Identification of risk loci for primary aldosteronism in genome-wide association studies.

Primary aldosteronism affects up to 10% of hypertensive patients and is responsible for treatment resistance and increased cardiovascular risk. Here we perform a genome-wide association study in a discovery cohort of 562 cases and 950 controls and identify three main loci on chromosomes 1, 13 and X; associations on chromosome 1 and 13 are replicated in a second cohort and confirmed by a meta-analysis involving 1162 cases and 3296 controls. The association on chromosome 13 is specific to men and stronger in bilateral adrenal hyperplasia than aldosterone producing adenoma. Candidate genes located within the two loci, CASZ1 and RXFP2, are expressed in human and mouse adrenals in different cell clusters. Their overexpression in adrenocortical cells suppresses mineralocorticoid output under basal and stimulated conditions, without affecting cortisol biosynthesis. Our study identifies the first risk loci for primary aldosteronism and highlights new mechanisms for the development of aldosterone excess.

Colocalization of Wnt/ß-Catenin and ACTH Signaling Pathways and Paracrine Regulation in Aldosterone-producing Adenoma.

CONTEXT: Aldosterone-producing adenomas (APAs) are a common cause of primary aldosteronism (PA). Despite the discovery of somatic mutations in APA and the characterization of multiple factors regulating adrenal differentiation and function, the sequence of events leading to APA formation remains to be determined. OBJECTIVE: We investigated the role of Wnt/ß-catenin and adrenocorticotropin signaling, as well as elements of paracrine regulation of aldosterone biosynthesis in adrenals with APA and their relationship to intratumoral heterogeneity and mutational status. METHODS: We analyzed the expression of aldosterone-synthase (CYP11B2), CYP17A1, ß-catenin, melanocortin type 2 receptor (MC2R), phosphorlyated cAMP response element-binding protein (pCREB), tryptase, S100, CD34 by multiplex immunofluorescence, and immunohistochemistry-guided reverse transcription-quantitative polymerase chain reaction. Eleven adrenals with APA and 1 with micronodular hyperplasia from patients with PA were analyzed. Main outcome measures included localization of CYP11B2, CYP17A1, ß-catenin, MC2R, pCREB, tryptase, S100, CD34 in APA and aldosterone-producing cell clusters (APCCs). RESULTS: Immunofluorescence revealed abundant mast cells and a dense vascular network in APA, independent of mutational status. Within APA, mast cells were localized in areas expressing CYP11B2 and were rarely colocalized with nerve fibers, suggesting that their degranulation is not controlled by innervation. In these same areas, ß-catenin was activated, suggesting a zona glomerulosa cell identity. In heterogeneous APA with KCNJ5 mutations, MC2R and vascular endothelial growth factor A expression was higher in areas expressing CYP11B2. A similar pattern was observed in APCC, with high expression of CYP11B2, activated ß-catenin, and numerous mast cells. CONCLUSION: Our results suggest that aldosterone-producing structures in adrenals with APA share common molecular characteristics and cellular environment, despite different mutation status, suggesting common developmental mechanisms.

Somatic mutations in adrenals from patients with primary aldosteronism not cured after adrenalectomy suggest common pathogenic mechanisms between unilateral and bilateral disease.

OBJECTIVE: Primary aldosteronism (PA) is the most common form of secondary and curable hypertension. Different germline and somatic mutations are found in aldosterone-producing adenoma (APA) and familial forms of the disease, while the causes of bilateral adrenal hyperplasia (BAH) remain largely unknown. Adrenalectomy is the recommended treatment for patients with APA; however, 6% of patients are not cured and show persistent PA after surgery suggesting BAH. The objective of this study was to analyze clinical data of patients with APA without biochemical success after adrenalectomy as well as the histological and genetic characteristics of their adrenal glands. DESIGN AND METHODS: Clinical data of 12 patients with partial and absent biochemical cure were compared to those from 39 PA patients with hormonal cure after surgery. Histological, morphological, and genetic characterization of the adrenals was carried out by CYP11B2 and CYP11B1 immunostaining and by CYP11B2-guided NGS. RESULTS: Patients with absent hormonal cure displayed a longer duration of arterial hypertension and lower lateralization index of aldosterone production. In ten patients, APAs expressing CYP11B2 were identified. No difference in histological and morphological characteristics was observed between patients with or without a hormonal cure. Somatic mutations in APA driver genes were identified in all CYP11B2 positive APAs; CACNA1D mutations were the most frequent genetic abnormality. CONCLUSIONS: Patients with partial and absent biochemical cure were diagnosed later and exhibited a lower lateralization index of aldosterone production, suggesting asymmetric aldosterone production in the context of BAH. Somatic mutations in adrenal glands from those patients indicate common mechanisms underlying BAH and APA.

Biocatalytic production of Ɛ-caprolactone using Geotrichum candidum cells immobilized on functionalized silica.

Immobilization of the Geotrichum candidum (CCT 1205) cell with functionalized silica creates promising biocatalysts for production of ɛ-caprolactone. The results obtained by immobilization of the whole cell on SiO2-NH2 and SiO2-SH supports indicate that the presence of reactive functional groups on the support may promote effective chemical bonds with the cell walls resulting the decreased dehydrogenases enzyme activity (5% yield in less than 2h) and consequently, increased Baeyer-Villiger monooxygenases enzyme activity with redacting of 25% of time reaction when is used SiO2-NH2 as support and 50% through use of SiO2-SH as support relative to free cells when cyclohexanone is used as a substrate. The catalysts SiO2-NH2-Geotrichum candidum and SiO2-SH-Geotrichum candidum were recycling and reused in the ɛ-caprolactone synthesis from cyclohexanone, and the biocatalysts promoted a quantitative conversion up to the eighth reaction cycle. KEY POINTS: • Immobilized microorganism is more efficient than free cell in the caprolactone synthesis. • The reaction times for amino and thiol groups in support were 3 h and 2 h, respectively. • These catalysts showed higher ɛ-caprolactone conversion at higher concentrations.

Concurrent imaging of vascularization and metabolism in a mouse model of paraganglioma under anti-angiogenic treatment.

Rationale: Deregulation of metabolism and induction of vascularization are major hallmarks of cancer. Using a new multimodal preclinical imaging instrument, we explored a sequence of events leading to sunitinib-induced resistance in a murine model of paraganglioma (PGL) invalidated for the expression of succinate dehydrogenase subunit B (Sdhb-/-). Methods: Two groups of Sdhb-/- tumors bearing mice were treated with sunitinib (6 weeks) or vehicle (3 weeks). Concurrent Positron Emission Tomography (PET) with 2' -deoxy-2'-[18F]fluoro-D-glucose (FDG), Computed Tomography (CT) and Ultrafast Ultrasound Imaging (UUI) imaging sessions were performed once a week and ex vivo samples were analyzed by western blots and histology. Results: PET-CT-UUI enabled to detect a rapid growth of Sdhb-/- tumors with increased glycolysis and vascular development. Sunitinib treatment prevented tumor growth, vessel development and reduced FDG uptake at week 1 and 2 (W1-2). Thereafter, imaging revealed tumor escape from sunitinib treatment: FDG uptake in tumors increased at W3, followed by tumor growth and vessel development at W4-5. Perfused vessels were preferentially distributed in the hypermetabolic regions of the tumors and the perfused volume increased during escape from sunitinib treatment. Finally, initial changes in total lesion glycolysis and maximum vessel length at W1 were predictive of resistance to sunitinib. Conclusion: These results demonstrate an adaptive resistance of Sdhb-/- tumors to six weeks of sunitinib treatment. Early metabolic changes and delayed vessel architecture changes were detectable and predictable in vivo early during anti-angiogenic treatment. Simultaneous metabolic, anatomical and functional imaging can monitor precisely the effects of anti-angiogenic treatment of tumors.

Genetic, Cellular, and Molecular Heterogeneity in Adrenals With Aldosterone-Producing Adenoma.

Aldosterone-producing adenoma (APA) cause primary aldosteronism-the most frequent form of secondary hypertension. Somatic mutations in genes coding for ion channels and ATPases are found in APA and in aldosterone-producing cell clusters. We investigated the genetic, cellular, and molecular heterogeneity of different aldosterone-producing structures in adrenals with APA, to get insight into the mechanisms driving their development and to investigate their clinical and biochemical correlates. Genetic analysis of APA, aldosterone-producing cell clusters, and secondary nodules was performed in adrenal tissues from 49 patients by next-generation sequencing following CYP11B2 immunohistochemistry. Results were correlated with clinical and biochemical characteristics of patients, steroid profiles, and histological features of the tumor and adjacent adrenal cortex. Somatic mutations were identified in 93.75% of APAs. Adenoma carrying KCNJ5 mutations had more clear cells and cells expressing CYP11B1, and fewer cells expressing CYP11B2 or activated ß-catenin, compared with other mutational groups. 18-hydroxycortisol and 18-oxocortisol were higher in patients carrying KCNJ5 mutations and correlated with histological features of adenoma; however, mutational status could not be predicted using steroid profiling. Heterogeneous CYP11B2 expression in KCNJ5-mutated adenoma was not associated with genetic heterogeneity. Different mutations were identified in secondary nodules expressing aldosterone synthase and in independent aldosterone-producing cell clusters from adrenals with adenoma; known KCNJ5 mutations were identified in 5 aldosterone-producing cell clusters. Genetic heterogeneity in different aldosterone-producing structures in the same adrenal suggests complex mechanisms underlying APA development.

Molecular mechanisms in primary aldosteronism

Primary aldosteronism (PA) is the most common form and an under-diagnosed cause of secondary arterial hypertension, accounting for up to 10% of hypertensive cases and associated to increased cardiovascular risk. PA is caused by autonomous overproduction of aldosterone by the adrenal cortex. It is mainly caused by a unilateral aldosterone-producing adenoma (APA) or bilateral adrenal hyperplasia. Excess aldosterone leads to arterial hypertension with suppressed renin, frequently associated to hypokalemia. Mutations in genes coding for ion channels and ATPases have been identified in APA, explaining the pathophysiology of increased aldosterone production. Different inherited genetic abnormalities led to the distinction of four forms of familial hyperaldosteronism (type I to IV) and other genetic defects very likely remain to be identified. Somatic mutations are identified in APA, but also in aldosterone-producing cell clusters (APCCs) in normal adrenals, in image-negative unilateral hyperplasia, in transitional lesions and in APCC from adrenals with bilateral adrenal hyperplasia (BAH). Whether these structures are precursors of APA or whether somatic mutations occur in a proliferative adrenal cortex, is still a matter of debate. This review will summarize our knowledge on the molecular mechanisms responsible for PA and the recent discovery of new genes related to early-onset and familial forms of the disease. We will also address new issues concerning genomic and proteomic changes in adrenals with APA and discuss adrenal pathophysiology in relation to aldosterone-producing structures in the adrenal cortex.