BACKGROUND:Aldosterone induces cardiac fibrotic remodeling and arrhythmogenic alterations. The lack of suitable preclinical models has hampered an in-depth investigation of the molecular mechanisms involved in aldosterone-induced cardiac damage. Our aim was to evaluate the effects of aldosterone on 3D human microtissue (hMT) cardiac organoids. METHODS:hMT were generated by coculturing human cardiac fibroblasts, aortic endothelial cells and induced pluripotent stem cell-derived cardiomyocytes. hMT were treated with aldosterone, the mineralocorticoid receptor antagonist eplerenone, and serum from patients with primary aldosteronism or matched subjects with essential hypertension. Immunofluorescence, histology, and Western blot analyses were used to assess fibrosis; a multielectrode array was used to record extracellular field potentials of spontaneously beating human cardiomyocytes. RESULTS:Levels of profibrotic markers increased after incubation with serum from primary aldosteronism patients, compared with untreated organoids and hMT incubated with essential hypertension patient-derived serum. Aldosterone treatment reproduced the same profibrotic effect in a dose-dependent manner, and coadministration of eplerenone blunted these effects. Aldosterone treatment increased corrected field potential duration (an estimate of the QT interval) and downregulated the expression levels of KCNQ1 and ATP2A2, responsible for the slow delayed rectifier potassium current and for calcium handling in the sarcoplasmic reticulum. Eplerenone cotreatment reverted these electrical alterations. CONCLUSIONS:3D hMT organoids offer a relevant in vitro model to study aldosterone-mediated cardiac effects. Aldosterone directly induces fibrosis and prolongation of the QT interval in this model, which may partially explain the increase of cardiovascular risk in patients with primary aldosteronism and underscores the benefit of mineralocorticoid receptor antagonist therapy.

Background High sodium intake is associated with arterial hypertension and cardiovascular disease, through mechanisms that go beyond hemodynamic changes, including endothelial dysfunction, oxidative stress, and induction of a proinflammatory milieu. The aim of this study was to assess the role of dietary sodium modulation on renal pathophysiology through evaluation of small RNA cargos on urinary extracellular vesicles. Methods Fourteen high‐risk normotensive subjects with normal kidney function were prospectively enrolled to undergo a low‐sodium diet followed by a high‐sodium diet (HSD). The urinary extracellular vesicles were isolated from a 24‐hour urine collection at the end of each diet phase and profiled by small RNA sequencing. Selected differentially expressed miRNAs were validated in human proximal tubular cell line (human kidney 2 cells) to assess miRNA‐mRNA target interactions. Results We identified 111 small RNA species, of which 30 were significantly different between the low‐sodium diet and HSD. Bioinformatic network analysis showed that pathways related to the innate and adaptive immune system, interleukin and interferon signaling were enriched in the HSD, whereas pathways related to PPARα (peroxisome proliferator‐activated receptor α) regulation were enriched in the low‐sodium diet. In human kidney 2 cells, the inhibition of miR‐320b, downregulated in the HSD, increased ICAM‐1 (intercellular adhesion molecule 1), with renal proinflammatory effects. The inhibition of miR‐10b‐5p, downregulated in the low‐sodium diet, increased PPARα, which has an antifibrotic and anti‐inflammatory role in the kidney. Conclusions Small RNA characterization from extracellular vesicles revealed that an HSD is associated with proinflammatory changes, potentially contributing to sodium‐induced low‐grade renal inflammation.

Reliable predictive biomarkers to reduce unnecessary coronary angiograms (CAGs) in non-ST-segment elevation myocardial infarction (NSTEMI) and unstable angina (UA) patients displaying high-risk features are still lacking. Here, we show that profiling patient-derived circulating extracellular vesicles (EVs) can not only improve their risk stratification but also reduce unnecessary CAGs. Analysis of EVs and their miR cargo revealed that CD62p+EVs enriched in miR-130a-3p correlated with the absence of non-critical coronary artery disease (CAD). Proteomic analysis identified nine proteins differentially enriched in patients with or without critical-CAD (NO CAD), irrespective of their diagnosis. Multivariate analysis identified miR-130a-3p (odds ratio [OR]:0.35 [0.19-0.67]), phospholipid transfer protein (OR 0.96 [0.94-0.98]), and subunit beta of mitochondrial trifunctional enzyme (OR:0.96 [0.94-0.98]) as predictors of NO CAD. Furthermore, EV-miR-130a-3p enrichment predicted the absence of multivessel disease (OR:0.46 [0.23-0.90]). These findings establish EV profiling as a valuable tool for stratifying and optimizing the clinical management of patients with acute coronary syndrome.

Obstructive sleep apnea (OSA) is a common disorder in the general population and individuals with hypertension. We reviewed the literature on the prevalence of OSA in hypertension and hypertension subgroups. The current literature shows a high prevalence of OSA in patients with nocturnal and resistant hypertension, up to more than 90% in patients with refractory hypertension. The prevalence of OSA in patients with primary aldosteronism is greater than 45%. We also conducted an Italian national survey to assess the diagnostic approach to OSA in centers associated with European and Italian Societies of Hypertension. The median rate of OSA diagnosis was 10 patients/year, with a higher rate in Excellence Centers. The most common criterion for OSA screening was the combination of hypertension, snoring, and daytime somnolence (90%), followed by hypertension and a nondipping profile (55%). Resistant hypertension was considered a criterion by only 23% of the specialists.

BACKGROUND:Sodium is involved in osmoregulation, fluid balance, and immune system function. High sodium (HS) consumption is linked to endothelial dysfunction, hypertension, and mortality. Extracellular vesicles (EVs), nano-sized particles reflecting cellular status, could link dietary sodium to vascular and immune changes. This study aimed to characterize circulating EVs and assess their functional role on endothelial cells after dietary sodium modulation. METHODS:Fifty subjects underwent 2 dietary sodium interventions, each lasting 5 to 7 days: sodium-restriction (10-40 mmol/d, 0.23-0.92 g/d) followed by sodium-loading (intake above 180 mmol/d, 4.14 g/d). Serum EVs were isolated after each phase and analyzed using a flow cytometry bead-based platform. Bioinformatics identified intracellular targets of EV surface antigens upregulated after the HS diet. Human microvascular endothelial cells were used to assess EV effects in vitro. RESULTS:In matched comparison, after HS-diet, EVs exhibited increased levels of CD14-CD25-CD40 (immune system markers), CD29-CD42-CD62P (coagulation and platelet activation markers), and CD31 (endothelial marker). Among targets identified by signaling network analysis, the incubation of human microvascular endothelial cells with patient-derived EVs after HS-diet resulted in decreased expression of AKT1, increased expression of MAPK3, and levels of active RhoA. It also resulted in increased expression of ICAM1, number of ICAM-1 (Intercellular adhesion molecule-1) positive human microvascular endothelial cells, and nitric oxide levels. CONCLUSIONS:Dietary sodium modulates the expression of EV surface antigens associated with vascular inflammation, platelet activation, and endothelial function. Circulating EVs may mediate the effects of HS condition by interacting with endothelial cells, through multiple pathways, including RhoA activation and increased ICAM-1 expression.

In calcific aortic valve disease (CAVD), mechanosensitive valvular cells respond to fibrosis- and calcification-induced tissue stiffening, further driving pathophysiology. No pharmacotherapeutics are available to treat CAVD because of the paucity of (i) appropriate experimental models that recapitulate this complex environment and (ii) benchmarking novel engineered aortic valve (AV)–model performance. We established a biomaterial-based CAVD model mimicking the biomechanics of the human AV disease-prone fibrosa layer, three-dimensional (3D)–bioprinted into 96-well arrays. Liquid chromatography–tandem mass spectrometry analyses probed the cellular proteome and vesiculome to compare the 3D-bioprinted model versus traditional 2D monoculture, against human CAVD tissue. The 3D-bioprinted model highly recapitulated the CAVD cellular proteome (94% versus 70% of 2D proteins). Integration of cellular and vesicular datasets identified known and unknown proteins ubiquitous to AV calcification. This study explores how 2D versus 3D-bioengineered systems recapitulate unique aspects of human disease, positions multiomics as a technique for the evaluation of high throughput–based bioengineered model systems, and potentiates future drug discovery.

BACKGROUND:About 10% of patients with arterial hypertension have a positive screening test for primary aldosteronism (PA) and 50% to 70% of them have a negative confirmatory test: the appropriate follow-up of these patients is currently unknown. We investigated the incidence of PA in patients with previous negative confirmatory testing, after at least a 2-year follow-up.METHODS:One hundred eighty-four patients with a previously elevated aldosterone-to-renin ratio followed by a negative confirmatory test were recruited in 2 hypertension centers (Torino and Munich). We repeated the screening test for PA and, if positive, the confirmatory test (seated saline infusion test or captopril challenge test). Primary end point of the study was the incidence of newly diagnosed overt PA, as defined by a positive confirmatory test.RESULTS:After a mean follow-up of 5 years, 20% of patients developed overt PA. When subtype diagnosis was offered systematically, one-third of patients displayed unilateral PA. Patients who developed PA showed worsening of blood pressure control and a higher rate of cardiac organ damage, despite similar implementation of antihypertensive therapy, compared with patients without PA. A mild progression of autonomous aldosterone secretion was evident even in patients without confirmed PA but with relatively stable control of blood pressure levels over time.CONCLUSIONS:About one-fifth of patients with a negative confirmatory test develop overt PA over time. A clinical follow-up of patients with a negative confirmatory test is advisable, along with the repetition of PA investigation, primarily in patients with worsening of blood pressure control.

BACKGROUND: Fewer than 50% of patients who develop aortic valve calcification have concomitant atherosclerosis, implying differential pathogenesis. Although circulating extracellular vesicles (EVs) act as biomarkers of cardiovascular diseases, tissue-entrapped EVs are associated with early mineralization, but their cargoes, functions, and contributions to disease remain unknown. METHODS: Disease stage–specific proteomics was performed on human carotid endarterectomy specimens (n=16) and stenotic aortic valves (n=18). Tissue EVs were isolated from human carotid arteries (normal, n=6; diseased, n=4) and aortic valves (normal, n=6; diseased, n=4) by enzymatic digestion, (ultra)centrifugation, and a 15-fraction density gradient validated by proteomics, CD63-immunogold electron microscopy, and nanoparticle tracking analysis. Vesiculomics, comprising vesicular proteomics and small RNA-sequencing, was conducted on tissue EVs. TargetScan identified microRNA targets. Pathway network analyses prioritized genes for validation in primary human carotid artery smooth muscle cells and aortic valvular interstitial cells. RESULTS: Disease progression drove significant convergence ( P <0.0001) of carotid artery plaque and calcified aortic valve proteomes (2318 proteins). Each tissue also retained a unique subset of differentially enriched proteins (381 in plaques; 226 in valves; q<0.05). Vesicular gene ontology terms increased 2.9-fold ( P <0.0001) among proteins modulated by disease in both tissues. Proteomics identified 22 EV markers in tissue digest fractions. Networks of proteins and microRNA targets changed by disease progression in both artery and valve EVs revealed shared involvement in intracellular signaling and cell cycle regulation. Vesiculomics identified 773 proteins and 80 microRNAs differentially enriched by disease exclusively in artery or valve EVs (q<0.05); multiomics integration found tissue-specific EV cargoes associated with procalcific Notch and Wnt signaling in carotid arteries and aortic valves, respectively. Knockdown of tissue-specific EV-derived molecules FGFR2 , PPP2CA , and ADAM17 in human carotid artery smooth muscle cells and WNT5A , APP , and APC in human aortic valvular interstitial cells significantly modulated calcification. CONCLUSIONS: The first comparative proteomics study of human carotid artery plaques and calcified aortic valves identifies unique drivers of atherosclerosis versus aortic valve stenosis and implicates EVs in advanced cardiovascular calcification. We delineate a vesiculomics strategy to isolate, purify, and study protein and RNA cargoes from EVs entrapped in fibrocalcific tissues. Integration of vesicular proteomics and transcriptomics by network approaches revealed novel roles for tissue EVs in modulating cardiovascular disease.

CONTEXT:Adrenal venous sampling (AVS) is the gold standard procedure for subtype diagnosis in patients with primary aldosteronism (PA). Cortisol is usually adopted for the normalization of aldosterone levels in peripheral and adrenal samples. However, asymmetrical cortisol secretion can potentially affect the lateralization index, leading to subtype misdiagnosis.OBJECTIVE:We aimed to assess the prevalence of asymmetrical cortisol secretion in patients undergoing AVS and whether variations in adrenal vein cortisol might influence AVS interpretations. We then evaluated the use of metanephrines for the normalization of aldosterone levels for lateralization index.METHODS:We retrospectively included 101 patients with PA who underwent AVS: 49 patients underwent unstimulated AVS, while 52 patients underwent both unstimulated and cosyntropin-stimulated AVS. Eighty-eight patients had bilateral successful AVS according to metanephrine ratio. We assessed the prevalence of asymmetrical cortisol secretion through the cortisol to metanephrine (C/M) lateralization index (LI). We then evaluated whether the use of aldosterone to metanephrine (A/M) LI can improve the diagnostic accuracy of AVS compared with aldosterone to cortisol (A/C) LI.RESULTS:Asymmetrical cortisol secretion is present in 18% of patients with PA. Diagnosis with A/M LI and A/C LI is discordant in 14% of patients: 9% had a diagnosis of unilateral PA with A/M LI instead of bilateral PA with A/C LI and 5% had a diagnosis of bilateral PA with A/M LI instead of unilateral PA.CONCLUSION:The assessment of metanephrine levels in AVS is useful for the determination of selectivity and lateralization, allowing an accurate diagnosis, especially in patients with asymmetrical cortisol secretion.

Circulating tumour-derived extracellular vesicles are supposed to contribute to the spreading of distant metastasis. In this study, we investigated the impact of circulating extracellular vesicles derived from tumour-endothelial cells (TEVs) in the expansion of the metastatic bulk. We focus on the role of immune cells in controlling this process using the 4T1 triple negative breast cancer (TNBC) syngeneic model. 4T1 cells were intravenously injected and exposed to circulating TEVs from day 7. The lung, spleen, and bone marrow (BM) were recovered and analysed. We demonstrated that circulating TEVs boost lung metastasis and angiogenesis. FACS and immunohistochemically analyses revealed a significant enrichment of Ly6G+/F4/80+/CD11b+ cells and Ly6G+/F4/80-/CD11b+ in the lung and in the spleen, while Ly6G+/F4/80-/CD11b+ in the BM, indicating the occurrence of a systemic and local immune suppression. TEV immune suppressive properties were further supported by the increased expression of PD-L1, PD-1, and iNOS in the tumour mass. In addition, in vitro experiments demonstrated an increase of CD11+ cells, PD-L1+ myeloid and cancer cells, upregulation of LAG3, CTLA4 and PD-1 in T-cells, release of ROS and NOS, and impaired T-cell-mediated cytotoxic effect in co-culture of TEVs-preconditioned PBMCs and cancer cells. Granulocyte-colony stimulating factor (G-CSF) level was increased in vivo, and was involved in reshaping the immune response. Mechanistically, we also found that mTOR enriched TEVs support G-CSF release and trigger the phosphorylation of the S6 (Ser235/236) mTOR downstream target. Overall, we provided evidence that circulating TEVs enriched in mTOR supported G-CSF release thereby granting tumour immune suppression and metastasis outgrowth.

Objective: Current guidelines recommend screening for PA in patients with hypertension on the basis of individual factors, that considered together are present in more than 50% of patients. Recently, some experts proposed to further expand the screening for PA to all patients with hypertension, potentially increasing the burden and the costs on the health care system. Design and method: We designed a study to build and validate prediction models based on supervised learning and a conventional scoring system to define the risk of PA in arterial hypertension and tailor the diagnostic workup to the individual risk of each patient. We developed a clinical score and supervised machine learning algorithms in a retrospective internal cohort of 4059 patients with hypertension, and an external cohort of 584 patients with hypertension. Primary aldosteronism was confirmed by confirmatory tests, in agreement with major international recommendations, and subtype diagnosis was achieved by computed tomography and adrenal vein sampling. Results: On the basis of 6 widely available parameters (male sex, systolic blood pressure, antihypertensive treatment, body mass index, lowest potassium, and organ damage) we developed a numerical scoring system (SToP-PA score) and 308 machine learning based models, selecting the one with the highest predicting performances. At internal validation we obtained high predictive performance with SToP-PA score (ptimised sensitivity of 90.7% for PA and 92.3% for unilateral PA) and even higher with machine learning based model (ptimised sensitivity of 96.6% for PA, and 100.0% for unilateral PA). The application of these models allowed the identification of a subgroup of patients with very low probability of having PA (0.6% with both models) and null probability of having unilateral PA. Finally, we validated our models within an independent external cohort, with performance not significantly different from internal validation. Conclusions: The SToP-PA score and the machine learning model accurately predict the individual pretest probability of PA in patients with hypertension, avoiding the screening for PA in up to 32.7% of patients using a machine learning algorithm, without omitting patients with unilateral UPA.

Besides the physiological regulation of water, sodium, and potassium homeostasis, aldosterone modulates several physiological and pathological processes in the cardiovascular system. At the vascular level, aldosterone excess stimulates endothelial dysfunction and infiltration of inflammatory cells, enhances the development of the atherosclerotic plaque, and favors plaque instability, arterial stiffness, and calcification. At the cardiac level, aldosterone increases cardiac inflammation, fibrosis, and myocardial hypertrophy. As a clinical consequence, high aldosterone levels are associated with enhanced risk of cardiovascular events and mortality, especially when aldosterone secretion is inappropriate for renin levels and sodium intake, as in primary aldosteronism. Several clinical trials showed that mineralocorticoid receptor antagonists reduce cardiovascular mortality in patients with heart failure and reduced ejection fraction, but inconclusive results were reported for other cardiovascular conditions, such as heart failure with preserved ejection fraction, myocardial infarction, and atrial fibrillation. In patients with primary aldosteronism, adrenalectomy or treatment with mineralocorticoid receptor antagonists significantly mitigate adverse aldosterone effects, reducing the risk of cardiovascular events, mortality, and incident atrial fibrillation. In this review, we will summarize the major preclinical and clinical studies investigating the cardiovascular damage mediated by aldosterone and the protective effect of mineralocorticoid receptor antagonists for the reduction of cardiovascular risk in patients with cardiovascular diseases and primary aldosteronism.

Extracellular vesicles (EVs) are nanosized membrane-bound structures released by cells that are able to transfer nucleic acids, protein cargos, and metabolites to specific recipient cells, allowing cell-to-cell communications in an endocrine and paracrine manner. Endothelial, leukocyte, and platelet-derived EVs have emerged both as biomarkers and key effectors in the development and progression of different stages of vascular damage, from earliest alteration of endothelial function, to advanced atherosclerotic lesions and cardiovascular calcification. Under pathological conditions, circulating EVs promote endothelial dysfunction by impairing vasorelaxation and instigate vascular inflammation by increasing levels of adhesion molecules, reactive oxygen species, and proinflammatory cytokines. Platelets, endothelial cells, macrophages, and foam cells secrete EVs that regulate macrophage polarization and contribute to atherosclerotic plaque progression. Finally, under pathological stimuli, smooth muscle cells and macrophages secrete EVs that aggregate between collagen fibers and serve as nucleation sites for ectopic mineralization in the vessel wall, leading to formation of micro- and macrocalcification. In this review, we summarize the emerging evidence of the pathological role of EVs in vascular damage, highlighting the major findings from the most recent studies and discussing future perspectives in this research field.

Reduced or absent compliance to anti-hypertensive treatment is a major obstacle to the achievement of blood pressure target in patients with arterial hypertension. Current available methods for therapeutic adherence assessment display low accuracy, limited applicability in clinical practice and/or high costs. We designed a prospective study to evaluate the accuracy of serial measurement of ARR to assess the therapeutic compliance to RAAS inhibitors. We prospectively enrolled 80 subjects: 40 patients with arterial hypertension and 40 normotensive controls. The ARR was evaluated at baseline and 2 and 8 week after initiation of a RAAS inhibitor in patients with hypertension, and at baseline and 2 weeks for the control group. Adherence to the prescribed therapy was confirmed by therapeutic drug monitoring. We observed a significant increase of renin levels and reduction of aldosterone levels after RAAS inhibitors initiation, with consequent reduction of ARR. Delta ARR (ΔARR), defined as relative change in ARR before and after treatment initiation, provided high accuracy for determination of therapeutic compliance, with an AUC of 0.900 at 2 weeks and 0.886 at 8 weeks. A cut-off of −48% of ΔARR provided 90% sensitivity and 75% specificity, at 2 and 8 weeks. In conclusion, the measurement of ΔARR is a powerful test, cheap and widely available to accurately identify the non-adherence to RAAS inhibitors treatment. Herein we propose the implementation of ΔARR in clinical practice through a multi-step flow-chart for the management of patients with uncontrolled blood pressure, with identification of those suspected of non-adherence, reserving therapeutic drug monitoring for non-adherence confirmation.

Primary aldosteronism (PA) is the cause of arterial hypertension in 4% to 6% of patients, and 30% of patients with PA are affected by unilateral and surgically curable forms. Current guidelines recommend screening for PA ≈50% of patients with hypertension on the basis of individual factors, while some experts suggest screening all patients with hypertension. To define the risk of PA and tailor the diagnostic workup to the individual risk of each patient, we developed a conventional scoring system and supervised machine learning algorithms using a retrospective cohort of 4059 patients with hypertension. On the basis of 6 widely available parameters, we developed a numerical score and 308 machine learning-based models, selecting the one with the highest diagnostic performance. After validation, we obtained high predictive performance with our score (optimized sensitivity of 90.7% for PA and 92.3% for unilateral PA [UPA]). The machine learning-based model provided the highest performance, with an area under the curve of 0.834 for PA and 0.905 for diagnosis of UPA, with optimized sensitivity of 96.6% for PA, and 100.0% for UPA, at validation. The application of the predicting tools allowed the identification of a subgroup of patients with very low risk of PA (0.6% for both models) and null probability of having UPA. In conclusion, this score and the machine learning algorithm can accurately predict the individual pretest probability of PA in patients with hypertension and circumvent screening in up to 32.7% of patients using a machine learning-based model, without omitting patients with surgically curable UPA.