The Elusive Nature of ABCC8-related Maturity-Onset Diabetes of the Young (ABCC8-MODY). A Review of the Literature and Case Discussion.

PURPOSE OF REVIEW: Maturity-onset diabetes of the young (MODY) are monogenic forms of diabetes resulting from genetic defects, usually transmitted in an autosomal dominant fashion, leading to ß-cell dysfunction. Due to the lack of homogeneous clinical features and univocal diagnostic criteria, MODY is often misdiagnosed as type 1 or type 2 diabetes, hence its diagnosis relies mostly on genetic testing. Fourteen subtypes of MODY have been described to date. Here, we review ABCC8-MODY pathophysiology, genetic and clinical features, and current therapeutic options. RECENT FINDINGS: ABCC8-MODY is caused by mutations in the adenosine triphosphate (ATP)-binding cassette transporter subfamily C member 8 (ABCC8) gene, involved in the regulation of insulin secretion. The complexity of ABCC8-MODY genetic picture is mirrored by a variety of clinical manifestations, encompassing a wide spectrum of disease severity. Such inconsistency of genotype-phenotype correlation has not been fully understood. A correct diagnosis is crucial for the choice of adequate treatment and outcome improvement. By targeting the defective gene product, sulfonylureas are the preferred medications in ABCC8-MODY, although efficacy vary substantially. We illustrate three case reports in whom a diagnosis of ABCC8-MODY was suspected after the identification of novel ABCC8 variants that turned out to be of unknown significance. We discuss that careful interpretation of genetic testing is needed even on the background of a suggestive clinical context. We highlight the need for further research to unravel ABCC8-MODY disease mechanisms, as well as to clarify the pathogenicity of identified ABCC8 variants and their influence on clinical presentation and response to therapy.

Double CYP11B1/CYP11B2 Immunohistochemistry and Detection of KCNJ5 Mutations in Primary Aldosteronism.

CONTEXT: The search for somatic mutations in adrenals resected from primary aldosteronism (PA) patients is being performed by Sanger sequencing, often implemented with immunohistochemistry (IHC)-guidance focused on aldosterone-producing (CYP11B2-positive) areas. OBJECTIVE: To investigate the impact of double IHC for CYP11B1 and CYP11B2 on Sanger and next generation sequencing (NGS). METHODS: We investigated 127 consecutive adrenal aldosterone producing adenoma from consenting surgically cured PA patients using double IHC for CYP11B1 and CYP11B2, Sanger sequencing and NGS. RESULTS: Double IHC for CYP11B2 and CYP11B1 revealed 3 distinct patterns: CYP11B2-positive adenoma (pattern 1), mixed CYP11B1/CYP11B2-positive adenoma (pattern 2), and adrenals with multiple small CYP11B2-positive nodules (pattern 3). Sanger sequencing allowed detection of KCNJ5 mutations in 44% of the adrenals; NGS revealed such mutations in 10% of those negative at Sanger and additional mutations in 61% of the cases. Importantly the rate of KCNJ5 mutations differed across patterns: 17.8% in pattern 1, 71.4% in pattern 2, and 10.7% in pattern 3 (χ2=22.492, p<0.001). CONCLUSIONS: NGS allowed detection of mutations in many adrenals that tested negative at Sanger sequencing. Moreover, the different distribution of KCNJ5 mutations across IHC patterns indicates that IHC-guided sequencing protocols selecting CYP11B2-positive areas could furnish results that might not be representative of the entire mutational status of the excised adrenal, which is important at a time when KCNJ5 mutations are suggested to drive management of APA patient.

Circulating extracellular vesicles and small non-coding RNAs cargo in idiopathic inflammatory myopathies reveal differences across myositis subsets.

OBJECTIVE: To investigate the epigenetic footprint of idiopathic inflammatory myopathies (IIM) through characterization of circulating extracellular vesicles (EVs) and the expression of EV-derived small non-coding RNAs (sncRNAs). METHODS: In this cross-sectional study, EVs were isolated by size-exclusion chromatography from plasma of patients with IIM and age- and sex-matched healthy donors (HD). EV-derived sncRNAs were sequenced and quantified using Next-Generation Sequencing (NGS). Following quality control and normalization, filtered count reads were used for differential microRNA (miRNA) and piwi-interacting RNA (piRNA) expression analyses. Putative gene targets enriched for pathways implicated in IIM were analyzed. Patients' clinical and laboratory characteristics at the time of sampling were recorded. RESULTS: Forty-seven IIM patients and 45 HD were enrolled. MiR-486-5p (p < 0.01), miR-122-5p, miR-192-5p, and miR-32-5p were significantly upregulated (p < 0.05 for all), while miR-142-3p (p < 0.001), miR-141-3p (p < 0.01), let-7a-5p (p < 0.05) and miR-3613-5p (p < 0.05) downregulated in EVs from IIM patients versus HD. MiR-486-5p was associated with raised muscle enzymes levels. Several target genes of up/downregulated miRNAs in IIM participate in inflammation, necroptosis, interferon and immune signaling. Six piRNAs were significantly dysregulated in IIM EVs versus HD (p < 0.05). Within IIM, miR-335-5p was selectively upregulated and miR-27a-5p downregulated in dermatomyositis (n = 21, p < 0.01). Finally, plasma EV levels were significantly increased in cancer-associated myositis (CAM, n = 12) versus non-CAM IIM (n = 35, p = 0.02) and HD (p < 0.01). EVs cargo in CAM was significantly enriched of let-7f-5p and depleted of miR-143-3p. CONCLUSION: Through an unbiased screening of EV-derived sncRNAs, we characterize miRNAs and piRNAs in the EVs cargo as potential biomarkers and modifiers of diverse IIM phenotypes.

Coexistence of Multiple Gene Variants in Some Patients with Erythrocytoses.

Background: Erythrocytosis is a relatively common condition; however, a large proportion of these patients (70%) remain without a clear etiologic explanation. Methods: We set up a targeted NGS panel for patients with erythrocytosis, and 118 sporadic patients with idiopathic erythrocytosis were studied. Results: In 40 (34%) patients, no variant was found, while in 78 (66%), we identified at least one germinal variant; 55 patients (70.5%) had 1 altered gene, 18 (23%) had 2 alterations, and 5 (6.4%) had 3. An altered HFE gene was observed in 51 cases (57.1%), EGLN1 in 18 (22.6%) and EPAS1, EPOR, JAK2, and TFR2 variants in 7.7%, 10.3%, 11.5%, and 14.1% patients, respectively. In 23 patients (19.45%), more than 1 putative variant was found in multiple genes. Conclusions: Genetic variants in patients with erythrocytosis were detected in about 2/3 of our cohort. An NGS panel including more candidate genes should reduce the number of cases diagnosed as "idiopathic" erythrocytosis in which a cause cannot yet be identified. It is known that HFE variants are common in idiopathic erythrocytosis. TFR2 alterations support the existence of a relationship between genes involved in iron metabolism and impaired erythropoiesis. Some novel multiple variants were identified. Erythrocytosis appears to be often multigenic.

Subtype Identification of Surgically Curable Primary Aldosteronism During Treatment With Mineralocorticoid Receptor Blockade.

BACKGROUND: Current guidelines and consensus documents recommend withdrawal of mineralocorticoid receptor antagonists (MRAs) before primary aldosteronism (PA) subtyping by adrenal vein sampling (AVS), but this practice can cause severe hypokalemia and uncontrolled high blood pressure. Our aim was to investigate if unilateral PA can be identified by AVS during MRA treatment. METHODS: We compared the rate of unilateral PA identification between patients with and without MRA treatment in large data sets of patients submitted to AVS while off renin-angiotensin system blockers and ß-blockers. In sensitivity analyses, the between-group differences of lateralization index values after propensity score matching and the rate of unilateral PA identification in subgroups with undetectable (≤2 mUI/L), suppressed (<8.2 mUI/L), and unsuppressed (≥8.2 mUI/L) direct renin concentration levels were also evaluated. RESULTS: Plasma aldosterone concentration, direct renin concentration, and blood pressure values were similar in non-MRA-treated (n=779) and MRA-treated (n=61) patients with PA, but the latter required more antihypertensive agents (P=0.001) and showed a higher rate of adrenal nodules (82% versus 67%; P=0.022) and adrenalectomy (72% versus 54%; P=0.01). However, they exhibited no significant differences in commonly used AVS indices and the area under the receiving operating characteristic curve of lateralization index, both under unstimulated conditions and postcosyntropin. Several sensitivity analyses confirmed these results in propensity score matching adjusted models and in patients with undetectable, or suppressed or unsuppressed renin levels. CONCLUSIONS: At doses that controlled blood pressure and potassium levels, MRAs did not preclude the identification of unilateral PA at AVS. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT01234220.

MicroRNAs in idiopathic inflammatory myopathies: state-of-the-art and future perspectives.

PURPOSE OF REVIEW: Idiopathic inflammatory myopathies (IIMs) are a group of rare autoimmune disorders characterized by muscle weakness and inflammation. MicroRNAs (miRNAs) are the main class of small noncoding RNAs regulating a wide range of physiological and pathological processes and play a role in mediating autoimmunity and inflammation. In this review, we summarize the latest knowledge on the role of miRNAs in systemic autoimmune diseases with particular focus on IIMs. RECENT FINDINGS: Study on miRNA expression in IIMs is helping in understanding the pathogenetic basis of the disease at a tissue and systemic level. Several miRNAs, even with a muscle-specific expression (myomiRs), have been shown to be involved in immune and nonimmune mechanisms of myofiber damage. MiRNAs modulate and orchestrate the local inflammatory infiltrate and could be used as potential biomarkers as they correlate with disease activity and response to therapy. SUMMARY: IIMs comprise different clinical phenotypes and still little is known about the molecular signature of each subset. Further research about miRNA profiling will provide additional insights in the disease characterization with an expected impact on the therapeutic strategies.

Circulating Small Noncoding RNA Profiling as a Potential Biomarker of Atherosclerotic Plaque Composition in Type 1 Diabetes.

OBJECTIVE: Cardiovascular disease (CVD) accounts for most deaths in patients with type 1 diabetes (T1D); however, the determinants of plaque composition are unknown. miRNAs regulate gene expression, participate in the development of atherosclerosis, and represent promising CVD biomarkers. This study analyzed the circulating miRNA expression profile in T1D with either carotid calcified (CCP) or fibrous plaque (CFP). RESEARCH DESIGN AND METHODS: Circulating small noncoding RNAs were sequenced and quantified using next-generation sequencing and bioinformatic analysis in an exploratory set of 26 subjects with T1D with CCP and in 25 with CFP. Then, in a validation set of 40 subjects with CCP, 40 with CFP, and 24 control subjects with T1D, selected miRNA expression was measured by digital droplet PCR. Putative gene targets enriched for pathways implicated in atherosclerosis/vascular calcification/diabetes were analyzed. The patients' main clinical characteristics were also recorded. RESULTS: miR-503-5p, let-7d-5p, miR-106b-3p, and miR-93-5p were significantly upregulated, while miR-10a-5p was downregulated in patients with CCP compared with CFP (all fold change >±1.5; P < 0.05). All candidate miRNAs showed a significant correlation with LDL-cholesterol, direct for the upregulated and inverse for the downregulated miRNA, in CCP. Many target genes of upregulated miRNAs in CCP participate in osteogenic differentiation, apoptosis, inflammation, cholesterol metabolism, and extracellular matrix organization. CONCLUSIONS: These findings characterize miRNAs and their signature in the regulatory network of carotid plaque phenotype in T1D, providing new insights into plaque pathophysiology and possibly novel biomarkers of plaque composition.

Circulating microRNA signatures associated with disease severity and outcome in COVID-19 patients.

Background: SARS-CoV-2 induces a spectrum of clinical conditions ranging from asymptomatic infection to life threatening severe disease. Host microRNAs have been involved in the cytokine storm driven by SARS-CoV-2 infection and proposed as candidate biomarkers for COVID-19. Methods: To discover signatures of circulating miRNAs associated with COVID-19, disease severity and mortality, small RNA-sequencing was performed on serum samples collected from 89 COVID-19 patients (34 severe, 29 moderate, 26 mild) at hospital admission and from 45 healthy controls (HC). To search for possible sources of miRNAs, investigation of differentially expressed (DE) miRNAs in relevant human cell types in vitro. Results: COVID-19 patients showed upregulation of miRNAs associated with lung disease, vascular damage and inflammation and downregulation of miRNAs that inhibit pro-inflammatory cytokines and chemokines, angiogenesis, and stress response. Compared with mild/moderate disease, patients with severe COVID-19 had a miRNA signature indicating a profound impairment of innate and adaptive immune responses, inflammation, lung fibrosis and heart failure. A subset of the DE miRNAs predicted mortality. In particular, a combination of high serum miR-22-3p and miR-21-5p, which target antiviral response genes, and low miR-224-5p and miR-155-5p, targeting pro-inflammatory factors, discriminated severe from mild/moderate COVID-19 (AUROC 0.88, 95% CI 0.80-0.95, p<0.0001), while high leukocyte count and low levels of miR-1-3p, miR-23b-3p, miR-141-3p, miR-155-5p and miR-4433b-5p predicted mortality with high sensitivity and specificity (AUROC 0.95, 95% CI 0.89-1.00, p<0.0001). In vitro experiments showed that some of the DE miRNAs were modulated directly by SARS-CoV-2 infection in permissive lung epithelial cells. Conclusions: We discovered circulating miRNAs associated with COVID-19 severity and mortality. The identified DE miRNAs provided clues on COVID-19 pathogenesis, highlighting signatures of impaired interferon and antiviral responses, inflammation, organ damage and cardiovascular failure as associated with severe disease and death.

A miR-125/Sirtuin-7 pathway drives the pro-calcific potential of myeloid cells in diabetic vascular disease.

AIMS/HYPOTHESIS: Ectopic calcification is a typical feature of diabetic vascular disease and resembles an accelerated ageing phenotype. We previously found an excess of myeloid calcifying cells in diabetic individuals. We herein examined molecular and cellular pathways linking atherosclerotic calcification with calcification by myeloid cells in the diabetic milieu. METHODS: We first examined the associations among coronary calcification, myeloid calcifying cell levels and mononuclear cell gene expression in a cross-sectional study of 87 participants with type 2 diabetes undergoing elective coronary angiography. Then, we undertook in vitro studies on mesenchymal stem cells and the THP-1 myeloid cell line to verify the causal relationships of the observed associations. RESULTS: Coronary calcification was associated with 2.8-times-higher myeloid calcifying cell levels (p=0.037) and 50% elevated expression of the osteogenic gene RUNX2 in mononuclear cells, whereas expression of Sirtuin-7 (SIRT7) was inversely correlated with calcification. In standard differentiation assays of mesenchymal stem cells, SIRT7 knockdown activated the osteogenic program and worsened calcification, especially in the presence of high (20 mmol/l) glucose. In the myeloid cell line THP-1, SIRT7 downregulation drove a pro-calcific phenotype, whereas SIRT7 overexpression prevented high-glucose-induced calcification. Through the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, high glucose induced miR-125b-5p, which in turn targeted SIRT7 in myeloid cells and was directly associated with coronary calcification. CONCLUSIONS/INTERPRETATION: We describe a new pathway elicited by high glucose through the JAK/STAT cascade, involving regulation of SIRT7 by miR-125b-5p and driving calcification by myeloid cells. This pathway is associated with coronary calcification in diabetic individuals and may be a target against diabetic vascular disease. DATA AVAILABILITY: RNA sequencing data are deposited in GEO (accession number GSE193510; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE193510 ).

Comparison of Cortisol, Androstenedione and Metanephrines to Assess Selectivity and Lateralization of Adrenal Vein Sampling in Primary Aldosteronism.

Success of adrenal vein sampling (AVS) is verified by the selectivity index (SI), i.e., by a step-up of cortisol levels between the adrenal vein and the infrarenal inferior vena cava samples, beyond a given cut-off. We tested the hypothesis that androstenedione, metanephrine, and normetanephrine, which have higher gradients than cortisol, could increase the rate of AVS studies judged to be bilaterally successful and usable for the clinical decision making. We prospectively compared within-patient, head-to-head, the selectivity index of androstenedione (SIA), metanephrine (SIM), and normetanephrine (SINM), and cortisol (SIC) in consecutive hypertensive patients with primary aldosteronism submitted to AVS. Main outcome measures were rate of bilateral success, SI values, and identification of unilateral PA. We recruited 136 patients (55 + 10 years, 35% women). Compared to the SIC, the SIA values were 3.5-fold higher bilaterally, and the SIM values were 7-fold and 4.4-fold higher on the right and the left side, respectively. With the SIA and the SIM the rate of bilaterally successful AVS increased by 14% and 15%, respectively without impairing the identification of unilateral PA. We concluded that androstenedione and metanephrine outperformed cortisol for ascertaining AVS success, thus increasing the AVS studies useable for the clinical decision making.

PAR-4/Ca2+-calpain pathway activation stimulates platelet-derived microparticles in hyperglycemic type 2 diabetes.

BACKGROUND: Patients with type 2 diabetes (T2DM) have a prothrombotic state that needs to be fully clarified; microparticles (MPs) have emerged as mediators and markers of this condition. Thus, we investigate, in vivo, in T2DM either with good (HbA1c ≤ 7.0%; GGC) or poor (HbA1c > 7.0%; PGC) glycemic control, the circulating levels of MPs, and in vitro, the molecular pathways involved in the release of MPs from platelets (PMP) and tested their pro-inflammatory effects on THP-1 transformed macrophages. METHODS: In 59 T2DM, and 23 control subjects with normal glucose tolerance (NGT), circulating levels of CD62E+, CD62P+, CD142+, CD45+ MPs were determined by flow cytometry, while plasma levels of ICAM-1, VCAM-1, IL-6 by ELISA. In vitro, PMP release and activation of isolated platelets from GGC and PGC were investigated, along with their effect on IL-6 secretion in THP-1 transformed macrophages. RESULTS: We found that MPs CD62P+ (PMP) and CD142+ (tissue factor-bearing MP) were significantly higher in PGC T2DM than GGC T2DM and NGT. Among MPs, PMP were also correlated with HbA1c and IL-6. In vitro, we showed that acute thrombin exposure stimulated a significantly higher PMP release in PGC T2DM than GGC T2DM through a more robust activation of PAR-4 receptor than PAR-1 receptor. Treatment with PAR-4 agonist induced an increased release of PMP in PGC with a Ca2+-calpain dependent mechanism since this effect was blunted by calpain inhibitor. Finally, the uptake of PMP derived from PAR-4 treated PGC platelets into THP-1 transformed macrophages promoted a marked increase of IL-6 release compared to PMP derived from GGC through the activation of the NF-kB pathway. CONCLUSIONS: These results identify PAR-4 as a mediator of platelet activation, microparticle release, and inflammation, in poorly controlled T2DM.

Low P66shc with High SerpinB3 Levels Favors Necroptosis and Better Survival in Hepatocellular Carcinoma.

Cell proliferation and escape from apoptosis are important pathological features of hepatocellular carcinoma (HCC), one of the tumors with the highest mortality rate worldwide. The aim of the study was to evaluate the expression of the pro-apoptotic p66shc and the anti-apoptotic SerpinB3 in HCCs in relation to clinical outcome, cell fate and tumor growth. p66shc and SerpinB3 were evaluated in 67 HCC specimens and the results were correlated with overall survival. Proliferation and cell death markers were analyzed in hepatoma cells overexpressing SerpinB3, under different stress conditions. p66shc-/- mice and xenograft models were also used to assess the effects of p66shc and SerpinB3 on tumor growth. In patients with HCC, the best survival was observed in the subgroup with p66shc levels below median values and SerpinB3 levels above median values. Mice p66shc-/- showed high levels of SerpinB3, while in HepG2 cells overexpressing SerpinB3, p66shc expression was trivial. HepG2 overexpressing SerpinB3 cells were more prone to die after oxidizing treatments, such as diamide or high concentration H2O2. These cells injected in nude mice developed tumors five times smaller than those from control HepG2 cells. Tumors originating from HepG2 overexpressing SerpinB3 cells showed decreased activated Caspase-8, with concomitant increase of RIP3K and decreased levels of cleaved RIP3K, typical features of necroptosis. In conclusion, in patients affected by HCC, the pattern characterized by p66shc downregulation and elevated SerpinB3 levels was associated with markedly better survival. This pattern favored necroptosis in experimental high-stress conditions.

High sodium intake, glomerular hyperfiltration, and protein catabolism in patients with essential hypertension.

AIMS: A blood pressure (BP)-independent metabolic shift towards a catabolic state upon high sodium (Na+) diet, ultimately favouring body fluid preservation, has recently been described in pre-clinical controlled settings. We sought to investigate the real-life impact of high Na+ intake on measures of renal Na+/water handling and metabolic signatures, as surrogates for cardiovascular risk, in hypertensive patients. METHODS AND RESULTS: We analysed clinical and biochemical data from 766 consecutive patients with essential hypertension, collected at the time of screening for secondary causes. The systematic screening protocol included 24 h urine (24 h-u-) collection on usual diet and avoidance of renin-angiotensin-aldosterone system-confounding medications. Urinary 24 h-Na+ excretion, used to define classes of Na+ intake (low ≤2.3 g/day; medium 2.3-5 g/day; high >5 g/day), was an independent predictor of glomerular filtration rate after correction for age, sex, BP, BMI, aldosterone, and potassium excretion [P = 0.001; low: 94.1 (69.9-118.8) vs. high: 127.5 (108.3-147.8) mL/min/1.73 m2]. Renal Na+ and water handling diverged, with higher fractional excretion of Na+ and lower fractional excretion of water in those with evidence of high Na+ intake [FENa: low 0.39% (0.30-0.47) vs. high 0.81% (0.73-0.98), P < 0.001; FEwater: low 1.13% (0.73-1.72) vs. high 0.89% (0.69-1.12), P = 0.015]. Despite higher FENa, these patients showed higher absolute 24 h Na+ reabsorption and higher associated tubular energy expenditure, estimated by tubular Na+/ATP stoichiometry, accordingly [Δhigh-low = 18 (12-24) kcal/day, P < 0.001]. At non-targeted liquid chromatography/mass spectrometry plasma metabolomics in an unselected subcohort (n = 67), metabolites which were more abundant in high versus low Na+ intake (P < 0.05) mostly entailed intermediates or end products of protein catabolism/urea cycle. CONCLUSION: When exposed to high Na+ intake, kidneys dissociate Na+ and water handling. In hypertensive patients, this comes at the cost of higher glomerular filtration rate, increased tubular energy expenditure, and protein catabolism from endogenous (muscle) or excess exogenous (dietary) sources. Glomerular hyperfiltration and the metabolic shift may have broad implications on global cardiovascular risk independent of BP.

Effects of Mineralocorticoid and AT1 Receptor Antagonism on The Aldosterone-Renin Ratio In Primary Aldosteronism-the EMIRA Study.

CONTEXT: While current guidelines recommend the withdrawal of mineralocorticoid receptor antagonist (MRA) and renin-angiotensin system blockers for the screening and detection of primary aldosteronism (PA), this can worsen hypokalemia and control of high blood pressure (BP) values. OBJECTIVE: To investigate whether aldosterone/renin ratio (ARR) values were affected by the MRA canrenone and/or by canrenone plus olmesartan treatment in patients with PA. DESIGN: Within-patient study. SETTING: The European Society of Hypertension center of excellence at the University of Padua. PATIENTS: Consecutive patients with an unambiguous diagnosis of PA subtyped by adrenal vein sampling. INTERVENTIONS: Patients were treated for 1 month with canrenone (50-100 mg orally), and for an additional month with canrenone plus olmesartan (10-20 mg orally). Canrenone and olmesartan were up-titrated over the first 2 weeks until BP values and hypokalemia were controlled. Patients with unilateral PA were adrenalectomized; those with bilateral PA were treated medically. MAIN OUTCOME MEASURES: BP, plasma levels of sodium and potassium, renin and aldosterone. RESULTS: Canrenone neither lowered plasma aldosterone nor increased renin; thus, the high ARR and true positive rate remained unaffected. Addition of the angiotensin type 1 receptor blocker raised renin and slightly lowered aldosterone, which reduced the ARR and increased the false negative rate. CONCLUSIONS: At doses that effectively controlled serum potassium and BP values, canrenone did not preclude an accurate diagnosis in patients with PA. Addition of the angiotensin type 1 receptor blocker olmesartan slightly raised the false negative rate. Hence, MRA did not seem to endanger the accuracy of the diagnosis of PA.

Transcription Factors Regulation in Human Peripheral White Blood Cells during Hypobaric Hypoxia Exposure: an in-vivo experimental study.

High altitude is a natural laboratory, within which the clinical study of human physiological response to hypobaric hypoxia (HH) is possible. Failure in the response results in progressive hypoxemia, inflammation and increased tissue oxidative stress (OxS). Thus, investigating temporal changes in key transcription factors (TFs) HIF-1α, HIF-2α, NF-κB and NRF2 mRNA levels, relative to OxS and inflammatory markers, may reveal molecular targets which contrast deleterious effects of hypoxia. Biological samples and clinical data from 15 healthy participants were collected at baseline and after rapid, passive ascent to 3830 m (24 h and 72 h). Gene expression was assessed by qPCR and ROS generation was determined by EPR spectroscopy. Oxidative damage and cytokine levels were estimated by immuno or enzymatic methods. Hypoxia transiently enhanced HIF-1α mRNA levels over time reaching a peak after 24 h. Whereas, HIF-2α and NRF2 mRNA levels increased over time. In contrast, the NF-κB mRNA levels remained unchanged. Plasma levels of IL-1ß and IL-6 also remained within normal ranges. ROS production rate and markers of OxS damage were significantly increased over time. The analysis of TF-gene expression suggests that HIF-1α is a lead TF during sub-acute HH exposure. The prolongation of the HH exposure led to a switch between HIF-1α and HIF-2α/NRF2, suggesting the activation of new pathways. These results provide new insights regarding the temporal regulation of TFs, inflammatory state, and ROS homeostasis involved in human hypoxic response, potentially also relevant to the mediation of diseases that induce a hypoxic state.

Design of a study to investigate the mechanisms of obstructive sleep apnoea by means of drug-induced sleep endoscopy.

Background Obstructive sleep apnoea (OSA) is an independent risk factor of hypertension and cardiovascular diseases. Recurrent episodes of upper airways collapse during sleep causing blood oxygen desaturation, hypercapnia, and micro-arousals, are known to activate the sympathetic nervous system (SNS). However, whether changes in the renin-angiotensin-aldosterone system and endothelial activation also occur remains contentious. Methods Based on routine use of drug-induced sleep endoscopy (DISE) for the work-up of OSA patients in our centre, we designed a prospective study to investigate the haemodynamic and humoral changes occurring during the apnoeic episodes reproduced in vivo in the course of DISE. Specifically, plasma aldosterone concentration and renin activity, C-terminal fragment of proendothelin-1, as a marker of endothelial damage, and free plasma catecholamines, will be measured at fixed times during DISE. The activity of catechol-O-methyltransferase (COMT), a key catecholamine-inactivating enzyme that has been scantly investigated thus far owing to the lack of commercially available kits, will be also determined by a newly developed high performance liquid chromatography method, which is herein described. Results and conclusions The aim of this study is to provide novel information on the haemodynamic, hormonal, and SNS changes, and also on COMT activity modification concomitantly occurring during apnoea, thus contributing substantively to the understanding of the pathophysiology of OSA.

Effects of mineralocorticoid and AT-1 receptor antagonism on the aldosterone-renin ratio (ARR) in primary aldosteronism patients (EMIRA Study): rationale and design.

Current guidelines recommend withdrawal of treatments that affect the aldosterone/renin ratio (ARR) when screening for primary aldosteronism (PA). However, abandonment of mineralocorticoid-receptor antagonist (MRA) and/or blockers of the renin-angiotensin system can deteriorate control of blood pressure (BP) and hypokalemia. Thus, in consecutive patients with an unambiguous diagnosis of PA in washout from confounding treatments and subtyped by AVS, we will compare within-patient plasma aldosterone and active renin concentration, and the ARR values, measured at baseline, and after a 1-month treatment with MRA alone and combined with an AT-1 receptor blocker (ARB). Patients on a regular salt intake will be treated with canrenone (50-100 mg orally) for 1 month, after which olmesartan (10 or 20 mg orally) will be added for another month with up-titration of both treatments over the first 2 weeks to control BP and hypokalemia, while background therapy will be maintained. The biochemical variables and the ARR will be assessed in an identical manner at baseline values and after each month of treatment. With a sample size of 40 patients, the study will have a 95% power to show a clinically significant 20% change in the ARR at a 5% α value using a two-sided paired t test. Hence, this study will allow us to determine if an MRA alone, or added to an ARB at doses that control BP and hypokalemia, affects or not the ARR, thus allowing to establish if these agents can be administered or must be forbidden during the screening of PA.

Synthesis and Biological Characterization of a New Norbormide Derived Bodipy FL-Conjugated Fluorescent Probe for Cell Imaging.

Background: Norbormide (NRB) is a selective rat toxicant endowed with vasoconstrictor activity confined to the rat peripheral arteries. In a recent work we used a fluorescent derivative of NRB (NRB-AF12), obtained by coupling the NBD fluorophore to the parent molecule via a linker, in order to gain information about the possible site of action of the unlabeled compound. We found that NRB-AF12 labeled intracellular organelles in both NRB-sensitive and -insensitive cells and we accordingly proposed its use as a scaffold for the development of a new class of fluorescent probes. In this study, we examined the fluorescent properties of a BODIPY FL-conjugated NRB probe (MC009) developed: (A) to verify if NRB distribution could be influenced by the attached fluorophore; (B) to improve the fluorescent performance of NRB-AF12. Methods: MC009 characteristics were investigated by confocal fluorescence microscopy, in freshly isolated rat caudal artery myocytes (FIRCAM) and in LX2 cells, representative of NRB-sensitive and insensitive cells, respectively. Main results: In both FIRCAM and LX2 cells MC009 stained endoplasmic reticulum, mitochondria, Golgi apparatus and lipid droplets, revealing the same intracellular distribution as NRB-AF12, and, at the same time, had both improved photostability and gave a more intense fluorescent signal at lower concentrations than was possible with NRB-AF12, which resulted in a better and finer visualization of intracellular structures. Furthermore, MC009 was effective in cellular labeling in both living and fixed cells. At the concentration used to stain the cells, MC009 did not show any cytotoxic effect and did not affect the regular progression of cell cycle and division. Conclusions: This study demonstrates that the distribution of fluorescently labeled NRB is not affected by the type of fluorophore attached to the parent compound, supporting the idea that the localization of the fluorescent derivatives may reasonably reflect that of the parent compound. In addition, we observed a marked improvement in the fluorescent properties of BODIPY FL-conjugated NRB (MC009) over its NBD-derived counterpart (NRB-AF12), confirming NRB as a scaffold for the development of new, high performance, non-toxic fluorescent probes for the labeling of intracellular structures in both living and fixed cells.