Lorenzo A. Calò

Reduced expression of regulator of G-protein signaling 2 (RGS2) in hypertensive patients increases calcium mobilization and ERK1/2 phosphorylation induced by angiotensin II.

Context RGS2 (regulators of G-protein signaling) is a negative regulator of Gαq protein signaling, which mediates the action of several vasoconstrictors. RGS2-deficient mouse line exhibits a hypertensive phenotype and a prolonged response to vasoconstrictors. Objective To compare RGS2 expression in peripheral blood mononuclear cells (PBMs) and cultured fibroblasts from normotensive subjects and hypertensive patients. Methods PBMs were isolated from 100 controls and 150 essential hypertensives. Additionally, fibroblasts were isolated from skin biopsy of 11 normotensives and 12 hypertensives and cultured up to the third passage. Quantitative mRNA and protein RGS2 expression were performed by real-time quantitative reverse transcriptase-polymerase chain reaction and by immunoblotting, respectively. Free Ca2+ measurement was performed in monolayers of 24-h serum-deprived cells, using FURA-2 AM. Phosphorylation of the extracellular signal-regulated kinases ERK1/2 was measured by immunoblotting. Polymorphism (C1114G) in the 3′ untranslated region of the RGS2 gene was investigated by direct sequencing and real-time polymerase chain reaction (PCR). Results RGS2 mRNA expression was significantly lower in PBM and in fibroblasts from hypertensives, in comparison to normotensives. C1114G polymorphism was associated with RGS2 expression, with the lowest values in GG hypertensives. The 1114G allele frequency was increased in hypertensives compared with normotensives. Angiotensin II-stimulated intracellular Ca2+ increase and ERK1/2 phosphorylation were higher in fibroblasts from hypertensive patients compared with control subjects, and in those with the G allele, independently of the blood pressure status. The angiotensin II-stimulated Ca2+ mobilization and ERK1/2 phosphorylation were negatively correlated with RGS2 mRNA expression. Conclusion Low expression of RGS2 contributes to increased G-protein-coupled signaling in hypertensive patients. The allele G is associated with low RGS2 expression and blood pressure increase in humans.

Forearm Nitric Oxide Balance, Vascular Relaxation, and Glucose Metabolism in NIDDM Patients

Endothelium-dependent and -independent vascular responses were assessed in 10 NIDDM patients and 6 normal subjects with no evidence of atherosclerotic disease. Changes in forearm blood flow and arteriovenous (AV) serum nitrite/nitrate (NO2−/NO3-) concentrations were measured in response to intra-arterial infusion of acetylcholine (ACh) (7.5, 15, 30 μg/min, endothelium-dependent response) and sodium nitroprusside (SNP) (0.3, 3, 10 μg/min, endothelium-independent response). Insulin sensitivity (determined by minimal model intravenous glucose tolerance test) was lower in NIDDM patients (0.82 ± 0.20 vs. 2.97 ± 0.29 104 min · μU−1 · ml−1; P < 0.01). Baseline forearm blood flow (4.8 ± 0.3 vs. 4.4 ± 0.3 ml · 100 ml−1 tissue · min−1; NS), mean blood pressure (100 ± 4 vs. 92 ± 4 mmHg; NS), and vascular resistance (21 ± 1 vs. 21 ± 1 units; NS), as well as their increments during ACh and SNP, infusion were similar in both groups. No difference existed in baseline NO2−/NO3− concentrations (4.09 ± 0.33 ]NIDDM patients] vs. 5.00 ± 0.48 μmol/1 ]control subjects]; NS), their forearm net balance (0.31 ± 0.08 ]NIDDM patients] vs. 0.26 ± 0.08 μmol/1 · 100 ml−1 tissue · min−1; NS), and baseline forearm glucose uptake. During ACh infusion, both NO2− and NO3− concentrations and net balance significantly increased in both groups, whereas glucose uptake increased only in control subjects. When data from NIDDM and control groups were pooled together, a correlation was found between the forearm AV NO2− and NO3− differences and blood flow (r = 0.494, P = 0.024). On the contrary, no correlation was evident between NO2− and NO3− concentrations or net balance and insulin sensitivity. In summary, 1) no difference existed in basal and ACh-stimulated NO generation and endothelium-dependent relaxation between uncomplicated NIDDM patients and control subjects; 2) in both NIDDM and control groups, forearm NO2− and NO3− net balance following ACh stimulation was related to changes in the forearm blood flow; and 3) ACh-induced increase in forearm blood flow was associated with an increase in glucose uptake only in control subjects but not in NIDDM patients. In conclusion, our results argue against a role of impaired NO generation and blood flow regulation in determining the insulin resistance of uncomplicated NIDDM patients; rather, it supports an independent insulin regulation of hemodynamic and metabolic effects.

The Role of Oxidized Low-Density Lipoproteins in Atherosclerosis: The Myths and the Facts

The oxidative modification hypothesis of atherosclerosis, which assigns to oxidized low-density lipoproteins (LDLs) a crucial role in atherosclerosis initiation and progression, is still debated. This review examines the role played by oxidized LDLs in atherogenesis taking into account data derived by studies based on molecular and clinical approaches. Experimental data carried out in cellular lines and animal models of atherosclerosis support the proatherogenic role of oxidized LDLs: (a) through chemotactic and proliferating actions on monocytes/macrophages, inciting their transformation into foam cells; (b) through stimulation of smooth muscle cells (SMCs) recruitment and proliferation in the tunica intima; (c) through eliciting endothelial cells, SMCs, and macrophages apoptosis with ensuing necrotic core development. Moreover, most of the experimental data on atherosclerosis-prone animals benefiting from antioxidant treatment points towards a link between oxidative stress and atherosclerosis. The evidence coming from cohort studies demonstrating an association between oxidized LDLs and cardiovascular events, notwithstanding some discrepancies, seems to point towards a role of oxidized LDLs in atherosclerotic plaque development and destabilization. Finally, the results of randomized clinical trials employing antioxidants completed up to date, despite demonstrating no benefits in healthy populations, suggest a benefit in high-risk patients. In conclusion, available data seem to validate the oxidative modification hypothesis of atherosclerosis, although additional proofs are still needed.

Plasma Free Fatty Acids and Endothelium-Dependent Vasodilation: Effect of Chain-Length and Cyclooxygenase Inhibition

Free fatty acids (FFA) are known to interfere with glucose metabolism. Moreover, it has been shown that they are able to impair the endothelium-dependent vasodilation. Therefore, we sought to determine whether their negative effect on endothelial function depends on their chain length or on their ability to modify PG production. Fourteen normal volunteers were studied under baseline conditions and then randomly allocated to two of the following four studies: 1) long chain triglyceride (LCT) emulsion and heparin infusion (n = 7), 2) infusion of an emulsion containing 56% medium chain triglycerides (MCT) and 44% LCT plus heparin (n = 7), 3) infusion of LCT and heparin preceded by an i.v. bolus of 900 mg lysine-salicylate (ASA; n = 7), and 4) after an i.v. bolus of ASA (n = 7). Basal forearm blood flow (FBF), endothelium-dependent vasodilation in response to intraarterial acetylcholine (Ach), and endothelium-independent vasodilation in response to intraarterial nitroprusside were assessed by venous occlusion plethysmography. Both LCT and MCT infusions significantly increased basal FBF from 1.58 +/- 0.35 to 2.60 +/- 0.76 and 2.28 +/- 0.56 mL/min 100 mL tissue, respectively (both P < 0.05). This increase was also observed for LCT plus heparin, but not after ASA alone. The percent increase in FBF during Ach was lowered during both LCT (252 +/- 34% of the ratio infused/control arm at maximal Ach dose) and MCT (255 +/- 41%) compared to the baseline conditions (436 +/- 44%; both P < 0.05). The response to Ach was also lower during LCT plus ASA, whereas it was similar to baseline with ASA alone. No differences were observed in the response to nitroprusside among the experimental conditions. In conclusion, 1) the effect of FFA on endothelium-dependent vasodilation is independent of their chain length; 2) both LCT and MCT increase baseline FBF, independently from cyclooxygenase inhibition; and 3) acute ASA administration does not affect endothelium-dependent vasodilation. The FFA effect on the endothelial response to Ach may contribute to altered endothelial function and, hence, to the development and progression of atherosclerotic cardiovascular disease.

Salivary Phosphate-Binding Chewing Gum Reduces Hyperphosphatemia in Dialysis Patients

In uremic patients, hyperphosphatemia is associated with cardiovascular calcification and increased cardiovascular mortality. Despite the use of phosphate binders, only half of hemodialysis (HD) patients achieve recommended serum phosphate levels. A hyperphosphoric salivary content, which correlates linearly with serum phosphate, has been reported in HD patients. We hypothesized that binding salivary phosphate during periods of fasting in addition to using phosphate binders with meals could improve the treatment of hyperphosphatemia. We assessed the phosphate-binding capacity of the natural polymer chitosan by (31)P nuclear magnetic resonance and established that 10 and 20% (wt/vol) middle viscosity chitosan solutions bind 30 and 50% of the phosphate contained in PBS, respectively. Thirteen HD patients with serum phosphate levels >6.0 mg/dl despite treatment with sevelamer hydrochloride chewed 20 mg of chitosan-loaded chewing gum twice daily for 2 wk at fast in addition to their prescribed phosphate-binding regimen. Salivary phosphate and serum phosphate significantly decreased during the first week of chewing; by the end of 2 wk, salivary phosphate decreased 55% from baseline (73.21 +/- 19.19 to 33.19 +/- 6.53; P < 0.00001), and serum phosphate decreased 31% from baseline (7.60 +/- 0.91 to 5.25 +/- 0.89 mg/dl; P < 0.00001). Salivary phosphate returned to baseline by day 15 after discontinuing the chewing gum, whereas serum phosphate levels took 30 d to return to baseline. Parathyroid hormone and serum calcium concentrations were not affected by the gum. In conclusion, adding salivary phosphate binding to traditional phosphate binders could be a useful approach for improving treatment of hyperphosphatemia in HD patients.

Oxidative stress in kidney transplant patients with calcineurin inhibitor-induced hypertension: effect of ramipril.

In patients with cyclosporine-induced hypertension, upregulation of the nitric oxide system and oxidative stress were shown, which could induce hypertension, remodeling, and chronic rejection by increasing nitric oxide catabolism. However, it is still debated whether cyclosporine and tacrolimus exert a different action. The aim of the current study was to compare the effects of cyclosporine and tacrolimus on markers of oxidative stress and endothelial dysfunction in kidney transplant patients with posttransplant hypertension. Monocyte p22phox, a NADH/NADPH system subunit, transforming growth factor-&bgr; (TGF-&bgr;), heme oxygenase-1 (HO-1), and endothelial NOS gene expression were measured in 16 patients. Angiotensin II is a potent stimulator of oxidative stress and angiotensin-converting enzyme inhibition may blunt this effect. Therefore, the same parameters were measured before and after 2 months of treatment with ramipril (5 mg/d). At baseline, in cyclosporine-and tacrolimus-treated patients, p22phox and TGF-&bgr; mRNA were similarly increased in comparison with normotensive healthy controls (0.90 ± 0.05 d.u. and 0.83 ± 0.05 in cyclosporine, 0.89 ± 0.07 and 0.84 ± 0.05 in tacrolimus; 0.53 ± 0.07 and 0.75 ± 0.03 in controls, respectively; p < 0.001). Endothelial NOS mRNA was increased in cyclosporine-and tacrolimus-treated patients in comparison with controls (0.92 ± 0.09, 0.96 ± 0.04, and 0.37 ± 0.05 respectively; p < 0.001), whereas no difference was found between patients and controls in HO-1 mRNA. Ramipril reduced blood pressure (from 140 ± 11/91 ± 7 mm Hg to 129 ± 6/85 ± 5 mm Hg in cyclosporine and from 138 ± 7/92 ± 7 mm Hg to 127 ± 10/82 ± 6 mm Hg in tacrolimus group; p < 0.02 with no difference between groups). Ramipril also reduced p22phox (to 0.83 ± 0.05 in cyclosporine, p < 0.03 and to 0.81 ± 0.08 in tacrolimus; p < 0.01) and TGF-&bgr; mRNA (to 0.72 ± 01 in cyclosporine, p < 0.02, and to 0.73 ± 0.05 in tacrolimus; p < 0.01) with no difference between groups, but it did not change HO-1 and ecNOS mRNA. Cyclosporine and tacrolimus induce a comparable oxidative stress in kidney transplant patients with posttransplant hypertension. The association of ramipril normalizes blood pressure and reduces the oxidative stress induced by both drugs.

Cyclosporin‐induced endothelial dysfunction and hypertension: are nitric oxide system abnormality and oxidative stress involved?

Abstract Hypertension is a major side effect of cyclosporin (CsA). While the mechanism(s) responsible are unclear, CsA‐induced endothelial dysfunction and CsA‐induced hypertension have been attributed to the CsA effect on the endothelial‐derived factors controlling vasomotor tone. Endothelial nitric oxide (NO) is crucial in the maintenance of a state of basal vasodilation, and recent studies have suggested an NO‐mediated counterregulatory mechanism protective from CsA‐induced vasoconstriction. Our study evaluates endothelial nitric oxide synthase (ecNOS) gene status (PCR analysis) and plasma levels of NO metabolites (ELISA) in kidney and heart transplant patients under chronic CsA treatment with CsA‐induced hypertension. Since CsA increases superoxide production, which metabolises NO, plasma hydroperoxides from cholesterol esters and from triglycerides and peroxynitrite were also evaluated (HPLC) as an index of the presence of superoxides and of “oxidative stress”. Quantification of monocyte ecNOS mRNA and NO metabolites plasma levels from patients and controls (C) demonstrated NO system upregulation in patients notwithstanding the hypertension. The mean ecNOS to β‐actin ratio was 1.80 ± 0.85 in patients vs 0.40 ± 0.09 in C (P < 0.04). NO metabolites were 34.03 ± 14.32 μM in patients vs 11.53 ± 5.64 μM in C (P < 0.001). Hydroperoxides from cholesterol esters and from triglycerides were also increased in patients, 3.4 ± 1.4 vs 1.3 ± 0.6 integrated area units (i. a. u.), P < 0.007 and 10.6 ± 6.4 vs 1.3 ± 0.8 i.a.u., P < 0.008, respectively, as well as the peroxynitrite plasma level, 0.32 ± 0.11 μM/l vs undetectable in C. This study confirms a CsA‐induced NO system upregulation in transplanted patients. However, the NO‐mediated counterregulatory system to CsA‐induced vasoconstriction, present in normals, could be canceled in patients by CsA‐induced superoxide (O2−) and free radical production which, by increasing NO metabolism, could contribute to CsA‐induced vasoconstriction and hypertension and predispose to atherosclerosis.

Increased urinary NO2−/NO3− and cyclic guanosine monophosphate levels in patients with bartter's syndrome: Relationship to vascular reactivity

Nitric oxide (NO) is a potent endogenous vasodilator and plays a pivotal role in the control of vascular tone by the formation of cyclic guanosine monophosphate (GMP). Patients affected by Bartters syndrome have lower than normal vascular reactivity with normohypotension and decreased peripheral resistances in spite of biochemical and hormonal abnormalities typical of hypertension, and it is possible that increased production of NO may be involved in maintaining this reduced vascular response and vasodilatation. We have examined this possibility by studying NO2-/NO3- and cyclic GMP urinary excretions to assess NO production in vivo in seven patients affected by Bartters syndrome compared with seven healthy controls. A group of five patients with hypokalemia other than Bartter syndrome (pseudo-Bartters) was also included in the study to evaluate the effect of hypokalemia on NO production. NO2-/NO3- urinary excretion (0.45 +/- 0.14 v 0.25 +/- 0.04 micromol/micromol urinary creatinine [controls], P < 0.005, v 0.28 +/- 0.05 [pseudo-Bartters], P < 0.01) and cyclic GMP urinary excretion (0.057 +/- 0.028 v 0.022 +/- 0.01 micromol/micromol of urinary creatinine [controls], P < 0.009, v 0.024 +/- 0.004 [pseudo-Bartters], P < 0.02) were increased in patients with Bartters syndrome in comparison with controls and pseudo-Bartters, and a linear correlation between these two parameters was also present (P < 0.001). We conclude that in Bartters syndrome the increased NO2-/NO3- and cyclic GMP urinary excretions point to an increased NO synthesis, which could account for the reduced vascular response of the disease, therefore adding its role in determining the vascular hyporeactivity of Bartters syndrome.

Silencing regulator of G protein signaling-2 (RGS-2) increases angiotensin II signaling: insights into hypertension from findings in Bartter's/Gitelman's syndromes.

Objective Regulator of G-protein signaling (RGS)-2 is a regulator of angiotensin II (Ang II) signaling. In Bartters syndrome/Gitelmans syndrome (BS/GS), we have demonstrated increased RGS-2 levels and blunted Ang II signaling which contribute to their reduced vasomotor tone and remodeling. The present study investigates the effect of silencing RGS-2 in fibroblasts from six BS/GS patients on intracellular Ca2+ ( ) mobilization and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, established Ang II-mediated responses. Methods Fibroblasts were RGS-2 silenced by transfecting chemically synthesized small interfering RNA. Silencing efficiency and Ang II-induced ERK 1/2 phosphorylation were evaluated by western blot and Ang II-induced using Fura-2 AM. Results RGS-2 expression in not silenced BS/GS fibroblasts from patients is increased compared with healthy controls [0.34 ± 0.02 vs. 0.19 ± 0.01 densitometric units (d.u.), P = 0.0005]. Silencing RGS-2 in BS/GS patients was achieved to the level of controls. Ang II-induced release and ERK 1/2 phosphorylation were reduced in not silenced cells from BG/GS patients compared with controls (112.16 ± 13.2 vs. 130.33 ± 13.64 mmol/l, P = 0.011 and 0.64 ± 0.08 vs. 0.91 ± 0.03 mmol/l, P < 0.006, respectively). Silencing RGS-2 in BS/GS patients increased Ang II-induced release and ERK 1/2 phosphorylation in silenced cells compared with not silenced cells [59.3 ± 10.8 (peak-basal) vs. 40.5 ± 14.1 nmol/l, P = 0.017 and 0.84 ± 0.06 vs. 0.64 ± 0.08 nmol/l, P < 0.03, respectively], whereas they were not different compared with controls (60.1 ± 4.3 and 0.91 ± 0.03 nmol/l). Integrating the response over time showed increased area under the curve (AUC) of BS/GS silenced cells compared with that of not silenced cells (P = 0.013). Conclusion This is the first report of silencing RGS-2 effect on Ang II signaling in a human clinical condition of altered vascular tone regulation and remodeling and establishes RGS-2 as a key regulatory element of Ang II signaling in humans.

Clinical significance of cytokine determination in synovial fluid.

Cytokines are a complex family of small regulatory proteins able to mediate intercellular communication and play a crucial role in immunologic and inflammatory reactions. Many reports have demonstrated that some cytokines, in particular tumor necrosis factor α (TNFα) and interleukin (IL)-1β, IL-6, and IL-8, so-called proinflammatory, may have a major role in the pathogenesis of joint diseases. Thus, high levels of these substances have been found in inflammatory arthropathies, in particular in those characterized by a more aggressive and destructive outcome, such as rheumatoid arthritis, gout, and infectious arthritis. In keeping with their role, the determination of cytokines in synovial fluid may be proposed for clinical purposes, including diagnostic and prognostic assessments. Furthermore, as some of these cytokines may reflect disease activity, their determination may also be useful in the evaluation of therapy.