United States

Programmed death-ligand 1 (PD‐L1) is a strong immunosuppressor that inhibits T-cell activation by binding to its receptor (PD-1) on T-cells. Blocking the PD-L1/PD-1 interaction by monoclonal antibodies (mAbs) is an effective therapy for several cancers via activation of T-cells. Destructive thyroiditis and hypothyroidism often develop during anti-PD-L1 (αPD-L1) therapy but the pathogenic mechanisms are not known. We evaluated in vivo, the role of PD-L1 and its interactions with inflammatory triggers in the development of autoimmune thyroiditis (AIT). NOD.H-2h4 mice were treated with either αPD-L1 or isotype control (CTR) for two weeks and AIT evaluated after six weeks. αPD-L1 treated mice showed significantly higher Abs (p = 6x10e-4) and T-cell responses (p = 0.02) to thyroglobulin (Tg) compared with CTR mice. Serum fT4 levels were lower (p = 0.01) and TSH higher (p = 0.05) in αPD-L1 treated vs. CTR mice. Seven of the eight αPD-L1 treated mice (and none of the CTR mice) developed thyroid lymphocytic infiltration characterized by the presence of the CD4+, CD8+, FoxP3+ T- cells and B220+ B- cells. To gain insight into the pathways regulated by PD-L1 in the thyroid we assessed gene expression in the thyroid of αPD-L1 treated mice using RNA-seq. PD-L1 blockade was associated with upregulation of pathways of lymphocyte activation (p = 1.3x10e-5), lymphocyte proliferation (p = 2x10e-5), chemotaxis (p = 3.8x10e-12), and inflammatory response (p = 7.9x10e-7). Specifically, blocking PD-L1 was associated with upregulation of cytokine/chemokine genes that have a critical role in Th1 differentiation: IFNγ, IL-12, IL-15, CCL1 to CCL5, CD300, CXCL10, CXCL11. Ingenuity Pathway Analysis identified IFNγ and TNF as the top regulators of these activated gene networks. These results indicate that blockade of PD-L1 in thyroid promoted the Th1 mediator, IFNγ, inducing activation and proliferation of thyroidal CD4+ and CD8+ T cells, triggering thyroid tissue damage and AIT. Since PD-L1 mediates immune responses to inflammatory stressors (e.g. viral infections), we evaluated PD-L1 interaction with a known inflammatory trigger of AIT, interferon alpha (IFNα). Treatment with αPD-L1 + IFNα accelerated AIT in NOD.H-2h4 mice that developed high Tg Ab and T-cell responses and thyroidal lymphocytic infiltration only two weeks after treatment. Moreover, thyroiditis was more severe in mice receiving αPD-L1 + IFNα than αPD-L1 alone, manifesting by higher T-cell responses (p = 5x10e-3), and significantly more profound hypothyroidism. These results suggest that PDL-1 has a protective role in the thyroid against inflammatory stressors, and that its blockade exposes the thyroid to inflammatory damage. Therefore, in patients undergoing αPD-L1 therapy, inflammatory conditions (e.g. infections) can be a &quot;second hit&quot; that accelerates the onset of thyroiditis and augments thyroidal inflammation.

ENDO 2022

Dissecting Thyroidal Pathways Activated by PD-L1 Blockade Identifies New Mechanisms for Thyroiditis Triggered by Immune Checkpoint Inhibitors

**Program**
ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

This year’s education program features:
more than 100 in-person and virtual sessions with another 70+ in-person only sessions;
six plenary sessions including plenaries on Envisioning Future Therapies for Type 1 Diabetes and Addressing Challenges in Fertility and Reproduction;
over 20 Meet the Professor sessions presented both in-person and live-streamed virtually and another 20+ presented live in Atlanta;
four Basic Science Pathways, including Diabetes and Metabolism; Neuroendocrinology; Nuclear Receptors and Signaling; and Reproductive Endocrinology; and
more than 30 oral sessions and a robust virtual poster hall for accepted scientific abstracts.

**What Programming Is Included in My Virtual Meeting Registration?**
As you build your schedule during the conference, you can select the sessions that are tagged with 'Livestreamed', so you can view virtually. These sessions are being livestreamed, so they are available in real time to our virtual audience. In addition, our in-person and livestreamed sessions will be recorded, so you can access them on demand after 72 hours. We will have over 200 hours available on demand for Standard and Premium pass registrants.

Product theaters and the Communications and Career Center will also include hybrid content delivery.

**Plenaries**
Spanning the spectrum of science, clinical care, and social implications, an outstanding slate of world-renowned speakers will showcase the most cutting-edge advances on research and medicine.

**Symposia**
Our ENDO 2022 lineup of sessions offers a unique opportunity to dive into topics from nearly every major therapeutic area of endocrinology and hormone science. Listen, learn, and be inspired by world-class faculty who are at the forefront of scientific discovery and high-quality health care.

**Basic Science Pathways**

These special “meeting-within-a-meeting” tracks integrate focused discussions, poster sessions, and networking opportunities to provide you with the latest research in:
Diabetes and Metabolism
Neuroendocrinology
Nuclear Receptors and Signaling
Reproductive Endocrinology

**Meet the Professor & Meet the Scientist**
Discover insights from experts on treating a variety of endocrine conditions and emerging science in the field of endocrinology, and learn the latest in patient care and scientific research.

ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

Programmed death-ligand 1 (PD‐L1) is a strong immunosuppressor that inhibits T-cell activation by binding to its receptor (PD-1) on T-cells. Blocking the PD-L1/PD-1 interaction by monoclonal antibodies (mAbs) is an effective therapy for several cancers via activation of T-cells. Destructive thyroiditis and hypothyroidism often develop during anti-PD-L1 (αPD-L1) therapy but the pathogenic mechanisms are not known. We evaluated in vivo, the role of PD-L1 and its interactions with inflammatory triggers in the development of autoimmune thyroiditis (AIT). NOD.H-2h4 mice were treated with either αPD-L1 or isotype control (CTR) for two weeks and AIT evaluated after six weeks. αPD-L1 treated mice showed significantly higher Abs (p = 6x10e-4) and T-cell responses (p = 0.02) to thyroglobulin (Tg) compared with CTR mice. Serum fT4 levels were lower (p = 0.01) and TSH higher (p = 0.05) in αPD-L1 treated vs. CTR mice. Seven of the eight αPD-L1 treated mice (and none of the CTR mice) developed thyroid lymphocytic infiltration characterized by the presence of the CD4+, CD8+, FoxP3+ T- cells and B220+ B- cells. To gain insight into the pathways regulated by PD-L1 in the thyroid we assessed gene expression in the thyroid of αPD-L1 treated mice using RNA-seq. PD-L1 blockade was associated with upregulation of pathways of lymphocyte activation (p = 1.3x10e-5), lymphocyte proliferation (p = 2x10e-5), chemotaxis (p = 3.8x10e-12), and inflammatory response (p = 7.9x10e-7). Specifically, blocking PD-L1 was associated with upregulation of cytokine/chemokine genes that have a critical role in Th1 differentiation: IFNγ, IL-12, IL-15, CCL1 to CCL5, CD300, CXCL10, CXCL11. Ingenuity Pathway Analysis identified IFNγ and TNF as the top regulators of these activated gene networks. These results indicate that blockade of PD-L1 in thyroid promoted the Th1 mediator, IFNγ, inducing activation and proliferation of thyroidal CD4+ and CD8+ T cells, triggering thyroid tissue damage and AIT. Since PD-L1 mediates immune responses to inflammatory stressors (e.g. viral infections), we evaluated PD-L1 interaction with a known inflammatory trigger of AIT, interferon alpha (IFNα). Treatment with αPD-L1 + IFNα accelerated AIT in NOD.H-2h4 mice that developed high Tg Ab and T-cell responses and thyroidal lymphocytic infiltration only two weeks after treatment. Moreover, thyroiditis was more severe in mice receiving αPD-L1 + IFNα than αPD-L1 alone, manifesting by higher T-cell responses (p = 5x10e-3), and significantly more profound hypothyroidism. These results suggest that PDL-1 has a protective role in the thyroid against inflammatory stressors, and that its blockade exposes the thyroid to inflammatory damage. Therefore, in patients undergoing αPD-L1 therapy, inflammatory conditions (e.g. infections) can be a "second hit" that accelerates the onset of thyroiditis and augments thyroidal inflammation.

technical paper

Introduction: Low levels of triiodothyronine (T3) are common in patients with heart failure (HF). It has been proposed that the decreased levels of T3 contribute to metabolic and cardiac dysfunction in HF. We aimed to evaluate the effects of treatment with low dose and high dose of T3 in an animal model of HF with preserved ejection fraction (HFpEF). Materials and Methods: We evaluated four groups: ZSF1 Lean (Lean-Ctrl, n=8), ZSF1 obese (rat model of metabolic syndrome with HFpEF, HFpEF-Ctrl, n=13), ZSF1 obese supplemented with a replacement dose of T3 (initially 0.04mg/ml, increased to 0.06mg/ml after 4 weeks; HFpEF-T3high, n=8), and ZSF1 Obese supplemented with a low-dose of T3 (0.03mg/mL; HFpEF-T3low, n=8). T3 was supplemented in drinking water from week 13 to 24. The animals were submitted to anthropometric and metabolic evaluation, echocardiography, VO2max evaluation, hemodynamic evaluation, single cardiomyocyte evaluation, and myocardial tissue collection at 24 weeks. Results: HFpEF-Ctrl animals had lower serum and myocardial thyroid hormone levels than Lean-Ctrl. Treatment with low dose and high dose of T3 did not correct serum T3 levels, but increased myocardial T3 levels, with normalization of myocardial T3 levels in the HFpEF-T3high group. In comparison to HFpEF-Ctrl, body weight was significantly decreased in HFpEF-T3low and HFpEF-T3high groups (616±39g, 572±24g and 535±18g respectively, p<0.001). An improvement in glucose metabolism was observed only in HFpEF-T3high, with a decreased AUC of glucose during OGTT and insulin tolerance test. Both treated groups presented an improvement in diastolic function (with decreased isovolumetric relaxation time) and systolic function (with increased peak systolic velocity and decreased ejection time) comparing to HFpEF-Ctrl. These results were supported by enhanced Ca2+ transients during diastole and sarcomere relaxation. Comparing with HFpEF-Ctrl, animals treated with T3 had higher myocardial expression of the calcium transporter RYR2, of the transcriptional coactivator PGC-1α, and of the α-myosin heavy chain (MHC), with a lower expression of β-MHC. VO2max was improved with low-dose and high-dose T3 compared with HFpEF-Ctrl. Comparing with HFpEF-Ctrl, heart rate was increased with high-dose (277.9±30.2 vs 313.5±35.8, p=0.049) but not significantly with low-dose (277.9±30.2 vs 299.8±30.9 bpm, p=0.13). Mortality was increased in the HFpEF-T3high comparing with the other groups (no animals died during the protocol in the Lean-Ctrl, HFpEF-Ctrl and HFpEF-T3low groups; three animals had sudden death in the HFpEF-T3high group). Conclusion: Treatment with T3 improves metabolic function, myocardial calcium handling, diastolic and systolic function, and exercise capacity in this animal model of HFpEF. While the low dose was well tolerated and safe, the high dose was associated with increased heart rate and a higher risk of sudden death. Modulation of thyroid hormones may be a potential therapeutic target in HFpEF; however such approaches must take into account a narrow therapeutic window of T3 in HFpEF.

Treatment of Heart Failure with Preserved Ejection Fraction (HFpEF) with Low and High Dose of Triiodothyronine: an Animal Model of Metabolic Syndrome with HFpEF

Thyroid cancer affects ethnic groups at different rates and severity. Despite this, thyroid cancer health disparities are still an understudied area. Our laboratory has shown a differential expression of vitamin D binding protein (DBP) in Filipino Americans (FA) versus European Americans (EA) [1]. Higher DBP levels correlate to a better prognosis. A study has revealed a DBP-dependent vitamin D receptor (VDR) promoter activation. In this study, we investigated whether VDR and DBP are expressed differentially in different ethnic groups, such as African Americans (AA), Hispanic Americans (HA), EA, and FA. We were also able to determine the differential DBP polymorphisms, VDR variant expressions, and DBP-VDR interactions within different ethnicities. By PCR-Restriction Fragment Length Polymorphism (PCR-RFLP), we determined a higher frequency of DBP gene polymorphism in FA versus EA. Analyzing the Cancer Genome Atlas (TCGA) thyroid cancer datasets with the UALCAN assay, we found the differential expressions of DBP and VDR genes based on cancer stages, sample types, race, and histological subtypes. By immunohistochemistry, we detected strong nuclear VDR (nVDR) and very low membranous VDR (mVDR) expression that correlated with low DBP in FA thyroid cancer tissues. In contrast, there was a higher expression of both mVDR and DBP in HA versus the other ethnicities. Co-immunoprecipitation analysis revealed a stronger DBP interaction with mVDR in FA compared to other ethnicities. Our data suggest that low DBP correlates with low mVDR in FA, whereas high DBP correlates with high mVDR in the other ethnicities. In conclusion, the strong interaction of DBP with mVDR in FA may implicate the potential role of DBP-mVDR crosstalk in aggressive thyroid cancer. Future research goals will be aimed at determining pathways involved in BDP-mVDR crosstalk in thyroid cancer health disparities.

B. Mull, R. Davis, I. Munir, M.C. Perez, A.A. Simental, S. Khan, Differential expression of Vitamin D binding protein in thyroid cancer health disparities, Oncotarget 12(7) (2021) 596-607.

Differential Interactions of Vitamin D Binding Protein and Vitamin D Receptor in Different Ethnic Groups with Aggressive Thyroid Cancer

Thyroid hormones (THs) thyroxine (T4) and triiodothyronine (T3) are under homeostatic and circadian control by the hypothalamic-pituitary-thyroid axis while their free plasma concentrations are strongly buffered by three TH binding proteins (THBPs): thyroxine-binding globulin (TBG), transthyretin (TTR), and albumin (ALB). The THBPs are crucial to the even delivery of THs from the blood to tissues, but their relative contributions and spatial kinetics within tissues are unclear. Many endocrine-disrupting chemicals (EDCs) can interfere with the regulation and actions of THs, some of which are structurally similar to THs with comparable or even higher binding affinities for THBPs. It has been suspected that by disrupting the binding of THs with THBPs, EDCs may alter the plasma TH concentrations and the delivery to tissues. Given that the majority of the binding sites on THBPs are unoccupied—thus available for EDC binding—it is uncertain whether TH homeostasis can be tangibly perturbed by EDC exposure. To answer these questions, we constructed a spatial multi-compartment human physiologically based kinetic (PBK) model of TH production, distribution, and metabolism. The model contains the thyroid, liver, and rest-of-body compartments with explicit descriptions of the reversible binding kinetics between THs and THBPs in the blood. The model was parameterized and validated extensively based on literature data. Here we reported several novel findings: (1) The contribution to TH tissue delivery by each THBP is not spatially constant across a tissue: ALB always contributes more THs to the arterial side of the tissue while TBG contributes more to the venous side.  (2) THBPs are essential for a uniform TH tissue delivery but are cross-compensatory in this role. (3) Contrary to the long-held assumption that ALB is the dominant contributor, TBG contributes more of the THs delivered to a tissue overall than ALB and TTR. (4) Bode plot analysis using control theory suggests that a circadian signal driving T4-to-T3 conversion in the extra-thyroidal tissue is crucial to establish the distinct phase and amplitude profiles of circulating T3 and T4 in humans. (5) Displacement of THs from THBPs by high-affinity EDCs has a negligible effect on free plasma THs even under high exposures. (6) Daily exposure to THBP-binding EDCs does not affect the circadian variations in tissue delivery of THs. The PBK model can be further elaborated in the future to make tissue-specific predictions of TH kinetics.

A Minimal Human Physiologically Based Kinetics Model of Thyroid Hormones and Effects of Endocrine-Disrupting Chemicals

Background: Functional Hypothalamic Amenorrhea (FHA) is characterized by suppression of central GnRH drive with secondary anovulation and hypoestrogenemia, hypercortisolism, and low leptin that results from a combination of psychosocial stress and metabolic imbalance due to excess physical activity and/or weight loss. Cardiovascular disease (CVD) is the leading killer of women at all ages and CVD mortality rates are unchanged or increasing in young women aged 35-44 years. We investigated relations between FHA and preclinical CVD measured by endothelial dysfunction in premenopausal women with and without FHA, and recently menopausal women not on hormone therapy.
Methods: We studied 31 women with FHA, 29 eumenorrheic controls, and 30 recently menopausal women not on hormone therapy. FHA was defined as amenorrhea ≥3 consecutive months, estradiol <50 pg/mL, FSH <10 mIU/L, and LH <10 mIU/L, and exclusion of other etiologies including polycystic ovary syndrome, prolactinoma, thyroid dysfunction, and pregnancy. Eumenorrheic controls had self-reported monthly menstrual cycles, were not on hormones, with a day 22-24 progesterone level >3 ng/ml to confirm ovulation. Preclinical CVD testing for eumenorrheic controls took place days 3-5 of menses. Recently menopausal women had natural menopause within 3 years, FSH >30, and not on hormone therapy. Preclinical CVD was measured using Endo PAT 2000 (Itamar® Medical Ltd) to calculate reactive hyperemic index (RHI) stress to rest pulse amplitude. RHI ≤1.67 indicates endothelial dysfunction. Statistical analysis included analysis of variance for normal distributed variables and Kruskal Wallis test for non-parametric variables.
Results: The mean age, BMI and estradiol of FHA, no FHA and recent menopause was 26.4 ± 6.2 y, 30.3 ± 3.7 and 53.3 ± 2.6, respectively (p<0.0001); BMI 21.6 ± 6.2 kg/m2, 21.8 ± 2.0, and 23.5 ± 4.2 (p=0.06); and estradiol 29.7±18.2, 46.4 ± 15.7, and 10.9 ± 14.4 pg/ml (p<0.0001), respectively. The median months of FHA amenorrhea was 10.8 months. There were no differences in testosterone (p=0.48), however FHA women had lower leptin (p=0.012) and higher cortisol (p=0.0005) compared to both no FHA and recently menopausal women. Preclinical CVD measured by RHI was significantly lower in FHA compared to no FHA and recently menopausal women (1.8 + 0.5 vs 2.2 + 0.5 vs 2.2 +0.6;p=0.007, respectively). Further 35% of FHA women had RHI ≤1.67 indicating endothelial dysfunction.
Conclusion: Our results demonstrate endothelial dysfunction in young women with FHA women as compared to eumenorrheic women and older recently menopausal women not on hormone therapy. Our findings suggest that FHA may be an important contributor to CVD mortality in younger women and that preclinical CVD in FHA is not likely due to hypoestrogenemia alone. Future studies will focus on identifying potential treatment targets for CVD prevention.

Functional Hypothalamic Amenorrhea and Preclinical Cardiovascular Disease

Background: The subtyping of primary aldosteronism (PA) requires interpretation of aldosterone and cortisol concentrations measured during adrenal vein sampling (AVS). The use of intravenous sedation during AVS is not standardized but may affect AVS outcomes.
Purpose: To characterize the impact of sedation on the outcomes of AVS, including cortisol and aldosterone concentrations, cannulation success and rates of lateralisation.
Materials and Methods: This is a retrospective cohort study based at a tertiary health centre. Consecutive patients who underwent AVS both before and after ACTH stimulation between June 2018 and July 2020 were included. AVS was performed without IV sedation in patient cohort 1 (n = 70), with sedation (midazolam and fentanyl) in cohort 2 (n = 34) and both before and after sedation in cohort 3 (n = 14). Comparisons were performed using the nonparametric (Mann Whitney U) test for continuous variables and the Chi square test for categorical variables.
Results. Overall, 118 patients (median age, 53 years; 67 men) were evaluated with no significant demographic differences between the three cohorts. Compared to cohort 1, patients in cohort 2 displayed significantly lower cortisol concentrations in the peripheral vein (median of 141 nmol/L vs 218 nmol/L), left adrenal vein (522 nmol/L vs 1284 nmol/L) and right adrenal vein (577 nmol/L vs 1578 nmol/L) in unstimulated AVS. Fewer patients were assessed to have successful right sided cannulation in cohort 2 (79% vs 93% in cohort 1, p = 0.047).  In cohort 3, cortisol concentrations were markedly lower after sedation, with the decrease being more marked in the adrenal veins compared to peripheral veins, hence leading to lower selectivity indices.  There were no clinically significant changes in aldosterone concentration or rates of lateralisation after sedation.
Conclusions. IV sedation reduces cortisol concentrations during unstimulated-AVS, leading to lower selectivity indices and fewer cannulations judged as successful. Importantly, radiologists are turning to point-of-care rapid cortisol tests to determine if the catheter is 'in' the adrenal vein in real-time.  Adrenal vein samples collected after sedation are more likely to be considered "unsuccessful" using these semi-quantitative assays. The need for IV sedation during AVS should be carefully assessed.

Impact of Intra-Procedural Sedation on Outcomes of Adrenal Vein Sampling

Background: Primary aldosteronism (PA) is broadly divided into unilateral and bilateral forms, and the most reliable subtyping tool available is adrenal vein sampling (AVS). Unilateral PA is generally diagnosed based on AVS lateralization indices (LI=[aldosterone/cortisol] ratio between the dominant and contralateral adrenal vein) ≥4, or even ≥2 when AVS is performed without cosyntopin stimulation. Long-term follow-up data from patients with PA treated with unilateral adrenalectomy have been minimal.
Objective: To assess the rates of residual and recurrent PA after AVS-guided unilateral adrenalectomy.
Methods: We enrolled patients with PA who underwent unilateral adrenalectomy between 09/2017 and 08/2021 in a single tertiary referral center and followed them longitudinally. Demographics, laboratory, imaging, and pathology data were collected. Aldosterone synthase (CYP11B2) immunohistochemistry and next generation sequencing were performed on available formalin-fixed paraffin-embedded adrenal tissue blocks.
Results: During the study period, 80 patients (52 men) with median age 53 (range, 19-76) years underwent unilateral adenectomy, and were followed for a median 80 days (range, 6-653 days). In total 14 (17.5%) patients had evidence of residual PA following surgery, including 3 who had complete biochemical failure, and 1 who had apparent cure of PA immediately post-operatively. All 14 patients had severe PA, with baseline plasma aldosterone concentrations between 23.4 and 496 ng/dL and suppressed renin. Baseline and post-cosyntropin stimulation LIs ranged from 2.7- 187.5 and 1.3-53.7. The LI was >4 only at baseline in 3 cases, and only after cosyntropin stimulation in 2 cases. Histology showed single adenomas, ranging from 0.7-5.3 cm in 7 (50%) patients, and multiple adenomas and/or nodular hyperplasia in 7 (50%) patients. Of the 7 cases analyzed for aldosterone-driver mutations so far, 4 harbored CACNA1D mutations, 2 ATP1A1 mutations, and 1 a KCNJ5 mutation.
Conclusions: Patients with severe PA and frank AVS aldosterone lateralization might have multifocal, bilateral disease. Long-term follow up and targeted medical therapy should be pursued after unilateral adrenalectomy for presumed unilateral PA.

Bilateral Primary Aldosteronism Is Not Excluded by High Adrenal Vein Sampling Lateralization Indices

Context: Accumulating evidence suggests that primary aldosteronism can manifest across a broad phenotypic spectrum and that its origins can at times be detected among normotensive individuals. Herein, we conducted deep-phenotyping studies to interrogate the pathophysiologic continuum of the primary aldosteronism phenotype among normotensive participants.

Methods: Normotensive participants with obesity, metabolic syndrome, and/or high-normal untreated blood pressure underwent dietary sodium loading and restriction protocols (to maximally suppress and stimulate angiotensin II-mediated aldosterone production), dexamethasone suppression and cosyntropin stimulation protocols (to maximally suppress and stimulate ACTH-mediated aldosterone production), and 24h ambulatory blood pressure monitoring. Measurement of aldosterone and 18-hybrid steroids via LC-MS/MS was conducted to quantify the degree of dysregulated CYP11B2 expression. Urinary extracellular vesicles (uEV) were isolated to measure renal tubular protein content associated with mineralocorticoid receptor activation. In order to characterize parameters across the severity spectrum of non-suppressible and renin-independent aldosterone production, participants with suppressed renin activity were categorized by tertile of 24h urinary aldosterone production following oral sodium suppression testing. Repeated measures linear mixed models were used to analyze trends for each parameter across tertiles of autonomous aldosterone production; for ease of presentation, the mean [SD] of the first vs. third tertile are presented for selected parameters.

Results: Following oral sodium loading, greater non-suppressible renin-independent aldosterone production was associated with greater kaliuresis, higher daytime ambulatory systolic blood pressure within the normal range, and a progressive impairment in the ability to suppress production of serum aldosterone (P-trend=0.009), 18-hydroxycorticosterone (9.3 [5.0] vs. 19.6 [6.5] ng/dL, P-trend=0.004), 18-hydroxycortisol (45.0 [25.4] vs. 70.9 [29.9] ng/dL, P-trend=0.03), and 18-oxocortisol (1.2 [0.03] vs. 3.4 [7.2], P-trend=0.01). Similarly, following dexamethasone suppression testing, there was a progressive inability to suppress 18-hydroxycorticosterone (8.3 [5.0] vs. 15.4 [8.2] ng/dL, P-trend=0.004), 18-hydroxycortisol (6.6 [3.8] vs. 10.2 [3.6] ng/dL, P-trend=0.03), and 18-oxocortisol (1.2 [0.01] vs. 1.4 [0.6] ng/dL, P-trend=0.01) in parallel with greater renin-independent aldosterone production. In contrast, following stimulation with cosyntropin, greater non-suppressible and renin-independent aldosterone production was associated with progressively enhanced production of 18-hydroxycorticosterone (77.6 [25.4] vs. 121.1 [31.1] ng/dL, P-trend=0.004), 18-hydroxycortisol (68.5 [28.4] vs. 86.2 [26.3] ng/dL, P-trend=0.03), and 18-oxocortisol (3.2 [2.8] vs. 7.3 [3.5] ng/dL, P-trend=0.01). Excretion of uEV's of pendrin (P=0.002) and aquaporin 2 (P=0.002) all increased with greater non-suppressible aldosterone production.

Conclusions: Dynamic phenotyping studies in normotensive individuals demonstrate a continuum of non-suppressible and renin-independent aldosterone production that parallels higher blood pressure, greater kaliuresis, increased 18-hybrid steroid production indicative of dysregulated CYP11B2 expression, and increased uEV excretion indicative of renal mineralocorticoid receptor activation. This pathophysiologic continuum of normotensive primary aldosteronism provides phenotypic support for histopathology studies showing ectopic CYP11B2 expression within adrenal glands of normotensive humans, and for epidemiologic studies demonstrating renin-independent aldosterone production in normotensive populations increases risk for developing hypertension.

Characterization of the Pathophysiologic Spectrum of Subclinical Primary Aldosteronism in Normotension

Background: Aldosterone-producing adenoma (APA) is a major cause of primary aldosteronism (PA) which is the most frequent form of secondary hypertension (1-2). The pathophysiology of PA is quite complex and still incompletely understood. Substance P (SP) encoded by the TAC1 gene belongs to a family of bioactive peptides named tachykinins. A recently published study indicated that SP, released by subcapsular nerve fibres, stimulates aldosterone production through activation of the neurokinin type 1 receptor (NK1R) in the human adrenal gland (3). The aim of our work was to investigate the presence of SP fibres and the NK1 receptor in a large series of APA in order to assess the potential role of this peptide in the pathophysiology of PA. Methods: APA tissues were studied by molecular and histological approaches. Expression of SP and NK1 receptor was searched for in a series of 51 APA by RT-Q-PCR and immunohistochemistry. Results: Quantitative RT-PCR data indicated that adenomas strongly express TAC1 mRNA. Immunohistochemistry showed the presence of SP-positive nerve fibres in APAs tissues. SP was also detected in a subpopulation of adenomatous cells. Adenomas strongly express mRNA encoding the NK1 receptor. The distribution of the NK1 receptor within APAs is similar to that of aldosterone synthase (CYP11B2), suggesting that SP may regulate aldosterone secretion by APAs. Conclusion: These results suggest that SP and its NK1 receptor may be involved in the pathophysiology of aldosterone hypersecretion by APAs. References: 1. Monticone S, Burrello J, Tizzani D, Bertello C, Viola A, Buffolo F, et al. Prevalence and Clinical Manifestations of Primary Aldosteronism Encountered in Primary Care Practice. J Am Coll Cardiol. 2017;69(14): 1811‑20. 2. Buffolo F, Monticone S, Tetti M, Mulatero P. Primary aldosteronism in the primary care setting. Curr Opin Endocrinol Diabetes Obes. 2018;25(3): 155‑9. 3. Wils J, Duparc C, Cailleux A-F, Lopez A-G, Guiheneuf C, Boutelet I, et al. The neuropeptide substance P regulates aldosterone secretion in human adrenals. Nat Commun. 2020;11(1): 2673.

Expression of the Neuropeptide Substance P and the NK1 Receptor in Aldosterone-Producing Adenomas

Background:
To promote ideal cardiovascular health, the American Heart Association recommends adhering to Life's Simple 7 (LS7)—achieving healthy targets for BMI, physical activity, dietary intake, blood pressure, fasting plasma glucose, and cholesterol, along with smoking abstinence. Retrospective data analyses have shown associations between poorer achievement of LS7 (lower score) and the development of hypertension and cardiovascular disease (CVD). However, less is known about the associations between LS7 and specific biomarkers linked to cardiovascular health.

Hypothesis:
We hypothesized that achievement of LS7 (higher score) would be associated with lower levels of aldosterone and inflammatory markers implicated in CVD in individuals with controlled dietary sodium intake.

Methodology:
The analysis included 379 normotensive and hypertensive individuals (age 18-66 years) from the HyperPATH (International Hypertensive Pathotype) consortium, who were maintained on at least 200 mEq of NaCl daily for one week and had a urinary salt balance of >150 mEq/24 hours at the time of sample collection. We calculated a 14-point summative LS7 score according to participants' baseline data. Based on the range of LS7 score in this population, we classified participants as 'inadequate' (3-6), 'average' (7-10), and 'optimal' (11-14). We then performed regression analyses (adjusted for age, sex, race, and serum creatinine) between the LS7 score groups (inadequate, average, or optimal) and biochemical measures, including levels of aldosterone, C-reactive protein (CRP), and interleukin 6 (IL-6).

Results:
When comparing across all three LS7 score groups, a higher LS7 score group was associated with lower aldosterone levels both in the serum (p=0.005, -0.97 [-1.63, -0.30]) and urine (p=0.003, -2.34 [-4.07, -0.825]). Additionally, a higher LS7 score group was associated with lower renin (p=0.008, -0.13 [-0.22, -0.03]) and a blunted increase in aldosterone with angiotensin-II infusion (p=0.019, -1.66 [-3.04, -0.27]). Serum potassium and serum cortisol were not associated with the LS7 scores. Being in the 'optimal' LS7 score group was associated with lower serum levels of CRP (p=0.001, -0.1 [-0.15, -0.04]) and IL-6 (p=0.026, -0.84 [-1.59, -0.10]). Notably, among the individual LS7 components, achieving target blood pressure and exercise goals most significantly impacted the associations between LS7 score and aldosterone levels.

Conclusions:
Achieving an optimal American Heart Association's LS7 score was associated with a lower activity of the renin-angiotensin-aldosterone system and lower levels of the inflammatory markers CRP and IL-6, which may help to explain previous reports of better cardiovascular outcomes in individuals with a higher LS7 score. These findings offer a possible link between ideal cardiovascular health targets and biomarkers known to play a central role in the development of CVD.

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Dissecting Thyroidal Pathways Activated by PD-L1 Blockade Identifies New Mechanisms for Thyroiditis Triggered by Immune Checkpoint Inhibitors

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Treatment of Heart Failure with Preserved Ejection Fraction (HFpEF) with Low and High Dose of Triiodothyronine: an Animal Model of Metabolic Syndrome with HFpEF

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