than in control subjects (7.1 ± 0.5 μmol/g Hb). The mean GSH:GSSG ratio was also lower (p = 0.0001) in the first (10.9 ± 4.5) than in the second group (20.2 ± 1.4). Circulating VCAM-1 and intraerythrocytic GSH concentrations were negatively correlated in non-insulin diabetic patients (r = 0.605, p = 0.01). Treatment with N-acetyl-L-cysteine decreased plasma VCAM-1 (p = 0.01) and intraerythrocytic GSSG (p = 0.006) but increased GSH concentrations (p = 0.04) and the GSH:GSSG ratio (p = 0.004) in non-insulin dependent diabetic patients. Our data indicate that the vascular endothelium is activated in non-insulin dependent diabetes. Antioxidant treatment counterbalanced such endothelial activation. Thus, antioxidant agents might protect against oxidant-related upregulation of endothelial adhesion molecules and slow down the progression of vascular damage in non-insulin dependent diabetes. [Diabetologia (1998) 41: 1392–1396]

Influence of reduced glutathione infusion on glucose metabolism in patients with non—insulin-dependent diabetes mellitus

To evaluate the relationship between oxidative stress and glucose metabolism, insulin sensitivity and intraerythrocytic reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio were measured in 10 non-insulin-dependent diabetes mellitus (NIDDM) patients and 10 healthy subjects before and after the intravenous administration of GSH. In particular, after baseline insulin sensitivity was assessed by a 2-hour euglycemic hyperinsulinemic clamp, either glutathione (1.35 g x m2 x min(-1)) or placebo (saline) were infused over a period of 1 hour. The same protocol was repeated at a 1-week interval, in cross-over, according to a randomized, single-blind design. In healthy subjects, baseline intraerythrocytic GSH/GSSG ratio (P < .0005) and total glucose uptake (P < .005) were significantly higher than in NIDDM patients. In the same subjects, GSH infusion significantly increased total glucose uptake (from 37.1 +/- 6.7 micromol kg(-1) x min(-1) to 39.5 +/- 7.7 micromol x kg(-1) x min(-1), P < .05), whereas saline infusion was completely ineffective. In addition, the mean intraerythrocytic GSH/GSSG ratio significantly increased after GSH infusion (from 21.0 +/- 0.9 to 24.7 +/- 1.3, P < .05). Similar findings were found in diabetic patients, in whom GSH infusion significantly increased both total glucose uptake (from 25.3 +/- 9.0 micromol x kg(-1) x min(-1) to 31.4 +/- 10.0 micromol x kg(-1) x min(-1), P < .001) and intraerythrocytic GSH/GSSG ratio (from 14.8 +/- 4.1 to 21.7 +/- 6.7, P < .01). Pooling diabetic patients and controls, significant correlations were found between intraerythrocytic GSH/GSSG ratio and total glucose uptake (r = .425, P < .05), as well as between increments of the same variables after GSH infusion (r = .518, P < .05). In conclusion, our data support the hypothesis that abnormal intracellular GSH redox status plays an important role in reducing insulin sensitivity in NIDDM patients. Accordingly, intravenous GSH infusion significantly increased both intraerythrocytic GSH/GSSG ratio and total glucose uptake in the same patients.

Polyol pathway activation and glutathione redox status in non-insulin-dependent diabetic patients.

The current study aimed to evaluate whether nicotinamide adenine dinucleotide phosphate (NADPH) alteration in erythrocytes from patients with non-insulin-dependent diabetes mellitus (NIDDM) is responsible for the impaired glutathione (GSH) redox status, and to assess if short-term inhibition of the polyol pathway normalizes NADPH levels and GSH redox status via an amelioration of the NADPH/total NADP (tNADP) ratio. For this purpose, erythrocyte NADPH and GSH levels were measured in 18 NIDDM patients at baseline and then after 1 week of random double-blind assignment to treatment with either tolrestat (an aldose reductase inhibitor, 200 mg daily) (n = 12) or placebo (n = 6). A group of 16 healthy volunteers served as the control. In the basal condition, mean GSH (P < .0001) and NADPH (P < .0001) levels and NADPH/tNADP (P < .0001) and GSH/ glutathione disulfide (GSSG) (P < .005) ratios were lower in NIDDM patients than in control subjects. Tolrestat treatment increased GSH levels (P < .05 v placebo and baseline) and the NADPH/tNADP ratio (P < .05 v placebo and baseline). Interestingly, tolrestat-induced changes in GSH and NADPH levels and in GSH/GSSG and NADPH/tNADP ratios were significant only in patients who showed a decreased NADPH/tNADP ratio at baseline (n = 8). In these latter patients, we also found a direct correlation between percentage increments in GSH levels and NADPH/tNADP ratios after tolrestat treatment (r = .71, P < .05). In conclusion, our findings support the hypothesis that polyol pathway activation decreases NADPH and GSH levels. Accordingly, short-term inhibition of this enzymatic route increased both the GSH level and the NADPH/tNADP ratio. These changes were observable only in the subgroup of patients with an abnormal NADPH/tNADP ratio at baseline. Polyol pathway inhibition could be useful for decreasing oxidative stress in NIDDM.

Role of Plasma and Urinary Endothelin-1 in Early Diabetic and Hypertensive Nephropathy

To evaluate the role of circulating and renal endothelin-1 (ET-1) in early diabetic nephropathy, plasma ET-1 levels and urinary ET-1 excretion were evaluated in lean, normotensive patients affected by non-insulin-dependent diabetes (NIDDM) either with (n = 9, NIDDM+) or without microalbuminuria (n = 18, NIDDM-); in never-treated, lean, essential hypertensive patients with (n = 12, EH+) or without microalbuminuria (n = 10, EH-); and in healthy volunteers (n = 12). Results showed higher plasma ET-1 levels in NIDDM+ (1.97 +/- 0.58 pg/mL) than in NIDDM- (1.59 +/- 0.14 pg/mL, P = .013), EH+ (1.40 +/- 0.21 pg/mL, P = .005), EH- (0.91 +/- 0.19 pg/mL, P < .0001), and controls (0.60 +/- 0.10 pg/mL, P < .0001). The circulating ET-1 concentration was also higher in EH+ than EH- and controls (P < .0001). Urinary ET-1 excretion did not differ (P = .387, NS) between NIDDM+ (48.5 +/- 20.1 pg/min) and NIDDM- (40.9 +/- 21.6 pg/min), but was significantly reduced (P < .0001) in both groups compared with controls (70.0 +/- 15.5 pg/min). Similar findings were observed in hypertensive subgroups. No correlations were found between urinary ET-1 and other variables, including plasma ET-1 levels, in all groups. In conclusion, NIDDM+ is accompanied by a significant increase in plasma ET-1 levels. A significant elevation of circulating ET-1 concentration was evident also in NIDDM-, suggesting that early abnormalities of ET-1 production might precede the microalbuminuric phase of diabetes-related renal damage.

Enhanced activity of sodium-lithium countertransport in patients with cardiac syndrome X: a potential link between cardiac and metabolic syndrome X.

OBJECTIVESnThis study was aimed at assessing both stimulated insulinemia and the sodium-lithium countertransport in a selected group of patients with cardiac syndrome X.nnnBACKGROUNDnHyperinsulinemia, which is frequently present in patients with cardiac syndrome X, is often associated with an enhanced activity of the sodium-lithium countertransport, an in vitro marker of sodium-hydrogen exchange.nnnMETHODSnFifteen patients with syndrome X and 14 matched controls were studied. After pharmacological washout, sodium-lithium countertransport was assessed from lithium-loaded red blood cells. Postload insulin levels were evaluated by a double-antibody radioimmunoassay.nnnRESULTSnMaximal velocity of sodium-lithium countertransport was higher in patients with syndrome X compared to controls (635+/-200 vs. 324+/-49 micromol/liter/h, p = 0.001). Fourteen of the 15 patients with syndrome X (93%) presented sodium-lithium countertransport values higher than the mean +2 SD of the control group. At 120 min, 12 patients with syndrome X (80%) had plasma levels of insulin >420 pmol/liter, which corresponds to the mean value +2 SD of controls (p = 0.006).nnnCONCLUSIONSnBoth enhanced activity of the sodium-lithium countertransport and stimulated hyperinsulinemia are present in the vast majority of patients with cardiac syndrome X. As enhanced activity of the sodium-lithium countertransport has the potential to cause both glucose intolerance and smooth muscle hyperreactivity, it might represent a common cause of the metabolic and vascular alterations frequently found in syndrome X.

Elevated plasma endothelin-1 levels as an additional risk factor in non-obese essential hypertensive patients with metabolic abnormalities.

Summary Circulating endothelin-1 concentration was evaluated in 93 lean patients with essential hypertension, of whom 16 had impaired glucose tolerance and hyperlipidaemia, 25 had impaired glucose tolerance, 28 had hyperlipidaemia and 24 had no metabolic abnormalities; we also studied 22 control subjects. All groups were age- and sex-matched. Plasma endothelin-1 levels were higher (p < 0.05) in hypertensive patients with impaired glucose tolerance and hyperlipidaemia than in the remaining groups, and were directly correlated with fasting insulin levels (r = 0.506, p = 0.045). Therefore, circulating endothelin-1 concentrations are elevated in hypertensive patients with a high-risk profile due to the presence of metabolic abnormalities, and might favour the development of vascular damage. [Diabetologia (1997) 40: 100–102]

Plasma endothelin-1 levels in patients with systemic sclerosis: influence of pulmonary or systemic arterial hypertension.

OBJECTIVES--To investigate the behaviour of circulating endothelin-1 (ET-1) in patients affected by systemic sclerosis and to elucidate the relationship between systemic and pulmonary plasma peptide and arterial pressure levels. METHODS--Plasma ET-1 concentrations were determined in 48 patients affected by systemic sclerosis (41 women, seven men; mean age 47.2 (SD 5.5) years) with or without systemic or pulmonary hypertension (or both). A group of 18 normal volunteers served as controls (15 women, three men; mean age 45.0 (10.1) years). RESULTS--Plasma ET-1 levels were significantly greater in patients affected by systemic sclerosis (1.65 (0.29) pg/ml) than in controls (0.63 (0.19) pg/ml) (p < 0.0001). Pulmonary artery systolic hypertension alone was present in 14 patients with systemic sclerosis (50.5 (8.49) mm Hg, range 37-67 mm Hg), and systemic hypertension alone (160.7 (5.9)/100.6 (3.2) mm Hg) was present in 11 patients. Both conditions were present in 12 patients, while 11 patients had systemic hypertension. There were no significant differences in plasma ET-1 levels between patients with pulmonary hypertension alone (1.62 (0.21) pg/ml) and those with systemic hypertension alone (1.65 (0.43) pg/ml). In particular, patients with normal pulmonary artery and systemic pressures (n = 11) had plasma ET-1 concentrations identical to those found in patients (n = 12) with both pulmonary and systemic hypertension (1.70 (0.15) v 1.64 (0.35) pg/ml, respectively). No correlations were observed between plasma ET-1 and either pulmonary or systemic pressures. CONCLUSION--Systemic sclerosis is characterised by increased plasma ET-1 levels, but neither pulmonary nor systemic hypertension are accompanied by further increase in plasma peptide levels.