Increasing Catecholamine Secretion Through NPY in Pheochromocytomas With False-Negative 123 I-MIBG Scintigraphy.

INTRODUCTION: 123 I-MIBG has been well established as a functional imaging tool, and 131 I-MIBG therapy is being considered for catecholamine-secreting tumors. Tumors with the characteristics of a noradrenergic biochemical phenotype, small, malignant, metastatic, extra-adrenal, bilateral, and hereditary, especially SDHx -related tumors, are reported to correlate with reduced MIBG uptake. However, the potential molecular mechanisms influencing MIBG uptake have been poorly studied. PATIENTS AND METHODS: To identify critical genes that may enhance MIBG accumulation in pheochromocytomas (PCCs), we performed RNA-seq analyses for 16 operated patients with PCCs (6 MIBG-negative and 10 MIBG-positive) combined with RT-qPCR for 27 PCCs (5 MIBG-negative and 22 MIBG-positive) and examined primary cultures of the surgical tissues. RESULTS: In the present study, 6 adrenal nodules of 66 nodules surgically removed from 63 patients with PCCs (9%) were MIBG negative. MIBG, a guanethidine analog of norepinephrine, can enter chromaffin cells through active uptake via the cellular membrane, be deposited in chromaffin granules, and be released via Ca 2+ -triggered exocytosis from adrenal chromaffin cells. When we compared expression of several catecholamine biosynthesis and secretion-associated genes between MIBG-negative and MIBG-positive tumors using transcriptome analyses, we found that neuropeptide Y, which is contained in chromaffin granules, was significantly increased in MIBG-negative tumors. NPY stimulated norepinephrine secretion dose-dependently in primary cell culture derived from MIBG-positive PCC. In our study, MIBG-negative PCCs were all norepinephrine-hypersecreting tumors. CONCLUSIONS: These data indicate that NPY upregulation in PCCs may stimulate chromaffin granule catecholamine secretion, which is associated with false-negative 123 I-MIBG scintigraphy.

The improvement of postoperative blood pressure and associated factors in patients with hormone-negative adrenal adenoma and hypertension.

OBJECTIVE: To investigate the effect of adrenal surgery on blood pressure (BP) improvements in patients with hormone-negative adrenal adenoma (HNA) concomitant with hypertension and analyze associated prognostic factors. METHODS: We retrospectively reviewed the clinical data of patients with HNA and hypertension and patients with aldosterone-producing adenoma (APA) and hypertension who underwent adrenal surgery at our center between 2019 and 2022. Hypertension outcomes were evaluated in all patients and subjects were divided into three groups according to follow-up BP and the administration of anti-hypertensive agents: a clinical curation group, an improvement group, and a no-improvement group. Logistic regression analysis was performed to predict factors associated with clinical curation in patients with HNA post-surgery. RESULTS: Of the 182 patients with HNA, clinical curation was achieved in 58 patients (31.9%), improvement in 72 (39.5%), and no improvement in 52 (28.6%). The clinical curation, improvement and no improvement rates in patients with APA were 64.8% (n = 118), 15.9% (n = 29), and 19.2% (n = 35). Multivariate logistic regression analysis indicated that a duration of hypertension ≤6 years and a plasma aldosterone level >160 pg/ml were both independent factors for the clinical curation of hypertension in patients with HNA after adrenal surgery. CONCLUSION: Adrenal surgery can cure or improve hypertension in most patients with HNA, especially in a short duration of hypertension and high plasma levels of aldosterone.

Animal and Cell Culture Models of PPGLs - Achievements and Limitations.

Research on rare tumors heavily relies on suitable models for basic and translational research. Paragangliomas (PPGL) are rare neuroendocrine tumors (NET), developing from adrenal (pheochromocytoma, PCC) or extra-adrenal (PGL) chromaffin cells, with an annual incidence of 2-8 cases per million. While most PPGL cases exhibit slow growth and are primarily treated with surgery, limited systemic treatment options are available for unresectable or metastatic tumors. Scarcity of appropriate models has hindered PPGL research, preventing the translation of omics knowledge into drug and therapy development. Human PPGL cell lines are not available, and few animal models accurately replicate the disease's genetic and phenotypic characteristics. This review provides an overview of laboratory models for PPGLs, spanning cellular, tissue, organ, and organism levels. We discuss their features, advantages, and potential contributions to diagnostics and therapeutics. Interestingly, it appears that in the PPGL field, disease models already successfully implemented in other cancers have not been fully explored.

Pancreatosteatosis in patients with adrenal incidentaloma: A risk factor for impaired glucose metabolism.

AIMS: Patients with adrenal incidentaloma (AI) are at increased risk of impaired glucose metabolism, which is known to be associated with pancreatosteatosis (PS). We aimed to investigate the risk of developing dysglycemia for patients with non-functioning AI (NFAI) versus those without, and whether the presence of PS predicts future dysglycemia in patients with NFAI. METHOD: In 80 patients with NFAI and 127 controls matched for age, sex, and body mass index, changes in fasting plasma glucose (FPG) and hemoglobin A1c(HbA1c) were evaluated at 2 years. PS was evaluated with data obtained from non-contrast abdominal computed tomography (CT) performed at the initial evaluation. RESULTS: Mean FPG levels increased significantly after 2 years in both groups (P < 0.001, for both), albeit significantly higher among patients than the controls (P = 0.002). The increases in HbA1c and FPG levels were significantly higher among patients with PS than without PS, in the adenoma group (p < 0.001, P = 0.00, respectively). The change in Hba1c levels was associated with the presence of PS in patients with NFAI (p < 0.001). CONCLUSIONS: Our findings suggest that the presence of PS may provide significant information in predicting newly developed dysglycemia in patients with NFAI.

Hair cortisol and endocannabinoid measurement in patients with adrenal incidentalomas: a case-control study.

PURPOSE: The role of endocannabinoids (ECs) in the regulation of the hypothalamic-pituitary-adrenocortical axis has already been studied; however, data are scarce in humans. The aim of our study was to analyze EC [anandamide (AEA) and 2-arachidonoylglycerol (2-AG)] and cortisol (F) levels in hair samples of patients with adrenal incidentalomas (AIs) in comparison with those found in controls and assess their association with the hormone profile. METHODS: Forty-four patients with AIs [32 with non-functioning AIs (NFAIs) and 12 with possible autonomous secretion (PACS)] and 44 controls were recruited. Basal and post-1 mg overnight dexamethasone suppression test (ODST) F, adrenocorticotropic hormone, dehydroepiandrosterone sulfate, and 24-h urinary free cortisol were analyzed. After hair collection, EC and F levels were measured by liquid chromatography tandem-mass spectrometry. RESULTS: There was no difference between the groups regarding age, sex, and metabolic status. Significantly decreased hair AEA and 2-AG levels were found in patients with AIs compared to controls (p < 0.001 and p = 0.002, respectively) as well as between NFAI or PACS and controls (p < 0.001 or p = 0.002 and p = 0.038 or p = 0.02, respectively). Among the AI patients, EC levels tended to be lower in the PACS group. AEA hair levels were negatively correlated with F levels post-1 mg ODST (rs = -0.257, p = 0.033). We found no significant difference comparing hair F between the groups. CONCLUSION: Our findings suggest that hair EC measurement could be a potential biomarker in the evaluation of patients with AIs, whereas hair F analysis is not a useful diagnostic test for mild hypercortisolemia.

A comprehensive characterisation of phaeochromocytoma and paraganglioma tumours through histone protein profiling, DNA methylation and transcriptomic analysis genome wide.

BACKGROUND: Phaeochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumours. Pathogenic variants have been identified in more than 15 susceptibility genes; associated tumours are grouped into three Clusters, reinforced by their transcriptional profiles. Cluster 1A PPGLs have pathogenic variants affecting enzymes of the tricarboxylic acid cycle, including succinate dehydrogenase. Within inherited PPGLs, these are the most common. PPGL tumours are known to undergo epigenetic reprograming, and here, we report on global histone post-translational modifications and DNA methylation levels, alongside clinical phenotypes. RESULTS: Out of the 25 histone post-translational modifications examined, Cluster 1A PPGLs were distinguished from other tumours by a decrease in hyper-acetylated peptides and an increase in H3K4me2. DNA methylation was compared between tumours from individuals who developed metastatic disease versus those that did not. The majority of differentially methylated sites identified tended to be completely methylated or unmethylated in non-metastatic tumours, with low inter-sample variance. Metastatic tumours by contrast consistently had an intermediate DNA methylation state, including the ephrin receptor EPHA4 and its ligand EFNA3. Gene expression analyses performed to identify genes involved in metastatic tumour behaviour pin-pointed a number of genes previously described as mis-regulated in Cluster 1A tumours, as well as highlighting the tumour suppressor RGS22 and the pituitary tumour-transforming gene PTTG1. CONCLUSIONS: Combined transcriptomic and DNA methylation analyses revealed aberrant pathways, including ones that could be implicated in metastatic phenotypes and, for the first time, we report a decrease in hyper-acetylated histone marks in Cluster 1 PPGLs.

The connection between tricarboxylic acid cycle enzyme mutations and pseudohypoxic signaling in pheochromocytoma and paraganglioma.

Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors originating from chromaffin cells, holding significant clinical importance due to their capacity for excessive catecholamine secretion and associated cardiovascular complications. Roughly 80% of cases are associated with genetic mutations. Based on the functionality of these mutated genes, PPGLs can be categorized into distinct molecular clusters: the pseudohypoxia signaling cluster (Cluster-1), the kinase signaling cluster (Cluster-2), and the WNT signaling cluster (Cluster-3). A pivotal factor in the pathogenesis of PPGLs is hypoxia-inducible factor-2α (HIF2α), which becomes upregulated even under normoxic conditions, activating downstream transcriptional processes associated with pseudohypoxia. This adaptation provides tumor cells with a growth advantage and enhances their ability to thrive in adverse microenvironments. Moreover, pseudohypoxia disrupts immune cell communication, leading to the development of an immunosuppressive tumor microenvironment. Within Cluster-1a, metabolic perturbations are particularly pronounced. Mutations in enzymes associated with the tricarboxylic acid (TCA) cycle, such as succinate dehydrogenase (SDHx), fumarate hydratase (FH), isocitrate dehydrogenase (IDH), and malate dehydrogenase type 2 (MDH2), result in the accumulation of critical oncogenic metabolic intermediates. Notable among these intermediates are succinate, fumarate, and 2-hydroxyglutarate (2-HG), which promote activation of the HIFs signaling pathway through various mechanisms, thus inducing pseudohypoxia and facilitating tumorigenesis. SDHx mutations are prevalent in PPGLs, disrupting mitochondrial function and causing succinate accumulation, which competitively inhibits α-ketoglutarate-dependent dioxygenases. Consequently, this leads to global hypermethylation, epigenetic changes, and activation of HIFs. In FH-deficient cells, fumarate accumulation leads to protein succination, impacting cell function. FH mutations also trigger metabolic reprogramming towards glycolysis and lactate synthesis. IDH1/2 mutations generate D-2HG, inhibiting α-ketoglutarate-dependent dioxygenases and stabilizing HIFs. Similarly, MDH2 mutations are associated with HIF stability and pseudohypoxic response. Understanding the intricate relationship between metabolic enzyme mutations in the TCA cycle and pseudohypoxic signaling is crucial for unraveling the pathogenesis of PPGLs and developing targeted therapies. This knowledge enhances our comprehension of the pivotal role of cellular metabolism in PPGLs and holds implications for potential therapeutic advancements.

Imaging Pathway of a Pediatric Patient with Succinate Dehydrogenase B-Deficient Paraganglioma.

Pheochromocytoma and paraganglioma are rare in children, at only 1 in every 50,000 cases. Even though some cases are sporadic, they have been connected to syndromes such as von Hippel-Lindau, multiple endocrine neoplasia types IIa and IIb, neurofibromatosis type 1, and hereditary pheochromocytoma-paraganglioma syndromes. A genetic mutation causes around 60% of pheochromocytomas and paragangliomas in children under 18. Methods: A 15-y-old child with a 6-y history of back discomfort is presented. The justification for using 2 functional imaging modalities, 68Ga-DOTATATE PET/CT and 123I-meta-iodobenzylguanidine SPECT/CT, is examined in this case study. We reviewed the patients' journey since the first referral for imaging. Results: Delaying the molecular imaging modalities has affected patients' overall diagnosis and applied treatment outcomes. Conclusion: This case study investigates the potential for the earlier use of various diagnostic modalities in conjunction with diagnostic testing to facilitate an earlier diagnosis. However, since this study is based solely on imaging and lacks access to the patient's clinical or family history, factors such as potential inequities in health-care facilities, health literacy, and socioeconomic status are not addressed. It is essential to acknowledge these influences as they contribute to the inequitable access to health-care settings in New Zealand.

TMEM127 suppresses tumor development by promoting RET ubiquitination, positioning, and degradation.

The TMEM127 gene encodes a transmembrane protein of poorly known function that is mutated in pheochromocytomas, neural crest-derived tumors of adrenomedullary cells. Here, we report that, at single-nucleus resolution, TMEM127-mutant tumors share precursor cells and transcription regulatory elements with pheochromocytomas carrying mutations of the tyrosine kinase receptor RET. Additionally, TMEM127-mutant pheochromocytomas, human cells, and mouse knockout models of TMEM127 accumulate RET and increase its signaling. TMEM127 contributes to RET cellular positioning, trafficking, and lysosome-mediated degradation. Mechanistically, TMEM127 binds to RET and recruits the NEDD4 E3 ubiquitin ligase for RET ubiquitination and degradation via TMEM127 C-terminal PxxY motifs. Lastly, increased cell proliferation and tumor burden after TMEM127 loss can be reversed by selective RET inhibitors in vitro and in vivo. Our results define TMEM127 as a component of the ubiquitin system and identify aberrant RET stabilization as a likely mechanism through which TMEM127 loss-of-function mutations cause pheochromocytoma.

Adrenal steroid metabolites and bone status in patients with adrenal incidentalomas and hypercortisolism.

BACKGROUND: Autonomous cortisol secretion (ACS), resulting from cortisol-producing adenomas (CPA), causes endogenous steroid-induced osteoporosis (SIOP). However, the risk of endogenous SIOP cannot be explained by cortisol excess alone, and how other steroid metabolites affect bone status is unclear. METHODS: ACS was diagnosed as serum cortisol ≥1.8 µg/dL after the 1-mg dexamethasone suppression test (DST-cortisol). Using liquid chromatography tandem mass spectrometry, 21 plasma steroid metabolites were measured in 73 patients with ACS and 85 patients with non-functioning adrenal tumors (NFAT). Expression of steroidogenic enzymes and relevant steroid metabolites were analyzed in some of CPA tissues. FINDINGS: Discriminant and principal component analyses distinguished steroid profiles between the ACS and NFAT groups in premenopausal women. Premenopausal women with ACS exhibited higher levels of a mineralocorticoid metabolite, 11-deoxycorticosterone (11-DOC), and lower levels of androgen metabolites, dehydroepiandrosterone-sulfate, and androsterone-glucuronide. In premenopausal women with ACS, DST-cortisol negatively correlated with trabecular bone score (TBS). Additionally, 11-DOC negatively correlated with lumbar spine-bone mineral density, whereas androsterone-glucuronide positively correlated with TBS. The CPA tissues showed increased 11-DOC levels with increased expression of CYP21A2, essential for 11-DOC synthesis. Adrenal non-tumor tissues were atrophied with reduced expression of CYB5A, required for androgen synthesis. INTERPRETATION: This study demonstrates that unbalanced production of adrenal steroid metabolites, derived from both adrenal tumor and non-tumor tissues, contributes to the pathogenesis of endogenous SIOP in premenopausal women with ACS. FUNDING: JSPS KAKENHI, Secom Science and Technology Foundation, Takeda Science Foundation, Japan Foundation for Applied Enzymology, AMED-CREST, JSTA-STEP, JST-Moonshot, and Ono Medical Research Foundation.

Prediction of metastatic pheochromocytoma and paraganglioma: a machine learning modelling study using data from a cross-sectional cohort.

BACKGROUND: Pheochromocytomas and paragangliomas have up to a 20% rate of metastatic disease that cannot be reliably predicted. This study prospectively assessed whether the dopamine metabolite, methoxytyramine, might predict metastatic disease, whether predictions might be improved using machine learning models that incorporate other features, and how machine learning-based predictions compare with predictions made by specialists in the field. METHODS: In this machine learning modelling study, we used cross-sectional cohort data from the PMT trial, based in Germany, Poland, and the Netherlands, to prospectively examine the utility of methoxytyramine to predict metastatic disease in 267 patients with pheochromocytoma or paraganglioma and positive biochemical test results at initial screening. Another retrospective dataset of 493 patients with these tumors enrolled under clinical protocols at National Institutes of Health (00-CH-0093) and the Netherlands (PRESCRIPT trial) was used to train and validate machine learning models according to selections of additional features. The best performing machine learning models were then externally validated using data for all patients in the PMT trial. For comparison, 12 specialists provided predictions of metastatic disease using data from the training and external validation datasets. FINDINGS: Prospective predictions indicated that plasma methoxytyramine could identify metastatic disease at sensitivities of 52% and specificities of 85%. The best performing machine learning model was based on an ensemble tree classifier algorithm that used nine features: plasma methoxytyramine, metanephrine, normetanephrine, age, sex, previous history of pheochromocytoma or paraganglioma, location and size of primary tumours, and presence of multifocal disease. This model had an area under the receiver operating characteristic curve of 0·942 (95% CI 0·894-0·969) that was larger (p<0·0001) than that of the best performing specialist before (0·815, 0·778-0·853) and after (0·812, 0·781-0·854) provision of SDHB variant data. Sensitivity for prediction of metastatic disease in the external validation cohort reached 83% at a specificity of 92%. INTERPRETATION: Although methoxytyramine has some utility for prediction of metastatic pheochromocytomas and paragangliomas, sensitivity is limited. Predictive value is considerably enhanced with machine learning models that incorporate our nine recommended features. Our final model provides a preoperative approach to predict metastases in patients with pheochromocytomas and paragangliomas, and thereby guide individualised patient management and follow-up. FUNDING: Deutsche Forschungsgemeinschaft.

Piceatannol Protects PC-12 Cells against Oxidative Damage and Mitochondrial Dysfunction by Inhibiting Autophagy via SIRT3 Pathway.

Oxidative stress has been identified as a major cause of cellular injury in a variety of neurodegenerative disorders. This study aimed to investigate the cytoprotective effects of piceatannol on hydrogen peroxide (H2O2)-induced pheochromocytoma-12 (PC-12) cell damage and explore the underlying mechanisms. Our findings indicated that piceatannol pre-treatment significantly attenuated H2O2-induced PC-12 cell death. Furthermore, piceatannol effectively improved mitochondrial content and mitochondrial function, including enhancing mitochondrial reactive oxygen species (ROS) elimination capacity and increasing mitochondrial transcription factor (TFAM), peroxisome-proliferator-activated receptor-γ coactivator-1α (PGC-1α) and mitochondria Complex IV expression. Meanwhile, piceatannol treatment inhibited mitochondria-mediated autophagy as demonstrated by restoring mitochondrial membrane potential, reducing autophagosome formation and light chain 3B II/I (LC3B II/I) and autophagy-related protein 5 (ATG5) expression level. The protein expression level of SIRT3 was significantly increased by piceatannol in a concentration-dependent manner. However, the cytoprotective effect of piceatannol was dramatically abolished by sirtuin 3 (SIRT3) inhibitor, 3-(1H-1,2,3-Triazol-4-yl) pyridine (3-TYP), which led to an exacerbated mitochondrial dysfunction and autophagy in PC-12 cells under oxidative stress. In addition, the autophagy activator (rapamycin) abrogated the protective effects of piceatannol on PC-12 cell death. These findings demonstrated that piceatannol could alleviate PC-12 cell oxidative damage and mitochondrial dysfunction by inhibiting autophagy via the SIRT3 pathway.

Low catestatin as a risk factor for cardiovascular disease - assessment in patients with adrenal incidentalomas.

Background: Catestatin (Cts) is a peptide derived from proteolytic cleavage of chromogranin A, which exhibits cardioprotective and anti-inflammatory properties. Cts has been proposed as a potential biomarker for cardiovascular (CV) disease. Objectives: examining Cts in patients with incidentally discovered adrenocortical adenomas (AI), and its associations with CV risk factors and blood pressure (BP). Materials and methods: In this cross-sectional study, 64 AI patients without overt CV disease other than primary hypertension were recruited along with 24 age-, sex-, and body-mass-index (BMI)-matched controls with normal adrenal morphology. Laboratory, 24-h ambulatory BP monitoring, echocardiography, and common carotid artery sonography examinations were performed. Results: Unadjusted Cts was higher in AI patients (median 6.5, interquartile range: 4.9-37 ng/ml) versus controls (4.5 (3.5 - 28)), p=0.048, however, the difference was insignificant after adjusting for confounding variables. Cts was lower in subjects with metabolic syndrome than in those without it (5.2 (3.9- 6.9) vs. 25.7 (5.8-115) ng/ml, p<0.01), and in men compared to women (4.9 (4-7.4) ng/ml vs. 7 (4.8-100), p=0.015). AI patients in the lower half of Cts levels compared to those in the upper had a higher prevalence of hypertension (OR 0.15, 95% CI: 0.041-0.5, p<0.001) and metabolic syndrome (OR 0.15, 95% CI 0.041-0.5, p<0.001). In AI patients Cts correlated positively with high-density lipoprotein cholesterol (Spearman's r=0.31), negatively with BMI (r=-0.31), and 10-year atherosclerotic CV disease risk (r=-0.42). Conclusions: Our data indicate associations between CV risk factors and Cts. More clinical research is needed to apply serum Cts as a biomarker.

Fucoidan and microtopography on polyvinyl alcohol hydrogels guided axons and enhanced neuritogenesis of pheochromocytoma 12 (PC12) cells.

Artificial nerve grafts that support axon growth hold promises in promoting nerve regeneration and function recovery. However, current artificial nerve grafts are insufficient to regenerate axons across long nerve gaps. Specific biochemical and biophysical cues are required to be incorporated to artificial nerve grafts to promote neural cell adhesion and guide neurite outgrowth. Polyvinyl alcohol (PVA) nerve conduits have been clinically approved, but the applicability of PVA nerve conduits is limited to short injuries due to low cell binding. In this study, we explored the incorporation of biochemical cues and topographical cues for promoting neuritogenesis and axon guidance. PVA was conjugated with extracellular matrix proteins and fucoidan, a bioactive sulfated polysaccharide, to improve cell adhesion. Micro-sized topographies, including 1.8 µm convex lenses, 2 µm gratings, and 10 µm gratings were successfully fabricated on PVA by nanofabrication, and the synergistic effects of topography and biochemical molecules on pheochromocytoma 12 (PC12) neuritogenesis and neurite alignment were studied. Conjugated fucoidan promoted the percentage of PC12 with neurite outgrowth from 0% to 2.8% and further increased to 5% by presenting laminin on the surface. Additionally, fucoidan was able to bind nerve growth factor (NGF) on the surface and allow for PC12 to extend neurites in NGF-free media. The incorporation of 2 µm gratings could double the percentage of PC12 with neurite outgrowth and neurite length, and guided the neurites to extend along the grating axis. The work presents a promising strategy to enhance neurite formation and axon guidance, presenting significant value in promoting nerve regeneration.

HIF and MYC signaling in adrenal neoplasms of the neural crest: implications for pediatrics.

Pediatric neural crest-derived adrenal neoplasms include neuroblastoma and pheochromocytoma. Both entities are associated with a high degree of clinical heterogeneity, varying from spontaneous regression to malignant disease with poor outcome. Increased expression and stabilization of HIF2α appears to contribute to a more aggressive and undifferentiated phenotype in both adrenal neoplasms, whereas MYCN amplification is a valuable prognostic marker in neuroblastoma. The present review focuses on HIF- and MYC signaling in both neoplasms and discusses the interaction of associated pathways during neural crest and adrenal development as well as potential consequences on tumorigenesis. Emerging single-cell methods together with epigenetic and transcriptomic analyses provide further insights into the importance of a tight regulation of HIF and MYC signaling pathways during adrenal development and tumorigenesis. In this context, increased attention to HIF-MYC/MAX interactions may also provide new therapeutic options for these pediatric adrenal neoplasms.

Effects of Curcumin on Axon Growth and Myelin Sheath Formation in an In Vitro Model.

Although the beneficial effects of curcumin, extracted from rhizomes of the ginger family genus Curcuma, on the repair and regeneration of nerves have been evaluated in vitro, there are few studies concerning its effects on axon myelination. Here, we used pheochromocytoma cells as an in vitro model of peripheral nerves. Pheochromocytoma cells were cultured alone or cocultured with Schwann cells and treated with increasing concentrations of curcumin. Cell growth was observed, and the expression levels of growth-associated protein 43 (GAP-43), microtubule-associated protein 2 (MAP-2), myelin basic protein (MBP), myelin protein zero (MPZ), Krox-20, and octamer binding factor 6 (Oct-6) were quantified. We found a significant increase in expression of all six proteins following curcumin treatment, with a corresponding increase in the levels of MBP, MPZ, Krox-20, and Oct-6 mRNA. Upregulation was greater with increasing curcumin concentration, showing a concentration-dependent effect. The results suggested that curcumin can promote the growth of axons by upregulating the expression of GAP-43 and MAP-2, stimulate synthesis and secretion of myelin-related proteins, and facilitate formation of the myelin sheath in axons by upregulating the expression of Krox-20 and Oct-6. Therefore, curcumin could be widely applied in future strategies for the treatment of nerve injuries.

Pathological Aspects and Immunohistochemical Evaluation of Troponin C in the Cardiovascular System of Dogs With Pheochromocytoma.

Functional pheochromocytomas secrete catecholamines and have been associated with cardiovascular lesions in dogs. This study aimed to describe the postmortem pathological findings in the cardiovascular system of dogs with pheochromocytoma and to evaluate the expression of cardiac troponin C in these dogs using immunohistochemical analysis. Twelve cases were identified, with a mean age of 10.6 years. The heart of all dogs was enlarged and with concentric hypertrophy of the left ventricular myocardium. Histological analysis showed cardiomyocyte necrosis and degeneration in the myocardium, with frequent bands of contraction, fibrosis, inflammation, and thickening of the medium-caliber arteries in the myocardium. There was a marked decrease or absence of immunolabeling in necrotic cardiomyocytes. We conclude that IHC for troponin C can be a useful tool for detecting myocardial necrosis in dogs with pheochromocytomas, including early cases of necrosis with only incipient cardiac changes where overt histologic abnormalities are not immediately apparent in the cardiomyocytes.

The Duration of Oxygen and Glucose Deprivation (OGD) Determines the Effects of Subsequent Reperfusion on Rat Pheochromocytoma (PC12) Cells and Primary Cortical Neurons.

Reperfusion is the fundamental treatment for ischaemic stroke; however, many ischaemic stroke patients cannot undergo reperfusion treatment. Furthermore, reperfusion can cause ischaemic reperfusion injuries. This study aimed to determine the effects of reperfusion in an in vitro ischaemic stroke model-oxygen and glucose deprivation (OGD) (0.3% O2)-with rat pheochromocytoma (PC12) cells and cortical neurons. In PC12 cells, OGD resulted in a time-dependent increase in cytotoxicity and apoptosis, and reduction in MTT activity from 2 h onwards. Reperfusion following shorter periods (4 and 6 h) of OGD recovered apoptotic PC12 cells, whereas after 12 h, OGD increased LDH release. In primary neurons, 6 h OGD led to significant increase in cytotoxicity, reduction in MTT activity and dendritic MAP2 staining. Reperfusion following 6 h OGD increased the cytotoxicity. HIF-1a was stabilised by 4 and 6 h OGD in PC12 cells and 2 h OGD onwards in primary neurons. A panel of hypoxic genes were upregulated by the OGD treatments depending on the duration. In conclusion, the duration of OGD determines the mitochondrial activity, cell viability, HIF-1a stabilization, and hypoxic gene expression in both cell types. Reperfusion following OGD of short duration is neuroprotective, whereas OGD of long duration is cytotoxic.

SDHB immunohistochemistry for prognosis of pheochromocytoma and paraganglioma: A retrospective and prospective analysis.

Introduction: Pheochromocytomas and paragangliomas (PCC/PGL) are rare neuroendocrine tumors and can secrete catecholamine. Previous studies have found that SDHB immunohistochemistry (IHC) can predict SDHB germline gene mutation, and SDHB mutation is closely associated with tumor progression and metastasis. This study aimed to clarify the potential effect of SDHB IHC as a predictive marker for tumor progression in PCC/PGL patients. Methods: We included PCC/PGL patients diagnosed in Ruijin Hospital, Shanghai Jiao Tong University School of Medicine from 2002 to 2014 for retrospective analysis and discovered that SDHB (-) staining patients had poorer prognoses. Then we examined SDHB protein expression by IHC on all tumors in the prospective series, which was composed of patients from 2015 to 2020 in our center. Results: In the retrospective series, the median follow-up was 167 months, and during follow-up, 14.4% (38/264) patients developed metastasis or recurrence, and 8.0% (22/274) patients died. Retrospective analysis revealed that 66.7% (6/9) of participants in the SDHB (-) group and 15.7% (40/255) of those in the SDHB (+) group developed progressive tumors (OR: 10.75, 95% CI: 2.72-52.60, P=0.001), and SDHB (-) was independently associated with poor outcomes after adjusting by other clinicopathological parameters (OR: 11.68, 95% CI: 2.58-64.45, P=0.002). SDHB (-) patients had shorter disease-free survival (DFS) and overall survival (OS) (P<0.001) and SDHB (-) was significantly associated with shorter median DFS (HR: 6.89, 95% CI: 2.41-19.70, P<0.001) in multivariate cox proportional hazard analysis. In the prospective series, the median follow-up was 28 months, 4.7% (10/213) patients developed metastasis or recurrence, and 0.5% (1/217) patient died. For the prospective analysis, 18.8% (3/16) of participants in the SDHB (-) group had progressive tumors compared with 3.6% (7/197) in the SDHB (+) group (RR: 5.28, 95% CI: 1.51-18.47, P=0.009), statistical significance remained (RR: 3.35, 95% CI: 1.20-9.38, P=0.021) after adjusting for other clinicopathological factors. Conclusions: Our findings demonstrated patients with SDHB (-) tumors had a higher possibility of poor outcomes, and SDHB IHC can be regarded as an independent biomarker of prognosis in PCC/PGL.

Metastatic Pheochromocytoma and Paraganglioma: Somatostatin Receptor 2 Expression, Genetics, and Therapeutic Responses.

CONTEXT: Pheochromocytomas and paragangliomas (PPGLs) with pathogenic mutations in the succinate dehydrogenase subunit B (SDHB) are associated with a high metastatic risk. Somatostatin receptor 2 (SSTR2)-dependent imaging is the most sensitive imaging modality for SDHB-related PPGLs, suggesting that SSTR2 expression is a significant cell surface therapeutic biomarker of such tumors. OBJECTIVE: Exploration of the relationship between SSTR2 immunoreactivity and SDHB immunoreactivity, mutational status, and clinical behavior of PPGLs. Evaluation of SSTR-based therapies in metastatic PPGLs. METHODS: Retrospective analysis of a multicenter cohort of PPGLs at 6 specialized Endocrine Tumor Centers in Germany, The Netherlands, and Switzerland. Patients with PPGLs participating in the ENSAT registry were included. Clinical data were extracted from medical records, and immunohistochemistry (IHC) for SDHB and SSTR2 was performed in patients with available tumor tissue. Immunoreactivity of SSTR2 was investigated using Volante scores. The main outcome measure was the association of SSTR2 IHC positivity with genetic and clinical-pathological features of PPGLs. RESULTS: Of 202 patients with PPGLs, 50% were SSTR2 positive. SSTR2 positivity was significantly associated with SDHB- and SDHx-related PPGLs, with the strongest SSTR2 staining intensity in SDHB-related PPGLs (P = .01). Moreover, SSTR2 expression was significantly associated with metastatic disease independent of SDHB/SDHx mutation status (P < .001). In metastatic PPGLs, the disease control rate with first-line SSTR-based radionuclide therapy was 67% (n = 22, n = 11 SDHx), and with first-line "cold" somatostatin analogs 100% (n = 6, n = 3 SDHx). CONCLUSION: SSTR2 expression was independently associated with SDHB/SDHx mutations and metastatic disease. We confirm a high disease control rate of somatostatin receptor-based therapies in metastatic PPGLs.