Cardiac recovery from pressure overload is not altered by thyroid hormone status in old mice.

Introduction: Thyroid hormones (THs) are known to have various effects on the cardiovascular system. However, the impact of TH levels on preexisting cardiac diseases is still unclear. Pressure overload due to arterial hypertension or aortic stenosis and aging are major risk factors for the development of structural and functional abnormalities and subsequent heart failure. Here, we assessed the sensitivity to altered TH levels in aged mice with maladaptive cardiac hypertrophy and cardiac dysfunction induced by transverse aortic constriction (TAC). Methods: Mice at the age of 12 months underwent TAC and received T4 or anti-thyroid medication in drinking water over the course of 4 weeks after induction of left ventricular pressure overload. Results: T4 excess or deprivation in older mice had no or only very little impact on cardiac function (fractional shortening), cardiac remodeling (cardiac wall thickness, heart weight, cardiomyocyte size, apoptosis, and interstitial fibrosis), and mortality. This is surprising because T4 excess or deprivation had significantly changed the outcome after TAC in young 8-week-old mice. Comparing the gene expression of deiodinases (Dio) 2 and 3 and TH receptor alpha (TRα) 1 and the dominant-negative acting isoform TRα2 between young and aged mice revealed that aged mice exhibited a higher expression of TRα2 and Dio3, while expression of Dio2 was reduced compared with young mice. These changes in Dio2 and 3 expressions might lead to reduced TH availability in the hearts of 12-month-old mice accompanied by reduced TRα action due to higher TRα2. Discussion: In summary, our study shows that low and high TH availability have little impact on cardiac function and remodeling in older mice with preexisting pressure-induced cardiac damage. This observation seems to be the result of an altered expression of deiodinases and TRα isoforms, thus suggesting that even though cardiovascular risk is increasing with age, the response to TH stress may be dampened in certain conditions.

Combination of nivolumab with standard induction chemotherapy in children and adults with EBV-positive nasopharyngeal carcinoma : Protocol of a prospective multicenter phase 2 trial.

BACKGROUND: Treatment of Epstein-Barr virus(EBV)-positive nasopharyngeal carcinoma (NPC) with cisplatin/5-fluorouracil (5-FU) induction chemotherapy, followed by radiochemotherapy and subsequent interferon­ß, has yielded high survival rates in children, adolescents, and young adults. A previous study has shown that reduction of radiation dose from 59.4 to 54.0 Gy appears to be safe in patients with complete response (CR) to induction chemotherapy. As immune checkpoint-inhibitors have shown activity in NPC, we hypothesize that the addition of nivolumab to standard induction chemotherapy would increase the rate of complete tumor responses, thus allowing for a reduced radiation dose in a greater proportion of patients. METHODS: This is a prospective multicenter phase 2 clinical trial including pediatric and adult patients with their first diagnosis of EBV-positive NPC, scheduled to receive nivolumab in addition to standard induction chemotherapy. In cases of non-response to induction therapy (stable or progressive disease), and in patients with initial distant metastasis, treatment with nivolumab will be continued during radiochemotherapy. Primary endpoint is tumor response on magnetic resonance imaging (MRI) and positron emission tomography (PET) after three cycles of induction chemotherapy. Secondary endpoints are event-free (EFS) and overall survival (OS), safety, and correlation of tumor response with programmed cell death ligand 1 (PD-L1) expression. DISCUSSION: As cure rates in localized EBV-positive NPC today are high with standard multimodal treatment, the focus increasingly shifts toward prevention of late effects, the burden of which is exceptionally high, mainly due to intense radiotherapy. Furthermore, survival in patients with metastatic disease and resistant to conventional chemotherapy remains poor. Primary objective of this study is to investigate whether the addition of nivolumab to standard induction chemotherapy in children and adults with EBV-positive NPC is able to increase the rate of complete responses, thus enabling a reduction in radiation dose in more patients, but also offer patients with high risk of treatment failure the chance to benefit from the addition of nivolumab. TRIAL REGISTRATION: EudraCT (European Union Drug Regulating Authorities Clinical Trials Database) No. 2021-006477-32.

Cardioprotection by Hypothyroidism Is Not Mediated by Favorable Hemodynamics-Role of Canonical Thyroid Hormone Receptor Alpha Signaling.

Hypothyroidism has been shown to reduce infarct size in rats, but the underlying mechanisms are unclear. We used isolated pressure-constant perfused hearts of control, hypothyroid and hyperthyroid mice and measured infarct size, functional parameters and phosphorylation of key molecules in cardioprotective signaling with matched heart rate. Compared with controls, hypothyroidism was cardioprotective, while hyperthyroidism was detrimental with enlarged infarct size. Next, we asked how thyroid hormone receptor α (TRα) affects ischemia/reperfusion (IR) injury. Thus, canonical and noncanonical TRα signaling was investigated in the hearts of (i) mice lacking TRα (TRα0), (ii) with a mutation in TRα DNA-binding domain (TRαGS) and (iii) in hyperthyroid TRα0 (TRα0hyper) and TRαGS mice (TRαGShyper). TRα0 mouse hearts were protected against IR injury. Furthermore, infarct size was reduced in the hearts of TRαGS mice that lack canonical TRα signaling but maintain noncanonical TRα action. Hyperthyroidism did not increase infarct size in TRα0 and TRαGS mouse hearts. These cardioprotective effects were not associated with increased phosphorylation of key proteins of RISK, SAFE and eNOS pathways. In summary, chronic hypothyroidism and the lack of canonical TRα signaling are cardioprotective in IR injury and protection is not due to favorable changes in hemodynamics.

Tentative Application of a Streamlined Protocol to Determine Organ-Specific Regulations of Deiodinase 1 and Dehalogenase Activities as Readouts of the Hypothalamus-Pituitary-Thyroid-Periphery-Axis.

In animal studies, both in basic science and in toxicological assessment of potential endocrine disruptors, the state of the thyroid hormone (TH) axis is often described and defined exclusively by the concentrations of circulating THs and TSH. Although it is known that the local, organ-specific effects of THs are also substantially regulated by local mechanisms such as TH transmembrane transport and metabolism of TH by deiodinases, such endpoint parameters of the axis are rarely assessed in these experiments. Currently developed in vitro assays utilize the Sandell-Kolthoff reaction, a photometric method of iodide determination, to test the effect of chemicals on iodotyrosine and iodothyronine deiodinases. Furthermore, this technology offers the possibility to determine the iodine content of various sample types (e.g., urine, ex vivo tissue) in a simple way. Here, we measured deiodinase type 1 and iodotyrosine dehalogenase activity by means of the Sandell-Kolthoff reaction in ex vivo samples of hypo- and hyperthyroid mice of two age groups (young; 3 months and old; 20 months). In thyroid, liver and kidney, organ-specific regulation patterns emerged across both age groups, which, based on this pilot study, may serve as a starting point for a deeper characterization of the TH system in relevant studies in the future and support the development of Integrated Approach for Testing and Assessment (IATA).

Canonical Thyroid Hormone Receptor ß Action Stimulates Hepatocyte Proliferation in Male Mice.

CONTEXT: 3,5,3'-L-triiodothyronine (T3) is a potent inducer of hepatocyte proliferation via the Wnt/ß-catenin signaling pathway. Previous studies suggested the involvement of rapid noncanonical thyroid hormone receptor (TR) ß signaling, directly activating hepatic Wnt/ß-catenin signaling independent from TRß DNA binding. However, the mechanism by which T3 increases Wnt/ß-catenin signaling in hepatocytes has not yet been determined. OBJECTIVE: We aimed to determine whether DNA binding of TRß is required for stimulation of hepatocyte proliferation by T3. METHODS: Wild-type (WT) mice, TRß knockout mice (TRß KO), and TRß mutant mice with either specifically abrogated DNA binding (TRß GS) or abrogated direct phosphatidylinositol 3 kinase activation (TRß 147F) were treated with T3 for 6 hours or 7 days. Hepatocyte proliferation was assessed by Kiel-67 (Ki67) staining and apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Activation of ß-catenin signaling was measured in primary murine hepatocytes. Gene expression was analyzed by microarray, gene set enrichment analysis (GSEA), and quantitative reverse transcription polymerase chain reaction. RESULTS: T3 induced hepatocyte proliferation with an increased number of Ki67-positive cells in WT and TRß 147F mice (9.2% ±â€…6.5% and 10.1% ±â€…2.9%, respectively) compared to TRß KO and TRß GS mice (1.2% ±â€…1.1% and 1.5% ±â€…0.9%, respectively). Microarray analysis and GSEA showed that genes of the Wnt/ß-catenin pathway-among them, Fzd8 (frizzled receptor 8) and Ctnnb1 (ß-catenin)-were positively enriched only in T3-treated WT and TRß 147F mice while B-cell translocation gene anti-proliferation factor 2 was repressed. Consequently, expression of Ccnd1 (CyclinD1) was induced. CONCLUSIONS: Instead of directly activating Wnt signaling, T3 and TRß induce key genes of the Wnt/ß-catenin pathway, ultimately stimulating hepatocyte proliferation via CyclinD1. Thus, canonical transcriptional TRß action is necessary for T3-mediated stimulation of hepatocyte proliferation.

Protective Effects of Thyroid Hormone Deprivation on Progression of Maladaptive Cardiac Hypertrophy and Heart Failure.

Purpose: Thyroid hormones (TH) play a central role for cardiac function. TH influence heart rate and cardiac contractility, and altered thyroid function is associated with increased cardiovascular morbidity and mortality. The precise role of TH in onset and progression of heart failure still requires clarification. Methods: Chronic left ventricular pressure overload was induced in mouse hearts by transverse aortic constriction (TAC). One week after TAC, alteration of TH status was induced and the impact on cardiac disease progression was studied longitudinally over 4 weeks in mice with hypo- or hyperthyroidism and was compared to euthyroid TAC controls. Serial assessment was performed for heart function (2D M-mode echocardiography), heart morphology (weight, fibrosis, and cardiomyocyte cross-sectional area), and molecular changes in heart tissues (TH target gene expression, apoptosis, and mTOR activation) at 2 and 4 weeks. Results: In diseased heart, subsequent TH restriction stopped progression of maladaptive cardiac hypertrophy and improved cardiac function. In contrast and compared to euthyroid TAC controls, increased TH availability after TAC propelled maladaptive cardiac growth and development of heart failure. This was accompanied by a rise in cardiomyocyte apoptosis and mTOR pathway activation. Conclusion: This study shows, for the first time, a protective effect of TH deprivation against progression of pathological cardiac hypertrophy and development of congestive heart failure in mice with left ventricular pressure overload. Whether this also applies to the human situation needs to be determined in clinical studies and would infer a critical re-thinking of management of TH status in patients with hypertensive heart disease.

Age-dependent response to T4 overtreatment and recovery on systemic and organ level.

Thyroid hormone (TH) metabolism and cellular TH action are influenced by ageing. To investigate the response to thyroxine (T4) overtreatment, a kinetic study was conducted in young and aged mice with chronic hyperthyroidism and hormone withdrawal. Five and 22 months old male mice were treated with T4 or PBS over 5 weeks, followed by observation for up to 12 days. Serial analysis was performed for thyroid function parameters, transcript levels of TH target genes, deiodinase type 1 (DIO1) activity as well as serum lipids at 12, 24, 72, 144, 216, and 288 h after cessation of T4 administration. Higher FT3 concentrations and higher renal DIO1 activities were noted in aged mice 12 h after T4 withdrawal and marked thyroid-stimulating hormone elevation was found in aged mice after 12 days compared to respective controls. A biphasic expression pattern occurred for TH target genes in all organs and a hypothyroid organ state was observed at the end of the study, despite normalization of TH serum concentrations after 72 h. In line with this, mirror-image kinetics were detected for serum cholesterol and triglycerides in aged and young mice. Recovery from TH overtreatment in mice involves short- and medium-term adaption of TH metabolism on systemic and organ levels. Increased renal DIO1 activity may contribute to higher T3 concentrations and prolonged thyrotoxicosis followed by hypothyroidism in an aged-mouse organism. Translation of these findings in the clinical setting seems warranted and may lead to better management of hyperthyroidism and prevention of T4 overtreatment in aged patients.

Noncanonical Thyroid Hormone Receptor α Action Mediates Arterial Vasodilation.

CONTEXT: Hypothyroidism impairs cardiovascular health and contributes to endothelial dysfunction with reduced vasodilation. How 3,5,3'-triiodothyronine (T3) and its receptors are involved in the regulation of vasomotion is not yet fully understood. In general, thyroid hormone receptors (TRs) either influence gene expression (canonical action) or rapidly activate intracellular signaling pathways (noncanonical action). OBJECTIVE: Here we aimed to characterize the T3 action underlying the mechanism of arterial vasodilation and blood pressure (BP) regulation. METHODS: Mesenteric arteries were isolated from male rats, wild-type (WT) mice, TRα knockout (TRα 0) mice, and from knockin mice with a mutation in the DNA-binding domain (TRα GS). In this mutant, DNA binding and thus canonical action is abrogated while noncanonical signaling is preserved. In a wire myograph system, the isolated vessels were preconstricted with norepinephrine. The response to T3 was measured, and the resulting vasodilation (Δ force [mN]) was normalized to maximum contraction with norepinephrine and expressed as percentage vasodilation after maximal preconstriction with norepinephrine (%NE). Isolated vessels were treated with T3 (1 × 10-15 to 1 × 10-5 mol/L) alone and in combination with the endothelial nitric oxide-synthase (eNOS) inhibitor L-NG-nitroarginine methyl ester (L-NAME) or the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. The endothelium was removed to determine the contribution of T3 to endothelium-dependent vasodilation. The physiological relevance of T3-induced vasodilation was determined by in vivo arterial BP measurements in male and female mice. RESULTS: T3 treatment induced vasodilation of mesenteric arteries from WT mice within 2 minutes (by 21.5 ±â€…1.7%NE). This effect was absent in arteries from TRα 0 mice (by 5.3 ±â€…0.6%NE, P < .001 vs WT) but preserved in TRα GS arteries (by 17.2 ±â€…1.1%NE, not significant vs WT). Inhibition of either eNOS or PI3K reduced T3-mediated vasodilation from 52.7 ±â€…4.5%NE to 28.5 ±â€…4.1%NE and 22.7 ±â€…2.9%NE, respectively. Removal of the endothelium abolished the T3-mediated vasodilation in rat mesenteric arteries (by 36.7 ±â€…5.4%NE vs 3.5 ±â€…6.2%NE). In vivo, T3 injection led to a rapid decrease of arterial BP in WT (by 13.9 ±â€…1.9 mm Hg) and TRα GS mice (by 12.4 ±â€…1.9 mm Hg), but not in TRα 0 mice (by 4.1 ±â€…1.9 mm Hg). CONCLUSION: These results demonstrate that T3 acting through noncanonical TRα action affects cardiovascular physiology by inducing endothelium-dependent vasodilation within minutes via PI3K and eNOS activation.

Age and Sex Influence Thyroid Hormone Effects in Target Tissues with Organ-Specific Responses.

Age and sex impact prevalence and clinical features of thyroid disease. Thyroid dysfunction occurs with a higher frequency in elderly patients and females. Moreover, age alters clinical presentation of hyper- and hypothyroidism and onset of thyroid hormone (TH) related co-morbidities leading to increased risk for underdiagnosis and maltreatment in the elderly. Rodent models allow further insights into mechanisms of age- and sex-dependent TH action in target tissues. In this review, we summarize findings from mouse studies showing distinct effects of age and sex on systemic versus organ-specific TH action and discuss their wider implication for clinical care.

Age effect on thyroid hormone brain response in male mice.

PURPOSE: Thyroid hormones (TH) are important for brain development and central nervous system (CNS) function. Disturbances of thyroid function occur with higher prevalence in the ageing population and may negatively impact brain function. METHODS: We investigated the age impact on behavior in young adult and old male mice (5 vs. 20 months) with chronic hypo- or hyper-thyroidism as well as in sham-treated controls. Expression of TH transporters and TH responsive genes was studied in CNS and pituitary by in situ hybridization and qRT-PCR, whereas TH serum concentrations were determined by immunoassay. RESULTS: Serum TH levels were lower in old compared with young hyperthyroid mice, suggesting a milder hyperthyroid phenotype in the aged group. Likewise, elevated plus maze activity was reduced in old hyperthyroid animals. Under hypothyroid conditions, thyroxine serum concentrations did not differ in young and old mice. Both groups showed a comparable decline in activity and elevated anxiety levels. However, an attenuated increase in hypothalamic thyrotropin releasing hormone and pituitary thyroid stimulating hormone transcript expression was found in old hypothyroid mice. Brain expression of monocarboxylate transporter 8 and organic anion transporting polypeptide 1c1 was not affected by age or TH status. CONCLUSIONS: In summary, ageing attenuates neurological phenotypes in hyperthyroid but not hypothyroid mice, which fits with age effects on TH serum levels in the animals. In contrast no changes in TH transporter expression were found in aged mouse brains with hyper- or hypo-thyroid state.

Thyroid Related Quality of Life in Elderly with Subclinical Hypothyroidism and Improvement on Levothyroxine is Distinct from that in Young Patients (TSAGE).

The aim of this study was to investigate in a longitudinal approach whether levothyroxine (LT4) substitution has a different impact on quality of life (QoL) and thyroid related QoL in younger (<40 years) and older subjects (>60 years) with elevated thyroid-stimulating hormone (TSH) concentrations. The study included male and female patients with newly diagnosed, untreated subclinical hypothyroidism defined by TSH>8 mU/l. Patients were recruited throughout Germany from 2013-2016 and evaluated by clinical assessment, blood sampling and questionnaires for health related QoL and thyroid-disease thyroid-related QoL (ThyPRO) at time of diagnosis and six months after initiation of LT4 treatment. We found significantly lower QoL in both young and old patients with subclinical hypothyroidism compared to age-matched healthy individuals. Higher scores on follow-up were found in all patients irrespective of age, indicating better QoL on LT4 therapy. Analysis of the ThyPRO questionnaire showed that old patients experienced less Emotional Susceptibility, Tiredness, and Impaired Day Life on LT4, while young patients reported less Cognitive Complaints, Emotional Susceptibility, and Impaired Day Life compared to baseline assessment. Hypothyroidism with TSH concentrations>8 mU/l is associated with impairment in general and ThyPRO QoL in young and old age. Older patients benefited from LT4 therapy and remarkably show similar degree of improvement as younger patients, albeit with some thematic variation in ThyPRO QoL. Our data confirm current recommendations on initiation of LT4 substitution and suggest that this should not be withheld in elderly with TSH concentration above 8-10 mU/l.