Francisco J. Fenoy

Role of Superoxide in Modulating the Renal Effects of Angiotensin II

Abstract—Angiotensin II is known to stimulate NADPH oxidase-dependent superoxide (O2−) generation, which may contribute to the acute renal vasoconstrictor and antinatriuretic actions of this peptide. To evaluate this hypothesis, the effects of a superoxide dismutase mimetic (tempol) or a NADPH inhibitor (apocynin) on the angiotensin renal actions were studied. Renal cortical nitric oxide (NO) was measured electrochemically in vivo. Tempol increased sodium excretion and NO levels. Apocynin raised renal blood flow, glomerular filtration rate, sodium excretion, and NO levels. These results indicate the presence of an endogenous NADPH oxidase-dependent O2− generation that may modulate renal function by scavenging NO. Angiotensin II infusion reduced renal blood flow, glomerular filtration, sodium excretion, and NO levels in a dose-dependent manner. The angiotensin receptor antagonist valsartan, tempol, or apocynin blunted the angiotensin effects on renal excretion and NO, suggesting that angiotensin receptors stimulation induces the NADPH oxidase-dependent O2− generation that might reduce NO bioavailability. This idea is supported by the finding that angiotensin increased O2− generation in renal homogenates, and this effect was prevented by valsartan, apocynin, or tempol. These results indicate that some of the acute renal effects of angiotensin II may be enhanced by an increased NADPH oxidase-derived O2− production that reduces renal NO bioavailability.

2-Methoxyestradiol attenuates hypertension and coronary vascular remodeling in spontaneously hypertensive rats.

OBJECTIVES Accumulating data provide evidence that some metabolites of 17beta-estradiol are biologically active and mediate multiple effects on the cardiovascular and renal systems. We investigated the effect of 2-methoxyestradiol (an active metabolite of estradiol with non-feminizing activity) on the development of hypertension and myocardial vascular remodeling in male and female ovarectomized SHR. METHODS Rats were divided into five groups: intact females, ovarectomized (OVX), OVX+ 2-methoxyestradiol (2ME), control males, and male+2ME. Systolic blood pressure was determined from 10 to 18 weeks. Structural changes in coronary vessels were quantified by an image analyzer. Immunoblotting of phosphorylated ERK1/2 and NADPH oxidase activity were performed on mesenteric arteries. RESULTS Treatment with 2ME reduced the increase in systolic blood pressure in male and ovarectomized rats to values not different from those obtained in intact females. Myocardial arterioles and small arteries showed significant increases in wall-to-lumen ratio and perivascular fibrosis in male and ovarectomized rats when compared with intact females. NADPH oxidase activity was increased in mesenteric arteries from males and ovarectomized females as compared with intact females. Finally, the expression of phosphorilated ERK1/2 were significantly higher in mesenteric arteries from male and ovariectomized animals than in those from intact females. Those effects of ovarectomy and gender differences were totally or partially prevented by treatment with 2-methoxyestradiol. CONCLUSIONS These data demonstrate that 2-methoxyestradiol protects the vasculature from hypertension-induced myocardial arterial remodeling in male and ovarectomized SHR, and that might be in part related to decreased superoxide generation and ERK1/2 activation.

Acute effects of 2-methoxyestradiol on endothelial aortic No release in male and ovariectomized female rats

The endogenous metabolites of 17beta-estradiol are thought to have protective vascular effects, especially in males and estrogen-deprived females. The present study evaluated the acute in vitro effects of the active metabolite 2-methoxyestradiol on endothelial NO release from ovariectomized female and intact male and female rat aortas. NO was measured electrochemically by differential normal pulse amperometry using carbon fiber microsensors, and also by fluorescence microscopy using 4,5-diaminofluorescein diacetate. 2-Methoxyestradiol alone induced a maintained increase in endothelial NO release in male and ovariectomized rats that was reduced by pretreatment with L-NAME. NO release induced by calcium ionophore alone (A23187) was lower in aortas from ovariectomized rats than from intact females, indicating that estrogen deprivation induces endothelial dysfunction. Pretreatment of aortas with 2-methoxyestradiol potentiated significantly the A23187-induced-NO release in ovariectomized as well as in males, but not in intact females. This potentiation was reduced or abolished by L-NAME. 2-Methoxyestradiol potentiated the vasodilatory effect of A23187 on intestinal arterioles, and also increased intestinal tissular laser-Doppler blood flow signal. These results demonstrate that 17beta-estradiol and its active metabolite 2-methoxyestradiol increase basal aortic endothelial NO production and also cause a potentiation of the calcium ionophore-stimulated NO release in male and ovariectomized, while it has no effects on intact females. 2-Methoxyestradiol appears to be a promising pharmacological agent capable of improving endothelial function in men and postmenopausal women.

Role of angiotensin II in modulating the hemodynamic effects of nitric oxide synthesis inhibition

This study examined the role of ANG II in modulating the increase of hematocrit and vascular permeability that follows nitric oxide (NO) synthesis blockade, that are contributing to the decrease in cardiac index (CI) in conscious, chronically catheterized rats. Pretreatment with losartan attenuated the N(omega)-nitro-L-arginine methyl ester (L-NAME)-induced increase in total peripheral resistance by 26% and also blunted the fall in CI (28%) and stroke volume. L-NAME produced an increase in hematocrit (4.5%) and in (125)I-labeled albumin content in the heart and small intestine in untreated rats, but the increase was prevented in rats pretreated with losartan. Furthermore, L-NAME induced a transient increase of plasma protein concentration and tissue intestinal blood flow, which was abolished in rats given losartan. The results of the present study indicate that the systemic hemodynamic responses, the fall in plasma volume, and the increase in albumin escape observed after inhibition of NO synthesis are in part the consequence of unmasking the actions of endogenous ANG II. These data suggest a physiological role for NO by restraint of the vascular actions of the renin-angiotensin system.

Endothelial dysfunction in gestational hypertension induced by catechol‐O‐methyltransferase inhibition

•  What is the central question of this study? Gestational hypertension is a major obstetric problem of placental origin, associated with reduced production of 2‐methoxyestradiol. In this study, we evaluated in rats the consequences of pharmacological inhibition of catechol‐O‐methyltransferase, the enzyme that produces 2‐methoxyestradiol from 2‐hydroxyestradiol. •  What is the main finding and its importance? The present study provides evidence that inhibition of catechol‐O‐methyltransferase in pregnant rats produces arterial hypertension and endothelial dysfunction due to reduced nitric oxide bioavailability. The results help us to understand gestational hypertension and support a possible new approach to early diagnosis and treatment of the disease.

Vascular and adrenal reninlike activity in chronically diabetic rats.

The aim of this work was to investigate, in an experimental model of diabetes mellitus, the levels of renin activity in vascular and adrenal tissues and their relationship to several circulating renin-angiotensin system components. Rats with chronic (12 weeks) streptozocin-induced diabetes showed a significant decrease in plasma renin activity (PRA), plasma renin concentration, and plasma aldosterone. However, plasma trypsin activatable inactive renin concentration was increased (11.65 +/- 1.40 vs 6.73 +/- 0.57 ng angiotensin I/ml/hr; p less than 0.001), as were aortic reninlike activity (p less than 0.001) and adrenal renin, both in the zona glomerulosa (p less than 0.01) and the fascicular-reticular-medullary portion (p less than 0.001) with respect to an age-matched control group. After bilateral nephrectomy, plasma renin-angiotensin system components (PRA and plasma active and inactive renin concentrations) as well as aortic and fascicular-reticular-medullary renin activity significantly decreased in both control and diabetic rats. However, glomerular renin activity increased in control nephrectomized rats to the levels observed in diabetic animals but did not change in diabetic nephrectomized rats. The parallel changes of aortic and fascicular-reticular-medullary renin activity and plasma inactive renin concentration in diabetes and nephrectomy suggest an interdependent relationship, whereas the increase of glomerular renin activity in diabetic and nephrectomized animals, both with low levels of PRA, suggests the existence of a local autonomic renin-angiotensin system regulated by plasma feedback. Tissue renin-angiotensin system alterations in diabetes could mean that a pathogenic factor is involved in long-term diabetic complications or that only a compensatory physiological process is at work.

2-methoxyestradiol plasma levels are associated with clinical severity indices and biomarkers of preeclampsia.

We investigated whether clinical severity indices and biomarkers for preeclampsia (PE) are associated with low plasmatic 2-methoxyestradiol (2ME) in the third trimester of gestation. Blood was collected from 53 women with PE and 73 control pregnant women before parturition. The concentration of 2ME was significantly higher in controls than in patients with PE (2906.43 ± 200.69 pg/mL vs 1818.41 ± 189.25 pg/mL). The risk of PE decreased as 2ME levels increased. The 2ME values were negatively correlated with systolic peak arterial pressure and proteinuria in PE. Additionally, those women with PE with lower 2ME had a more serious clinical situation and needed a more aggressive therapy. Finally, 2ME levels (in patients with PE and total population) were significantly correlated with concentrations of soluble fms-like tyrosine kinase 1 and placental growth factor . Summarizing, patients with PE had lower 2ME levels that were correlated with different clinical indices and biomarkers of severity, indicating that 2ME could be taken into account for the clinical management of this syndrome.

Predicting serious complications in patients with cancer and pulmonary embolism using decision tree modelling: the EPIPHANY Index

Background:Our objective was to develop a prognostic stratification tool that enables patients with cancer and pulmonary embolism (PE), whether incidental or symptomatic, to be classified according to the risk of serious complications within 15 days.Methods:The sample comprised cases from a national registry of pulmonary thromboembolism in patients with cancer (1075 patients from 14 Spanish centres). Diagnosis was incidental in 53.5% of the events in this registry. The Exhaustive CHAID analysis was applied with 10-fold cross-validation to predict development of serious complications following PE diagnosis.Results:About 208 patients (19.3%, 95% confidence interval (CI), 17.1–21.8%) developed a serious complication after PE diagnosis. The 15-day mortality rate was 10.1%, (95% CI, 8.4–12.1%). The decision tree detected six explanatory covariates: Hestia-like clinical decision rule (any risk criterion present vs none), Eastern Cooperative Group performance scale (ECOG-PS; <2 vs ⩾2), O2 saturation (<90 vs ⩾90%), presence of PE-specific symptoms, tumour response (progression, unknown, or not evaluated vs others), and primary tumour resection. Three risk classes were created (low, intermediate, and high risk). The risk of serious complications within 15 days increases according to the group: 1.6, 9.4, 30.6%; P<0.0001. Fifteen-day mortality rates also rise progressively in low-, intermediate-, and high-risk patients: 0.3, 6.1, and 17.1%; P<0.0001. The cross-validated risk estimate is 0.191 (s.e.=0.012). The optimism-corrected area under the receiver operating characteristic curve is 0.779 (95% CI, 0.717–0.840).Conclusions:We have developed and internally validated a prognostic index to predict serious complications with the potential to impact decision-making in patients with cancer and PE.

Fetal Val108/158Met catechol-O-methyltransferase (COMT) polymorphism and placental COMT activity are associated with the development of preeclampsia.

OBJECTIVE To evaluate the association between fetal and maternal catechol-O-methyltransferase (COMT) Val158Met and methyl tetrahydrofolate reductase (MTHFR) C677T functional polymorphisms and preeclampsia, examining its influence on placental COMT and in maternal 2-methoxyestradiol (2-ME) plasma levels. DESIGN Prospective case-control study. SETTING University hospital. PATIENT(S) A total of 53 preeclamptic and 72 normal pregnant women. INTERVENTION(S) Maternal and cord blood samples and placental tissue samples were obtained. MAIN OUTCOME MEASURE(S) Maternal and fetal COMT and MTHFR polymorphisms were genotyped. Maternal plasma 2-ME and homocysteine levels, and expression and activity of placental COMT were measured. RESULT(S) The odds ratio for the risk of preeclampsia for fetal COMT Met/Met was 3.22, and it increased to 8.65 when associated with fetal MTHFR TT. Placental COMT activity and expression were influenced by genotype, but COMT activity in preeclamptic placentas did not differ from control pregnancies. There was no association between any genotypes and maternal 2-ME. Homocysteine levels were higher in women with preeclampsia than in normal pregnancies, and were inversely correlated with 2-ME plasma levels, indicating that its altered metabolism may lower COMT activity in vivo. CONCLUSION(S) Fetal Met-Met COMT genotype reduces COMT placental expression and activity in vitro and increases preeclampsia, risk but it does not explain the difference in maternal 2-ME levels between preeclamptic and normal pregnancies. However, the preeclamptic patients had elevated homocysteine levels that correlated inversely with 2-ME, indicating that an altered methionine-homocysteine metabolism may contribute to reduce COMT activity in vivo and explain the decreased levels of 2-ME in preeclamptic women.

Effect of captopril on norepinephrine vascular contractility

In isolated aortic rings and in vitro perfused mesenteric arteries of Wistar rats the vasoconstrictor responses to norepinephrine (NE) were not affected by captopril (2 X 10(-4) M). However, captopril (1 mg/kg i.v.) in pithed Wistar rats attenuated significantly the increases in diastolic blood pressure induced by NE. In pithed rats the effect of captopril on NE diastolic blood pressure responses disappeared either in the presence of an angiotensin II (5 ng X kg-1 X min) infusion or when the rats were previously nephrectomized. These findings suggest that the effect of captopril on vascular responses to norepinephrine is mediated by an inhibition of the renin-angiotensin system and not by an antagonistic effect on alpha-adrenergic receptors.