Domenica Schiavone

CYP11B2 Gene Polymorphisms in Idiopathic Hyperaldosteronism

Primary aldosteronism is characterized by autonomous production of aldosterone and arterial hypertension, and it occurs in 2 principal forms: aldosterone-producing adenoma (APA) and idiopathic hyperaldosteronism (IHA). APA can be cured through removal of the adenoma, whereas IHA leads to hypertension that must be treated with medication. The origin of the autonomous aldosterone production in IHA is poorly understood, but genetic factors may contribute to its cause. To test the hypothesis that variants of the aldosterone synthase gene may contribute to susceptibility to IHA, we compared genotypes at 3 polymorphic sites in the CYP11B2 gene in patients with IHA (n=90) with those found in patients with APA (n=38), in patients with essential hypertension (n=72), and in normotensive individuals (n=102). We observed significant linkage disequilibrium among the 3 polymorphisms with 2 frequent haplotypes in all groups studied. One haplotype (C2R) was found to be increased in frequency in the IHA group (47%) compared with the other groups, which had a similar haplotype frequency (36%). The 3 polymorphisms studied have been implicated in either essential hypertension or excess aldosterone production in previous studies. Because of the strong linkage disequilibrium, the observed results could be due to the action of any 1 of the 3 alleles or to another allele in linkage disequilibrium with them. Our results suggest that variations in the CYP11B2 gene may contribute to dysregulation of aldosterone synthesis and lead to susceptibility to IHA.

Prevalence and Characteristics of Familial Hyperaldosteronism: The PATOGEN Study (Primary Aldosteronism in TOrino-GENetic forms)

Primary aldosteronism (PA) is the most frequent cause of secondary hypertension, and patients display an increased prevalence of cardiovascular events compared with essential hypertensives. To date, 3 familial forms of PA have been described and termed familial hyperaldosteronism types I, II, and III (FH-I to -III). The aim of this study was to investigate the prevalence and clinical characteristics of the 3 forms of FH in a large population of PA patients. Three-hundred consecutive PA patients diagnosed in our unit were tested by long-PCR of the CYP11B1/CYP11B2 hybrid gene that causes FH-I, and all of the available relatives of PA patients were screened to confirm or exclude PA and, thus, FH-II. Urinary 18-hydroxycortisol and 18-oxocortisol were measured in all of the familial PA patients. Two patients were diagnosed with FH-I (prevalence: 0.66%), as well as 21 of their relatives, and clinical phenotypes of the 2 affected families varied markedly. After exclusion of families who refused testing and those who were not informative, 199 families were investigated, of which 12 were diagnosed with FH-II (6%) and an additional 15 individuals had confirmed PA; clinical and biochemical phenotypes of FH-II families were not significantly different from sporadic PA patients. None of the families displayed a phenotype compatible with FH-III diagnosis. Our study demonstrates that familial forms of hyperaldosteronism are more frequent than previously expected and reinforces the recommendation of the Endocrine Society Guidelines to screen all first-degree hypertensive relatives of PA patients.

18-Hydroxycorticosterone, 18-Hydroxycortisol, and 18-Oxocortisol in the Diagnosis of Primary Aldosteronism and Its Subtypes

CONTEXT Diagnosis of primary aldosteronism (PA) is made by screening, confirmation testing, and subtype diagnosis (computed tomography scan and adrenal vein sampling). However, some tests are costly and unavailable in most hospitals. OBJECTIVE The aim of the study was to evaluate the role of serum 18-hydroxycorticosterone (s18OHB), urinary and serum 18-hydroxycortisol (u- and s18OHF), and urinary and serum 18-oxocortisol (u- and s18oxoF) in the diagnosis of PA and its subtypes, aldosterone-producing adenoma (APA) and bilateral adrenal hyperplasia (BAH). PATIENTS The study included 62 patients with low-renin essential hypertension (EH), 81 patients with PA (20 APA, 61 BAH), 24 patients with glucocorticoid-remediable aldosteronism, 16 patients with adrenal incidentaloma, and 30 normotensives. INTERVENTION AND MAIN OUTCOME MEASURES We measured s18OHB, s18OHF, and s18oxoF before and after saline load test (SLT) and 24-h u18OHF and u18oxoF. RESULTS PA patients displayed significantly higher levels of s18OHB, u18OHF, and u18oxoF compared to EH and normal subjects; APA patients displayed s18OHB, u18OHF, and u18oxoF levels significantly higher than BAH patients. Similar results were obtained for s18OHF and s18oxoF. SLT significantly reduced s18OHB, s18OHF, and s18oxoF in all groups, but steroid reduction was much less for APA patients compared to BAH and EH. The s18OHB/aldosterone ratio after SLT more than doubled in EH but remained unchanged in APA patients. CONCLUSIONS u18OHF, u18oxoF, and s18OHB measurements in patients with a positive aldosterone/plasma renin activity ratio correlate with confirmatory tests and adrenal vein sampling in PA patients. If verified, these steroid assays would refine the diagnostic workup for PA.

LXR-activating oxysterols induce the expression of inflammatory markers in endothelial cells through LXR-independent mechanisms

AIMS Liver X receptors alpha and beta (LXRalpha, LXRbeta) are key regulators of cholesterol homeostasis. The effects of LXR ligands on endothelial cells are largely unknown. While oxysterol LXR agonists can increase the endothelial-leukocyte interaction, synthetic LXR agonists are anti-atherogenic and anti-inflammatory. Mechanistic differences may underlie such findings. METHODS AND RESULTS LXRalpha and LXRbeta were found to be expressed in human endothelial cells. While synthetic LXR agonists could blunt the LPS-induced up-regulation of adhesion molecules (ICAM-1, VCAM-1, E-Selectin), 22-hydroxycholesterol and 24,25-epoxycholesterol enhanced such response. Microarray profiling further showed that the endothelial gene expression fingerprints of 22-hydroxycholesterol and T0901317 largely differed and unexpectedly shared only a restricted number of genes. Indeed, 22-hydroxycholesterol down-regulated eNOS and up-regulated a vast cohort of inflammatory mediators such as adhesion molecules, cytokines, enzymes and transcription factors. Other LXR-activating oxysterols such as 24,25-epoxycholesterol, 25-hydroxycholesterol and 27-hydroxycholesterol could also stimulate the endothelial expression of inflammatory markers, although significant differences were observed. These effects persisted in LXR-silenced cells, confirming the mechanistic dissociation of oxysterol and LXR pathways. Furthermore, the oxysterol-induced expression of inflammatory markers was not secondary to cell apoptosis and may relate to oxidative stress. CONCLUSIONS LXR-activating oxysterols comprehensively activate the expression of endothelial inflammation markers independently from LXRs. At proper dosage, synthetic LXR agonists are safe on endothelial cells and may even transrepress inflammatory reactions.

Vasoactive hormones induce nitric oxide synthase mRNA expression and nitric oxide production in human endothelial cells and monocytes

Isoform-2 nitric oxide synthase (NOS-2) mRNA expression and nitric oxide (NO) production are induced in endothelial cells and monocytes by cytokines such as gammaIFN and LPS. We evaluated NOS-2 and isoform-3 NOS (NOS-3) mRNA expression and NO production in human monocytes and human umbilical vein endothelial cells (HUVEC), under basal conditions and after incubation with physiologic concentrations of vasoactive hormones. NOS mRNA expression was detected by reverse transcription polymerase chain reaction (RT-PCR) and NO production by electronic paramagnetic resonance spectroscopy (EPR). We showed that NOS-2 mRNA expression and NO production were induced by stimulation with epinephrine, dopamine, endothelin-1, and angiotensin II, both in monocytes and HUVEC. NOS-3 mRNA expression and NO production were detected under basal conditions in monocytes and HUVEC and were not modified by the presence of vasoactive hormones. Human endothelial cells and monocytes express the NOS-2 and NOS-3 mRNA and the inducible NOS-2 mRNA expression increases after vasoactive hormone stimulation.

α1-Adrenergic Receptor Subtypes in Human Peripheral Blood Lymphocytes

We investigated the expression of alpha1-adrenergic receptor subtypes in intact human peripheral blood lymphocytes using reverse transcription-polymerase chain reaction (RT-PCR) and radioligand binding assay techniques combined with antibodies against the three subtypes of alpha1-adrenergic receptors (alpha1A, alpha1B, and alpha1D). RT-PCR amplified in peripheral blood lymphocytes a 348-bp alpha1A-adrenergic receptor fragment, a 689-bp alpha1B-adrenergic receptor fragment, and a 540-bp alpha1D-adrenergic receptor fragment. Radioligand binding assay with [3H]prazosin as radioligand revealed a high-affinity binding with a dissociation constant value of 0. 65+/-0.05 nmol/L and a maximum density of binding sites of 175. 3+/-20.5 fmol/10(6) cells. The pharmacological profile of [3H]prazosin binding to human peripheral blood lymphocytes was consistent with the labeling of alpha1-adrenergic receptors. Antibodies against alpha1A-, alpha1B-, and alpha1D-receptor subtypes decreased [3H]prazosin binding to a different extent. This indicates that human peripheral blood lymphocytes express the three alpha1-adrenergic receptor subtypes. Of the three different alpha1-adrenergic receptor subtypes, the alpha1B is the most represented and the alpha1D, the least. Future studies should clarify the functional relevance of alpha1-adrenergic receptors expressed by peripheral blood lymphocytes. The identification of these sites may represent a step for evaluating whether they represent a marker of alpha1-adrenergic receptors in cardiovascular disorders or for assessing responses to drug treatment on these receptors.

Blood pressure and heart rate in young thalassemia major patients

The analysis of blood pressure (BP) and heart rate (HR) variability is currently used to investigate the mechanisms responsible for cardiovascular control; therefore, we assessed whether an impairment of 24-h BP and HR profiles and sympathovagal interaction modulating cardiovascular function was present in patients with thalassemia major (TM) in preclinical phase of heart disease. Nine beta-thalassemic patients 18 years old without clinical signs of cardiac failure and 9 age- and sex-matched controls were studied. Twenty-four-hour-ambulatory BP and HR were measured using the SpaceLabs 90207 device. A truncated Fourier series with four harmonics was used to describe the diurnal blood pressure profile. Mean 24-h ambulatory systolic BP, diastolic BP, and mean arterial pressure were significantly lower in TM patients than in normal subjects (P < .05). A significantly higher nighttime HR value was found in TM patients (P < .05). More than 40% of the TM patients did not show a significant diurnal BP and HR rhythm. In TM patients, the overall amplitude of systolic BP, diastolic BP, and HR was significantly lower than in controls (P < .01). The night/day differences of systolic BP, diastolic BP, and HR were significantly lower in TM patients than in normals (P < .01). Furthermore, we performed power spectral analysis on short-term continuous finger BP and HR data in supine position and during passive head-up tilt. Total spectral power of systolic BP was significantly lower in patients than controls (P < .05). Low-frequency (LF) power of systolic BP and diastolic BP and LF/high-frequency (HF) ratio of HR were significantly lower during tilt in TM patients compared to controls (P < .05). High-frequency power of HR was significantly higher in patients than controls (P < .05). The baroreflex gain assessed by alpha-index was the same in supine position but was higher in TM patients during passive tilt (P < .05). An inverse relationship between LF/HF ratio of HR and hemoglobin levels in TM patients was found. Finally, plasma norepinephrine levels were significantly lower in thalassemics (P < .005). In young TM patients in a preclinical stage of heart disease, these findings demonstrated abnormal 24-h BP and HR rhythms and a decreased short-term variability of BP and HR, in particular in the LF range, showing a diminished sympathetic activity.

Bradykinin B2 receptor gene (-58T/C) polymorphism influences baroreflex sensitivity in never-treated hypertensive patients.

Background Most evidence currently favours a fundamental role of the autonomic nervous system in the pathogenesis of essential hypertension. Recent studies suggest that about 40% of baroreflex variation, an index of cardiac autonomic control, is influenced by genetic factors. Methods and results The aim of this study was to investigate the effect of a common polymorphic variant of the bradykinin B2 receptor gene (B2R; −58T/C) on the autonomic regulation of baroreflex sensitivity (BRS) in 129 mild–moderate never-treated hypertensive patients. No significant differences were found for clinical and biochemical parameters among genotypes. BRS increased with the number of B2R T alleles. B2R genotype was a strong independent predictor of BRS, accounting for 12% of its variation. We suggest that a decrease in the transcription of the bradykinin B2R gene in the presence of the B2R −58C allele could reduce BRS via the diminished effect of bradykinin. Conclusions B2R genotype can explain part of the BRS variation that is unaccounted for by simple anthropometric variables and common risk factors.

Evaluation of Serotonin Levels in Human Aqueous Humor

Purpose: Serotonin is biochemically present in the iris and ciliary body of animals and humans. Controversial findings are reported about the concentrations of serotonin in aqueous humor with respect to plasma in humans. The aim of this study was to evaluate the levels of serotonin both in aqueous humor and plasma in human subjects. Methods: In 50 patients with glaucoma or cataract, plasma and aqueous humor serotonin levels were measured by HPLC with electrochemical detection. Serotonin plasma levels were also measured in 25 healthy subjects as controls. Results: In all patients with cataract or glaucoma, the aqueous humor serotonin concentration is significantly lower than that in plasma [1.14±0.29 (SEM) vs. 5.33±1.03 ng/ml, p < 0.01]. Furthermore, in the same patients and in 25 healthy controls, serotonin plasma levels were similar. Conclusion: Our study shows that serotonin is present in human aqueous humor and its concentration is 4 times lower than in plasma.

Alpha1-adrenergic receptor subtypes in peripheral blood lymphocytes of essential hypertensives.

Objective The expression of α1-adrenergic receptor subtypes in peripheral blood lymphocytes was investigated in 28 essential hypertensive patients as well as in the peripheral blood lymphocytes and aorta of spontaneously hypertensive rats (SHR) and normotensive Wistar–Kyoto (WKY) rats. Methods α1-Adrenergic receptors were quantified by radioligand binding assays, employing [3H]-prazosin as the radioligand in association with compounds displaying different degrees of selectivity for α1A-, α1B- and α1D-adrenergic receptor subtypes. Results The affinity of [3H]-prazosin binding was similar in peripheral blood lymphocytes of different stage essential hypertensive and normotensive subjects or of SHR and age-matched normotensive WKY rats as well as in the aortas of SHR and WKY rats. The radioligand binding assay revealed no change in the expression of α1-adrenergic receptors in peripheral blood lymphocytes of essential hypertensives compared with normotensive subjects; a moderate decrease of α1B-adrenergic receptors and an increase of α1D-adrenergic receptors. The relative densities of the α1-adrenergic receptor subtypes were similar in the three groups of essential hypertensives. In peripheral blood lymphocytes and in aorta of SHR, [3H]-prazosin binding was significantly reduced compared with normotensive WKY rats. The expression of α1-adrenergic receptor subtypes in peripheral blood lymphocytes of SHR was similar to that found in peripheral blood lymphocytes of essential hypertensives. Conclusions Changes of lymphocyte α1-adrenergic receptor subtypes in essential hypertensives are similar to those observed in lymphocytes and vascular tissues of animal models of hypertension. This suggests that assays of lymphocyte α1-adrenergic receptors may represent an indirect marker of their involvement in essential hypertension.