Silvia Orisio

Nitric Oxide Synthesis by Cultured Endothelial Cells Is Modulated by Flow Conditions

In the present study, we examined the hypothesis that dynamic characteristics of flow modulate the production of vasoactive mediators, namely nitric oxide (NO) and endothelin-1 (ET-1), by human umbilical vein endothelial cells (HUVECs). Cells were exposed for 6 hours in a cone-and-plate apparatus to different types of flow: steady laminar, with shear stresses of 2, 8, and 12 dyne/cm2, pulsatile laminar, with shear stress from 8.2 to 16.6 dyne/cm2 and a frequency of 2 Hz; periodic laminar, with square wave cycles of 15 minutes and shear stress from 2 to 8 dyne/cm2, and turbulent, with shear stress of 8 dyne/cm2 on average. A second culture dish was kept in a normal incubator as a static control for each experiment. Laminar flow induced synthesis of NO by HUVECs that was dependent on shear-stress magnitude. Laminar shear stress at 8 dyne/cm2 also upregulated the level of NO synthase mRNA. As observed with steady laminar flow, pulsatile flow also induced an increase in NO release by endothelial cells. When HUVECs were subjected to step-change increases of laminar shear, a further increase of NO synthesis was observed, compared with steady laminar shear of the same magnitude. Turbulent flow did not upregulate NO synthase mRNA or increase NO release. Both laminar and turbulent shear stress reduced, although not significantly, ET-1 mRNA and ET-1 production compared with the static condition. These results indicate that local blood flow conditions modulate the production of vasoactive substances by endothelial cells. This may affect vascular cell functions such as nonthrombogenicity, regulation of blood flow, and vascular tone.

Blocking both type A and B endothelin receptors in the kidney attenuates renal injury and prolongs survival in rats with remnant kidney

Renal disease progression in the rat is associated with a time-dependent upregulation of renal endothelin-1 (ET-1) gene expression and synthesis. We have previously demonstrated that endothelin A receptor subtype (ETA) blockade in rats with remnant kidney reduced signs of disease activity, suggesting that ET-1 exerts part of its deleterious effects on the kidney through ETA. No data are available so far on the role of ETB receptor in progressive renal injury. We first studied renal ETA and ETB receptor gene expression in rats with remnant kidney on days 7, 30, and 120 after the surgical procedure. While renal expression of ETA was unaffected, ETB receptor gene was significantly upregulated with time in rats with remnant kidney, being 3.5-fold and sixfold higher than shamoperated rats at days 30 and 120. We also evaluated whether bosentan, a nonpeptidic ETA and ETB receptor antagonist, offered better protection against renal disease progression than reported for ETA-selective blockers and whether it improved survival in animals with renal ablation. Two groups of rats with renal mass reduction (n = 11 each) were given bosentan 100 mg/kg/d orally or its vehicle (carboxymethyl cellulose) beginning day 7 after the surgical procedure and were followed until the death of the vehicle-treated animals. Sham-operated animals comprised the control group. Bosentan partially prevented increases in blood pressure and proteinuria, but had a remarkable protective effect on renal function and significantly prolonged animal survival. These data suggest that blocking both renal ETA and ETB receptors might have major implications in the treatment of human progressive nephropathies.

Anti-Phospholipase A2 Receptor Antibody Titer Predicts Post-Rituximab Outcome of Membranous Nephropathy

Rituximab induces nephrotic syndrome (NS) remission in two-thirds of patients with primary membranous nephropathy (MN), even after other treatments have failed. To assess the relationships among treatment effect, circulating nephritogenic anti-phospholipase A2 receptor (anti-PLA2R) autoantibodies and genetic polymorphisms predisposing to antibody production we serially monitored 24-hour proteinuria and antibody titer in patients with primary MN and long-lasting NS consenting to rituximab (375 mg/m(2)) therapy and genetic analyses. Over a median (range) follow-up of 30.8 (6.0-145.4) months, 84 of 132 rituximab-treated patients achieved complete or partial NS remission (primary end point), and 25 relapsed after remission. Outcomes of patients with or without detectable anti-PLA2R antibodies at baseline were similar. Among the 81 patients with antibodies, lower anti-PLA2R antibody titer at baseline (P=0.001) and full antibody depletion 6 months post-rituximab (hazard ratio [HR], 7.90; 95% confidence interval [95% CI], 2.54 to 24.60; P<0.001) strongly predicted remission. All 25 complete remissions were preceded by complete anti-PLA2R antibody depletion. On average, 50% anti-PLA2R titer reduction preceded equivalent proteinuria reduction by 10 months. Re-emergence of circulating antibodies predicted disease relapse (HR, 6.54; 95% CI, 1.57 to 27.40; P=0.01), whereas initial complete remission protected from the event (HR, 6.63; 95% CI, 2.37 to 18.53; P<0.001). Eighteen patients achieved persistent antibody depletion and complete remission and never relapsed. Outcome was independent of PLA2R1 and HLA-DQA1 polymorphisms and of previous immunosuppressive treatment. Therefore, assessing circulating anti-PLA2R autoantibodies and proteinuria may help in monitoring disease activity and guiding personalized rituximab therapy in nephrotic patients with primary MN.

Ameliorating Hypertension and Insulin Resistance in Subjects at Increased Cardiovascular Risk. Effects of Acetyl-L-Carnitine Therapy

Insulin resistance, a key component of the metabolic syndrome, is a risk factor for diabetes mellitus and cardiovascular disease. Acetyl-l-carnitine infusion acutely ameliorated insulin sensitivity in type 2 diabetics with insulin resistance. In this sequential off-on-off pilot study, we prospectively evaluated the effects of 24-week oral acetyl-l-carnitine (1 g twice daily) therapy on the glucose disposal rate (GDR), assessed by hyperinsulinemic euglycemic clamps, and components of the metabolic syndrome in nondiabetic subjects at increased cardiovascular risk a priori segregated into 2 groups with GDR ≤7.9 (n=16) or >7.9 (n=16) mg/kg per minute, respectively. Baseline GDR and systolic blood pressure were negatively correlated (n=32; P=0.001; r=−0.545), and patients with GDR ≤7.9 mg/kg per minute had higher systolic/diastolic blood pressure than those with higher GDR. Acetyl-l-carnitine increased GDR from 4.89±1.47 to 6.72±3.12 mg/kg per minute (P=0.003, Bonferroni-adjusted) and improved glucose tolerance in patients with GDR ≤7.9 mg/kg per minute, whereas it had no effects in those with higher GDRs. Changes in GDR were significantly different between groups (P=0.017, ANCOVA). Systolic blood pressure decreased from 144.0±13.6 to 135.1±8.4 mm Hg and from 130.8±12.4 to 123.8±10.8 mm Hg in the lower and higher GDR groups, respectively (P<0.05 for both; P<0.001 overall) and progressively recovered toward baseline over 8 weeks posttreatment. Total and high molecular weight adiponectin levels followed specular trends. Diastolic blood pressure significantly decreased only in those with higher GDRs. Treatment was well tolerated in all of the patients. Acetyl-l-carnitine safely ameliorated arterial hypertension, insulin resistance, impaired glucose tolerance, and hypoadiponectinemia in subjects at increased cardiovascular risk. Whether these effects may translate into long-term cardioprotection is worth investigating.

Evidence against a pathogenetic role for endothelin in pre‐eclampsia

Objective To assess whether increased placental or systemic endothelin synthesis has a pathogenic role in pre‐eclampsia (gestational proteinuric hypertension).

Human placenta expresses endothelin gene and corresponding protein is excreted in urine in increasing amounts during normal pregnancy

Systemic and renal hemodyanmic changes in normal pregnancy have been attributed in part to altered vascular synthesis of vasodilatory prostaglandins. Besides vasodilatory substances, endothelium also generates vasoconstrictors, including endothelin. We evaluated the capacity of placental tissue from normal pregnant women to express endothelin gene and to generate endothelin. Placental tissue expressed a single 2.3 kb preproendothelin messenger ribonucleic acid and produced comparable amounts of endothelin 3, Big endothelin 1, and endothelin 1 and a minor quantity of endothelin 2. To investigate the possible influence of placental endothelin production on plasma levels of the peptide, plasma endothelin concentrations were measured in normal pregnant women at delivery and were found to be numerically higher than those measured in nonpregnant subjects. Urinary excretion of endothelin, taken as a marker of the renal synthesis of the peptide, tended to increase, although not significantly, in the first 14 weeks of pregnancy. This trend continued throughout pregnancy, resulting in a significant increase from the second trimester to delivery.

Variations of the angiotensin II type 1 receptor gene are associated with extreme human longevity

Longevity phenotype in humans results from the influence of environmental and genetic factors. Few gene polymorphisms have been identified so far with a modest effect on lifespan leaving room for the search of other players in the longevity game. It has been recently demonstrated that targeted disruption of the mouse homolog of the human angiotensin II type 1 receptor (AT1R) gene (AGTR1) translates into marked prolongation of animal lifespan (Benigni et al., J Clin Invest 119(3):524–530, 2009). Based on the above study in mice, here we sought to search for AGTR1 variations associated to reduced AT1 receptor protein levels and to prolonged lifespan in humans. AGTR1 was sequenced in 173 Italian centenarians and 376 younger controls. A novel non-synonymous mutation was detected in a centenarian. Two polymorphisms in AGTR1 promoter, rs422858 and rs275653, in complete linkage disequilibrium, were significantly associated with the ability to attain extreme old age. We then replicated the study of rs275653 in a large independent cohort of Japanese origin (598 centenarians and semi-supercentenarians, 422 younger controls) and indeed confirmed its association with exceptional old age. In combined analyses, rs275653 was associated to extreme longevity either at recessive model (P = 0.007, odds ratio (OR) 3.57) or at genotype level (P = 0.015). Significance was maintained after correcting for confounding factors. Fluorescence activated cell sorting analysis revealed that subjects homozygous for the minor allele of rs275653 had less AT1R-positive peripheral blood polymorphonuclear cells. Moreover, rs275653 was associated to lower blood pressure in centenarians. These findings highlight the role of AGTR1 as a possible candidate among longevity-enabling genes.

Single Strand Conformation Polymorphism (SSCP) as a quick and reliable method to genotype M235T polymorphism of angiotensinogen gene.

OBJECTIVES Conflicting results on the relationship between M235T polymorphism of angiotensinogen (AGT) gene and diabetic nephropathy are reported in the literature, probably due to the small number of subjects, to different inclusion criteria and the different genotype analysis methods used. The aim of the present study was to set up a fast, cheap and reliable method to allow the genotyping of M235T polymorphism in a large number of subjects. DESIGN AND METHODS We developed in our laboratory a new specifically designed PCR-SSCP method for M235T genotyping whose specificity was compared with that of Allele Specific PCR (ASPCR) and Mutagenically Separated PCR (MS-PCR). The exact M235T genotype was estabilished by direct sequencing. The new PCR-SSCP method was then used to genotype a population of 1171 hypertensive, normoalbuminuric type II diabetes mellitus patients. The patients were also genotyped for ACE I/D polymorphism. For comparison a group of hypertensive non diabetic patients (n = 88) were also screened. RESULTS The PCR-SSCP method identified the M235T polymorphism with no misinterpretation at variance with ASPCR and MS-PCR methods that showed a preferential amplification of the T allele. The rare Y248C polymorphism of the AGT gene was also detected by PCR-SSCP. In diabetic hypertensive patients the prevalence of TT genotype was higher than in normotensive healthy controls and equivalent to that found in hypertensive non diabetic patients. CONCLUSIONS The PCR-SSCP method for detection of M235T polymorphism is a powerful and sensitive tool for rapid, cheap and efficient screening of a large number of samples. The results obtained with this method demonstrate an association of the TT genotype of AGT gene with hypertension, both in diabetic and non diabetic patients.

Turnour necrosis factor stimulates endothelin-1 gene expression in cultured bovine endothelial cells

We have studied the effect of human recombinant tumour necrosis factor-α (TNF-α) on gene expression and production of endothelin-1 in cultured bovine aortic endothelial cells. TNF-α (10 and 100 ng ml−1) increased in a time dependent manner the preproendothelin-1 mRNA levels in respect to unstimulated endothelial cells. TNF-α induced endothelin-1 gene expression was associated with a parallel increase in the release of the corresponding peptide in the culture medium. These findings suggest that the enhanced synthesis and release of endothelin-1 occurring in conditions of increased generation of TNF, may act as a modulatory factor that counteracts the hypotensive effect and the excessive platelet aggregation and adhesion induced by TNF.