Receptor Levels Research Articles

Abstract PO4-25-09: HER2-Low and Ultra-Low expression in young women with luminal breast cancer in México

Abstract Background and objective HER2-Low in breast cancer is a biomarker with new approaches for prognosis and medical treatment decisions. However, HER2-Ultra-Low is an area of novel research. Our objective was to describe the clinicopathologic characteristics and HER2 expression status of a group of Mexican young patients with luminal breast cancer. Methods One hundred and fifty-seven young patients (< 40 years) with luminal breast cancer diagnosed from 2014 to 2020 were included. Clinicopathologic features, hormone receptors, HER2 expression, and tumor infiltrating lymphocytes (TILs) were assessed. We categorized patients in three groups: HER2 negative (n=95, 60.5%), HER2-Low (n=33, 21.0%), and HER2-Ultra-Low (n=29, 18.5%). Descriptive statistics were used to report demographic, clinical and histopathological characteristics. Kaplan-Meier curves and log-rank or Mantel-Cox tests were used to determine survival. Statistical significance was established using a P value < 0.05. This analysis was performed using R (version 4.0.4) through R Studio interface (version 1.4.1106). Results The median of age at diagnosis was 36.1 years [range 32.8-38.8], with a median of follow-up of 5.08 years [range 3.58-6.52]. The most frequent histologic type was ductal infiltrating carcinoma (81%), and 49.4% presented grade two of differentiation. T2, N0 and clinical stage II were the most common (45.2%, 44.5, and 48.1%, respectively), and fourteen patients (9.0%) presented clinical stage IV. We found statistically significant differences in Ki67 and total TILs quantification between HER2 negative and HER2-Low. Progesterone receptor levels were higher for HER2 negative and Ultra-Low in comparison to HER2-Low. The total population had a 5-year overall survival of 84.9%. We found statistically significant differences in the median of survival between HER2 groups, with a shorter survival for HER2-Low. Conclusion To date, there are no studies that describe the clinical profile and OS of HER2-Low and Ultra-Low in young patients with luminal breast cancer in México. These preliminary findings could indicate the need for further investigation in prognosis, outcomes, and specific treatment in these patients. This study contributes to better understand the clinicopathologic profile of the HER2-Ultra-Low group and its differences with the HER2-Low group. Citation Format: Jesus Edgardo Hernandez Hernandez, César Octavio Lara Torres, Bryan Moreno Moran, Cynthia Villareal Garza, Fany Iris Porras Reyes, Víctor Manuel Pérez Sánchez, Alejandro Mohar. HER2-Low and Ultra-Low expression in young women with luminal breast cancer in México [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-25-09.

Single-nucleus RNA-seq identifies one galanin neuronal subtype in mouse preoptic hypothalamus activated during recovery from sleep deprivation

The preoptic area of the hypothalamus (POA) is essential for sleep regulation. However, the cellular makeup of the POA is heterogeneous, and the molecular identities of the sleep-promoting cells remain elusive. To address this question, this study compares mice during recovery sleep following sleep deprivation to mice allowed extended sleep. Single-nucleus RNA sequencing (single-nucleus RNA-seq) identifies one galanin inhibitory neuronal subtype that shows upregulation of rapid and delayed activity-regulated genes during recovery sleep. This cell type expresses higher levels of growth hormone receptor and lower levels of estrogen receptor compared to other galanin subtypes. single-nucleus RNA-seq also reveals cell-type-specific upregulation of purinergic receptor (P2ry14) and serotonin receptor (Htr2a) during recovery sleep in this neuronal subtype, suggesting possible mechanisms for sleep regulation. Studies with RNAscope validate the single-nucleus RNA-seq findings. Thus, the combined use of single-nucleus RNA-seq and activity-regulated genes identifies a neuronal subtype functionally involved in sleep regulation.

SiRNA Mediated Downregulation of RhoA Expression Reduces Oxidative Induced Apoptosis in Retinal Ganglion Cells.

Glaucoma is the second leading cause of blindness. Apoptosis of retinal ganglion cells (RGCs) is an important mechanism of glaucomatous optic injury. Rho kinase expression is significantly increased in apoptotic RGCs. This study aimed to investigate the role of RhoA, a Rho GTPase, on the survival of RGCs and further to explore its potential therapeutic applications. RGCs were treated with siRhoA for 24 hours in vitro. Knockdown of RhoA was confirmed with quantitative RT-PCR. Oxidative stress was induced by treating the RGCs with 200 μM of H2O2 for 1 hour, and apoptosis of RGCs was quantified with TUNEL assay in situ, and with flow cytometry. The mRNA expression levels of RhoA, Nogo receptor, caspase 3 and Bcl-2 were evaluated by quantitative RT-PCR, and the protein levels of RhoA, ROCK1, ROCK2, Nogo receptor, caspase 3 and Bcl-2 were evaluated by Western blot. We found siRhoA treatment efficiently downregulated the expression of RhoA in RGCs and protected against H2O2-induced injury in RGCs in vitro. Apoptosis of RGC cells under oxidative stress was quantified in situ using TUNEL assay and confirmed with flow cytometry (FCM). With the knockdown of RhoA, the expression of ROCK1, ROCK2, Nogo Receptor, Casepase-3 were decreased, while the expression of Bcl-2 was increased in both mRNA and protein level. Our data indicated that siRhoA prevented H2O2-induced apoptosis in RGC cells by modulating the RhoA/ROCK pathway. The results suggested that siRhoA may exert potentially effective neuroprotection for RGCs by reducing injury.

Sex Differences of the Endothelin System in the Cardio-Renal Axis of a Lupus Prone Mouse Model

Systemic Lupus Erythematosus (SLE) is the most common form of lupus autoimmunity. It is characterized by overactivation of toll-like receptor 7 (TLR7) and immune complex deposition in target organs including the kidney and vasculature. A lupus diagnosis disproportionately affects women at a rate of 9:1 compared to men. Clinically, patients present with heterogenous symptoms including chronic hypertension, heart failure, renal failure, vascular disease, and neuropathies. Despite a variety of clinical presentations of lupus, elevated endothelin-1 (ET-1) has been detected in both male and female lupus patients' plasma compared to healthy controls. However, the exact sex differences in ET-1 in the development of lupus and its associated cardiovascular complications remain unknown. Therefore, we hypothesized that acceleration of SLE through TLR7 activation promotes sex-dependent regulation of the ET system within the cardiorenal axis. To investigate this, female and male lupus-prone B6.Nba2 mice were treated with a TLR7/8 agonist (R848; 100ug/30ul) or acetone as a control topically twice a week for 4 weeks. After the R848 treatment, mice were followed for 12 more weeks until the 16-week terminal timepoint where the kidney and heart tissues were collected and tissue ET-1, endothelin receptor A (ETA) and endothelin receptor B (ETB) were analyzed by ELISA, RTqPCR, and immunoblotting. At the RNA level, cardiac Ednra expression trended towards upregulation in females, whereas males remained unchanged compared to their respective controls. At the protein level, both sexes tended to decrease ETA receptor levels. Renal Ednra expression trended towards downregulation in females but was unchanged in treated males, which was opposite of renal protein ETA where females were unchanged, and males trended towards reduction. Interestingly, Ednrb and Edn1 trended towards an increase in both the cardiac and renal system in both sexes. However, at the protein level only female cardiac ET-1 was significantly elevated (p<0.0001) while renal ET-1 levels were unchanged. Males demonstrated a significant increase in both cardiac (p=0.0004) and renal ET-1 (p=0.0127). Analysis of female cardiac ETB receptor levels yielded a significant reduction (p=0.0025) and an increasing trend in renal levels. Both cardiac and renal ETB was unchanged in males. The sex differences in the cardiorenal ET system regulation warrants further exploration and possible therapeutic treatments into cardiorenal lupus associated pathologies. Veterans Affairs Biomedical Laboratory Research & Development Career Development Award (CDA-2) 1IK2BX005605-01 to J.P.V.B, Medical University of South Carolina College of Medicine Program Project Grant to and J.P.V.B., and Medical University of South Carolina Core Centers for Clinical Research Project Award P30AR072582 to M.A.C and J.P.V.B. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Dynamic modulation of nodulation factor receptor levels by phosphorylation-mediated functional switch of a RING-type E3 ligase during legume nodulation

The precise control of receptor levels is crucial for initiating cellular signaling transduction in response to specific ligands; however, such mechanisms regulating nodulation factor (NF) receptor (NFR)-mediated perception of NFs to establish symbiosis remain unclear. In this study, we unveil the pivotal role of the NFR-interacting RING-type E3 ligase 1 (NIRE1) in regulating NFR1/NFR5 homeostasis to optimize rhizobial infection and nodule development in Lotus japonicus. We demonstrated that NIRE1 has a dual function in this regulatory process. It associates with both NFR1 and NFR5, facilitating their degradation through K48-linked polyubiquitination before rhizobial inoculation. However, following rhizobial inoculation, NFR1 phosphorylates NIRE1 at a conserved residue, Tyr-109, inducing a functional switch in NIRE1, which enables NIRE1 to mediate K63-linked polyubiquitination, thereby stabilizing NFR1/NFR5 in infected root cells. The introduction of phospho-dead NIRE1Y109F leads to delayed nodule development, underscoring the significance of phosphorylation at Tyr-109 in orchestrating symbiotic processes. Conversely, expression of the phospho-mimic NIRE1Y109E results in the formation of spontaneous nodules in L. japonicus, further emphasizing the critical role of the phosphorylation-dependent functional switch in NIRE1. In summary, these findings uncover a fine-tuned symbiotic mechanism that a single E3 ligase could undergo a phosphorylation-dependent functional switch to dynamically and precisely regulate NF receptor protein levels.

The added value of ultrasound imaging biomarkers to clinicopathological factors for the prediction of high-risk Oncotype DX recurrence scores in patients with breast cancer.

The Oncotype DX (ODX) recurrence score (RS), a 21-gene assay, has been proven to recognize patients at high risk of recurrence (RS ≥26) who would benefit from chemotherapy. However, it has limited availability and high costs. Our study thus aimed to identify ultrasound (US) imaging biomarkers and develop a prediction model for identifying patients with a high ODX RS. In this retrospective study, consecutive patients with T1-3N0-1M0 breast cancer who were hormone receptor positive and human epidermal growth factor receptor 2 (HER2) negative who had an available ODX RS were reviewed. Patients treated from May 2012 and December 2015 were placed into a training cohort, and those treated from January 2016 to January 2017 were placed in a validation cohort. Clinicopathologic data were collected, and preoperative US scans were analyzed. Univariable and multivariable regression analyses were performed to evaluate the independent predictors for a high-risk of breast cancer in the training cohort, and a nomogram was developed and evaluated with the area under the receiver operating characteristic curve (AUC), calibration curve, and decision curve analysis (DCA). A total of 363 patients were in the training cohort and 160 in the validation cohort, with the proportion with a high RS (RS 26-100) being 14% and 13.1%, respectively. Echogenic halo, enhanced posterior echo, low level of progesterone receptor (PR), and high Ki-67 index were identified as independent risk factors for high RS (all P values <0.05). The nomogram was constructed based on the combined model, which showed a better discrimination ability than did the clinicopathological model [combined model: AUC =0.95, 95% confidence interval (CI): 0.93-0.97; clinicopathological model: AUC =0.89, 95% CI: 0.86-0.92; P=0.001] and greater clinical benefit according to DCA. Furthermore, the nomogram was found to be effective in the validation cohort (AUC =0.90, 95% CI: 0.84-0.94), especially in patients with stage T1N0M0 disease (AUC =0.91, 95% CI: 0.84-0.95). US features may serve as valuable imaging biomarkers for the prediction of high recurrence risk in patients with T1-3N0-1M0 breast cancer and hormone receptor (HR)-positive and HER2-negative status. A nomogram incorporating PR status, Ki-67 index, and US imaging biomarkers showed a good discrimination ability in the early selection of patients at high risk of recurrence, especially in those with stage T1N0M0 disease.

Effect of Chloroquine on Type 2 Inflammatory Response in MC903-Induced Atopic Dermatitis Mice.

Atopic dermatitis (AD) is a chronic, non-infectious inflammatory dermatosis. Chloroquine (CQ) has long been proven to possess anti-inflammatory properties. This paper aims to investigate the impact of CQ on type 2 inflammatory response in MC903-induced AD mice. An AD mouse model was established via MC903 induction. After CQ treatment, AD mice were intraperitoneally injected with polyinosinic: polycyclic acid [poly (I:C)] or Nigericin. Dermatitis severity was scored, and the thickness of the left ear was measured. The pathological changes in mouse skin tissues were observed by H&E staining. The number of mast cells was counted via TB staining. The content of peripheral blood T-helper 2 (Th2) cells and levels of immunoglobulin E (IgE), thymic stromal-derived lymphopoietin (TSLP), interleukin (IL)-4, IL-13, interferon (IFN)-γ, IL-1β, and IL-18 were assessed by flow cytometry and ELISA. The levels of toll-like receptor 3 (TLR3), NLRP3, ASC, and cleaved caspase-1 proteins in skin tissues were determined by Western blot. CQ treatment abated dermatitis severity and left ear thickness in AD mice, alleviated skin damage, reduced mast cell number, diminished IgE, TSLP, IL-4, and IL-13 levels, and peripheral blood Th2 cell content, with no significant changes in IFN-γ level. CQ alleviated type 2 inflammatory response in AD mice by inhibiting the activation of TLR3. CQ suppressed NLRP3 inflammasome activation. Activating TLR3/NLRP3 annulled CQ-mediated alleviation on type 2 inflammatory response in AD mice. CQ alleviated type 2 inflammatory response in AD mice by inhibiting TLR3 activation and NLRP3 inflammasome activation.

Continuous TNF-α exposure in mammary epithelial cells promotes cancer phenotype acquisition via EGFR/TNFR2 activation.

Tumor necrosis factor alpha (TNF-α), an abundant inflammatory cytokine in the tumor microenvironment (TME), is linked to breast cancer growth and metastasis. In this study, we established MCF10A cell lines incubated with TNF-α to investigate the effects of continuous TNF-α exposure on the phenotypic change of normal mammary epithelial cells. The established MCF10A-LE cell line, through long-term exposure to TNF-α, displayed cancer-like features, including increased proliferation, migration, and sustained survival signaling even in the absence of TNF-α stimulation. Unlike the short-term exposed cell line MCF10A-SE, MCF10A-LE exhibited elevated levels of epidermal growth factor receptor (EGFR) and subsequent TNF receptor 2 (TNFR2), and silencing of EGFR or TNFR2 suppressed the cancer-like phenotype of MCF10A-LE. Notably, we demonstrated that the elevated levels of NAD(P)H oxidase 4 (NOX4) and the resulting increase in reactive oxygen species (ROS) were associated with EGFR/TNFR2 elevation in MCF10A-LE. Furthermore, mammosphere-forming capacity and the expression of cancer stem cell (CSC) markers increased in MCF10A-LE. Silencing of EGFR reversed these effects, indicating the acquisition of CSC-like properties via EGFR signaling. In conclusion, our results reveal that continuous TNF-α exposure activates the EGFR/TNFR2 signaling pathway via the NOX4/ROS axis, promoting neoplastic changes in mammary epithelial cells within the inflammatory TME.

Human Soluble Prorenin Receptor Expressed in Adipose Tissue Improves Insulin Sensitivity and Endothelial Function in Obese Female Mice

Increased circulating levels of the soluble prorenin receptor (sPRR), a component of the renin angiotensin system (RAS), plays a role in obesity, glucose, and insulin balance. Additionally, studies show that elevated plasma sPRR in diabetic patients is correlated with hyperglycemia in women but not men. Hence, the current study sought to understand the functional role of circulating human sPRR (HsPRR) produced by the adipose tissue (Adi) on adipogenesis and obesity, glucose homeostasis and insulin sensitivity. Adi-HsPRR mice were generated by breeding human sPRR-Myc-tag transgenic mice with mice expressing Adiponectin/Cre. The mouse model was validated by detecting myc-tagged HsPRR in western blot at 28kD. Adi-HsPRR male and female mice were kept on a low-fat (10% kcal from fat; n=4-9/group) or high-fat diet (60% kcal from fat; n=8-13/group) for 20 weeks. Body weight was recorded weekly and body composition (EchoMRI) was assessed monthly. Intraperitoneal glucose tolerance test (ipGTT) was performed at 12 weeks and insulin tolerance test (ITT) at 14 weeks. At 16 weeks, mice were implanted with radio telemeter to measure blood for 5 consecutive days. At the end of the study, tissues and plasma were collected for biochemical analysis and vascular studies. Adipose HsPRR expression did not change circulating sPRR between groups in mice fed a low-fat diet mouse (M: 3.5±0.5 vs 3.9±0.2 ng/ml, F: 2.3±0.2 vs 3.0±0.2 ng/ml) and high fat-fed females (4.3±0.3 vs 5.4±0.5 ng/ml) but increased plasma sPRR in HFD Adi-HsPRR male mice compared to HFD CTL (5.3±0.4 vs 8.7±0.7 ng/ml p&lt;0.05). Yet, Adi-HsPRR expression did not change body weight and composition, glucose, and insulin balance, nor blood pressure and vascular function in male mice regardless the diet. However, Adi-HsPRR improved insulin sensitivity (438±22 vs 360±14 AUC p&lt;0.05) and endothelium-dependent vascular relaxation (11.7±2.6 vs 19.4±2.5 % p&lt;0.05) in Adi-HsPRR female mice fed an HFD. Blood pressure was similar between groups in males and females on both LFD and HFD. Further analysis showed that Adi-HsPRR increased vasodilator agent, Ang 1-7 (5.1±1.6 vs 16.2±3.0 pmol/L p&lt;0.05) in HFD females which was unchanged in HFD males (10.2±2.4 vs 5.2±1.7 pmol/L). Moreover, Adi-HsPRR reduced gene expression of SREBP1C (1.3±0.2 vs 0.7±0.1 2−ΔΔCT p&lt;0.05) and CD36 (1.5±0.3 vs 0.6±0.1 2−ΔΔCT p&lt;0.05), in gonadal white adipose of HFD females but not in males. Additionally, PPAR gamma mRNA was reduced in HFD Adi-HsPRR females (1.1±0.1 vs 0.6±0.1 2−ΔΔCT p&lt;0.05). Our findings suggest that HsPRR in adipose tissue exerts sex-specific protective effects on insulin sensitivity and endothelial function during obesity, despite similar plasma sPRR levels between control and Adi-HsPRR mice. Thus, our model could be a useful tool to investigate potential antidiabetic effect of adipose sPRR in obese women. This work was supported by National Institutes of Health grants (R01-HL-142969 and R01-HL-1647 to ASL); the National Institute of General Medical Sciences (P30 GM127211); and the University of Kentucky, Center for Clinical and Translational Sciences (UL1TR001998). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Efficacy and safety of pyrotinib combined with albumin-bound paclitaxel as first-line treatment for HER2-positive metastatic breast cancer in patients previously treated with adjuvant and/or neoadjuvant trastuzumab therapy: The stage 1 results of a single-arm, phase 2 prospective clinical trial.

It has been observed that the prognosis of patients with HER2-positive metastatic breast cancer has improved significantly with HER2-targeted agents. However, there is still a lack of evidence regarding first-line anti-HER2 treatment options for patients who have received adjuvant and/or neoadjuvant trastuzumab for HER2-positive metastatic breast cancer. Besides, there are no reliable markers that can predict the efficacy of anti-HER2 treatment in these patients. Patients who have received adjuvant and/or neoadjuvant trastuzumab for HER2-positive metastatic breast cancer were enrolled. Pyrotinib plus albumin-bound paclitaxel were used as first-line treatment. The primary endpoint was the objective response rate (ORR). The safety profile was also assessed. In order to explore predictive biomarkers using Olink technology, blood samples were collected dynamically. From December 2019 to August 2023, the first stage of the study involved 27 eligible patients. It has not yet reached the median PFS despite the median follow-up being 17.8 months. Efficacy evaluation showed that the ORR was 92.6%, and the DCR was 100%. Adverse events of grade 3 or higher included diarrhoea (29.6%), leukopenia (11.1%), neutropenia (25.9%), oral mucositis (3.7%), and hand-foot syndrome (3.7%). Toll-like receptor 3 (TLR3) and Proto-oncogene tyrosine-protein kinase receptor (RET) were proteins with significant relevance to PFS in these patients. This study demonstrates that pyrotinib plus albumin-bound paclitaxel as a first-line treatment regimen shows good efficacy and manageable safety for patients who have received adjuvant and/or neoadjuvant trastuzumab for HER2-positive metastatic breast cancer. Besides, a significant association was identified between the expression levels of TLR3 and RET and the PFS in patients.

Identification of endophenotypes supporting outcome prediction in hemodialysis patients based on mechanistic markers of statin treatment

BackgroundStatins are widely used to reduce the risk of cardiovascular disease (CVD). Patients with end-stage renal disease (ESRD) on hemodialysis have significantly increased risk of developing CVD. Statin treatment in these patients however did not show a statistically significant benefit in large trials on a patient cohort level. MethodsWe generated gene expression profiles for statins to investigate the impact on cellular programs in human renal proximal tubular cells and mesangial cells in-vitro. We subsequently selected biomarkers from key statin-affected molecular pathways and assessed these biomarkers in plasma samples from the AURORA cohort, a double-blind, randomized, multi-center study of patients on hemodialysis or hemofiltration that have been treated with rosuvastatin. Patient clusters (phenotypes) were created based on the identified biomarkers using Latent Class Model clustering and the associations with outcome for the generated phenotypes were assessed using Cox proportional hazards regression models. The multivariable models were adjusted for clinical and biological covariates based on previously published data in AURORA. ResultsThe impact of statin treatment on mesangial cells was larger as compared with tubular cells with a large overlap of differentially expressed genes identified for atorvastatin and rosuvastatin indicating a predominant drug class effect. Affected molecular pathways included TGFB-, TNF-, and MAPK-signaling and focal adhesion among others. Four patient clusters were identified based on the baseline plasma concentrations of the eight biomarkers. Phenotype 1 was characterized by low to medium levels of the hepatocyte growth factor (HGF) and high levels of interleukin 6 (IL6) or matrix metalloproteinase 2 (MMP2) and it was significantly associated with outcome showing increased risk of developing major adverse cardiovascular events (MACE) or cardiovascular death. Phenotype 2 had high HGF but low Fas cell surface death receptor (FAS) levels and it was associated with significantly better outcome at 1 year. ConclusionsIn this translational study, we identified patient subgroups based on mechanistic markers of statin therapy that are associated with disease outcome in patients on hemodialysis.

Obstructive Sleep Apnea Syndrome and Obesity Indicators, Circulating Blood Lipid Levels, and Adipokines Levels: A Bidirectional Two-Sample Mendelian Randomization Study.

This investigation sought to elucidate the genetic underpinnings that connect obesity indicators, circulating blood lipid levels, adipokines levels and obstructive sleep apnea syndrome (OSAS), employing a bidirectional two-sample Mendelian randomization (MR) analysis that utilizes data derived from extensive genome-wide association studies (GWAS). We harnessed genetic datasets of OSAS available from the FinnGen consortium and summary data of four obesity indices (including neck circumference), seven blood lipid (including triglycerides) and eleven adipokines (including leptin) from the IEU OpenGWAS database. We primarily utilized inverse variance weighted (IVW), weighted median, and MR-Egger methods, alongside MR-PRESSO and Cochran's Q tests, to validate and assess the diversity and heterogeneity of our findings. After applying the Bonferroni correction, we identified significant correlations between OSAS and increased neck circumference (Odds Ratio [OR]: 3.472, 95% Confidence Interval [CI]: 1.954-6.169, P= 2.201E-05) and decreased high-density lipoprotein (HDL) cholesterol levels (OR: 0.904, 95% CI: 0.858-0.952, P= 1.251E-04). Concurrently, OSAS was linked to lower leptin levels (OR: 1.355, 95% CI: 1.069-1.718, P= 0.012) and leptin receptor levels (OR: 0.722, 95% CI: 0.530-0.996, P= 0.047). Sensitivity analyses revealed heterogeneity in HDL cholesterol and leptin indicators, but further multiplicative random effects IVW method analysis confirmed these correlations as significant (P< 0.05) without notable heterogeneity or horizontal pleiotropy in other instrumental variables. This investigation compellingly supports the hypothesis that OSAS could be a genetic predisposition for elevated neck circumference, dyslipidemia, and adipokine imbalance. These findings unveil potential genetic interactions between OSAS and metabolic syndrome, providing new pathways for research in this domain. Future investigations should aim to delineate the specific biological pathways by which OSAS impacts metabolic syndrome. Understanding these mechanisms is critical for developing targeted prevention and therapeutic strategies.

Mitochondrial involvement in the metabolic defects evoked by hepatocyte-specific BBSome deficiency

The BBSome, a protein complex of 8 Bardet-Biedl syndrome (BBS) proteins including BBS1, has emerged as an important regulator of metabolic function. BBSome deficient human and mice display several metabolic features including obesity and type 2 diabetes. Here, we show that mice lacking the BBSome specifically in liver which were generated by crossing the Bbs1fl/fl mice with liver-specific (albumin) AlbCre mice exhibit significantly higher lipid droplets in liver, by Oil Red-O staining, and in primary cultured hepatocytes (5.7%±0.4% vs 2.4%±0.6%, control), and by Transmission electron microscope (TEM). AlbCre/Bbs1flox also have elevated fasting glucose (169± 14 vs 136±8 mg/dL, p&lt;0.05) and impaired insulin sensitivity. In addition, insulin-induced activation of AKT was significantly decreased in the liver (2.6±0.07 vs 6.6± 0.34 AU, p&lt;0.05), but not in white adipose tissue and skeletal muscle, of AlbCre/Bbs1flox mice. Using immunohistochemistry, we found that insulin receptor level in plasma membrane was decreased by 24.2±6.8% in primary cultured hepatocytes derived from AlbCre/Bbs1flox mice which explains the insulin resistance. Our previous work implicating the BBSome in the regulation of mitochondria led us to hypothesize that mitochondria dysfunction contributes to the development of metabolic defects in AlbCre/Bbs1flox mice. TEM studies revealed that mitochondria were significantly larger and longer in hepatocytes of AlbCre/Bbs1flox mice than controls. This was associated with decreased levels of Dynamin-related protein 1 (DRP1), which critically regulates mitochondrial function through fission. Next, we asked whether the mitochondrial defects may explain the insulin receptor mistraffcking in BBSome deficient hepatocytes. To test this, hepatocytes were infected with feline immunodeficiency virus that express an active mutant form of DRP1 (FIV-DRP1S637A). Interestingly, insulin receptor localization in the plasma membrane was significantly higher in hepatocytes of Bbs1fl/fl/AlbCre­ mice infected with FIV-DRP1S637A pointing to mitochondrial defects as the underlying cause of insulin receptor mislocalization induced by hepatic BBSome deficiency. Taken together, these findings demonstrate that BBSome regulation of metabolic function involve mitochondria. NIH and VA. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sex-Specific Regulation of Local Gonadal Hormone Signaling During Pathological Cardiac Remodeling in Spontaneously Hypertensive Rats

Hypertensive heart disease is characterized by cardiac fibrosis in the left ventricle (LV). Angiotensin II (Ang II) promotes cardiac fibrosis through direct actions on cardiac fibroblasts. Gonadal hormones are known to impact cardiac fibrosis either through direct actions on fibroblasts or through modulation of Ang II receptors. We have previously shown that Ang II infusion alters collagen gene expression in a sex-specific manner. The present study seeks to further understand our observed sex difference by investigating the impact of Ang II on gonadal hormone regulation in the left ventricle (LV) and cardiac fibroblasts (CFs) following a fibrogenic stimulus. For whole LV analysis, male and female SHR (15-week-old) were infused with Ang II (400ng/kg/min, s.c.) or vehicle for 2 weeks via an osmotic mini pump (n=8-11 per group per sex). For CF-specific analysis, primary CFs were isolated from adult male and female SHR LV and cultured on CytoSoft cell culture plates (8 kPa stiffness) coated with Type I Collagen. CFs were brought to P1 for expansion and treated with TGFb1 (10ug/uL) or vehicle (PBS + 0.1% BSA) for 48 hours (n=7-8 per group per sex). Col1a1, AT1aR, estrogen receptors α and β (ER-α, ER-β), androgen receptor (AR), 5α-reductase (5α-R), and aromatase gene expression in the left ventricle was assessed via RT-qPCR and data analyzed by Two-Way ANOVA. Ang II infusion significantly increased ER-α gene expression in male, but not female LV (sex x Ang II interaction p=0.0303). ER-β gene expression was also significantly increased in the male LV (p=0.0066), but not females. AR gene expression remained unchanged in the Ang II-infused male LV but was significantly reduced in female LV when compared to control (sex x Ang II interaction p=0.0084). Ang II significantly increased 5α-R gene expression in male (p=0.0024), but not female LV. Aromatase gene expression tended to increase in both male and female Ang II-infused animals when compared to control. Ang II-induced changes in LV gonadal hormone gene expression did not significantly correlate with Col1a1 gene expression. In vitro stimulation of CFs with TGFb1 results in a significant reduction in ER-α (p&lt;0.0001) and AR (p&lt;0.0001) in both males and females. ER-β tended to decrease in males stimulated with TGFb1, but not females, relative to control. Taken together, the opposing findings between LV and CF may indicate cell-specific regulation of gonadal hormone receptors. It may be that upregulation of local estrogen signaling in the remodeling LV in males serves to offset the pathological effects of Ang II stimulation. Future studies will evaluate the consequences of changes in gonadal hormone receptor levels at the cellular and tissue level. NHLBI R01HL153112. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Iron deficiency and all-cause mortality after myocardial infarction

BackgroundData on the clinical significance of iron deficiency (ID) in patients with myocardial infarction (MI) are conflicting. This may be related to the use of various ID criteria.We aimed to compare the association of different ID criteria with all-cause mortality after MI. MethodsConsecutive patients hospitalized for their first MI at a large tertiary heart center were included. We evaluated the association of different iron metabolism parameters measured on the first day after hospital admission with all-cause mortality. ResultsFrom the 1,156 patients included (aged 64±12 years, 25 % women), 194 (16.8 %) patients died during the median follow-up of 3.4 years. After multivariate adjustment, iron level ≤13 µmol/L (HR 1.67, 95 % CI 1.19–2.34) and the combination of iron level ≤12.8 µmol/L and soluble transferrin receptor (sTfR) ≥3 mg/L (HR 2.56, 95 % CI 1.64–3.99) termed as PragueID criteria were associated with increased mortality risk and had additional predictive value to the GRACE score. Compared to the model including iron level, the addition of sTfR improved risk stratification (net reclassification improvement 0.61, 95 % CI 0.52–0.69) by reclassifying patients into a higher-risk group. No association between ferritin level and mortality was found. 51 % of patients had low iron levels, and 58 % fulfilled the PragueID criteria. ConclusionIron deficiency is common among patients with the first MI. The PragueID criteria based on iron and soluble transferrin receptor levels provide the best prediction of mortality and should be evaluated in future interventional studies for the identification of patients potentially benefiting from intravenous iron therapy.

Activation of M4 muscarinic receptors in the striatum reduces tic-like behaviours in two distinct murine models of Tourette syndrome.

Current pharmacotherapies for Tourette syndrome (TS) are often unsatisfactory and poorly tolerated, underscoring the need for novel treatments. Insufficient striatal acetylcholine has been suggested to contribute to tic ontogeny. Thus, we tested whether activating M1 and/or M4 receptors-the two most abundant muscarinic receptors in the striatum-reduced tic-related behaviours in mouse models of TS. Studies were conducted using CIN-d and D1CT-7 mice, two TS models characterized by early-life depletion of striatal cholinergic interneurons and cortical neuropotentiation, respectively. First, we tested the effects of systemic and intrastriatal xanomeline, a selective M1/M4 receptor agonist, on tic-like and other TS-related responses. Then, we examined whether xanomeline effects were reduced by either M1 or M4 antagonists or mimicked by the M1/M3 agonist cevimeline or the M4 positive allosteric modulator (PAM) VU0467154. Finally, we measured striatal levels of M1 and M4 receptors and assessed the impact of VU0461754 on the striatal expression of the neural marker activity c-Fos. Systemic and intrastriatal xanomeline reduced TS-related behaviours in CIN-d and D1CT-7 mice. Most effects were blocked by M4, but not M1, receptor antagonists. VU0467154, but not cevimeline, elicited xanomeline-like ameliorative effects in both models. M4, but not M1, receptors were down-regulated in the striatum of CIN-d mice. Additionally, VU0467154 reduced striatal c-Fos levels in these animals. Activation of striatal M4, but not M1, receptors reduced tic-like manifestations in mouse models, pointing to xanomeline and M4 PAMs as novel putative therapeutic strategies for TS.

The Invasion Factor ODZ1 Is Upregulated through an Epidermal Growth Factor Receptor-Induced Pathway in Primary Glioblastoma Cells.

We have previously shown that the transmembrane protein ODZ1 promotes cytoskeletal remodeling of glioblastoma (GBM) cells and invasion of the surrounding parenchyma through the activation of a RhoA-ROCK pathway. We also described that GBM cells can control the expression of ODZ1 through transcriptional mechanisms triggered by the binding of IL-6 to its receptor and a hypoxic environment. Epidermal growth factor (EGF) plays a key role in the invasive capacity of GBM. However, the molecular mechanisms that enable tumor cells to acquire the morphological changes to migrate out from the tumor core have not been fully characterized. Here, we show that EGF is able to induce the expression of ODZ1 in primary GBM cells. We analyzed the levels of the EGF receptor (EGFR) in 20 GBM primary cell lines and found expression in 19 of them by flow cytometry. We selected two cell lines that do or do not express the EGFR and found that EGFR-expressing cells responded to the EGF ligand by increasing ODZ1 at the mRNA and protein levels. Moreover, blockade of EGF-EGFR binding by Cetuximab, inhibition of the p38 MAPK pathway, or Additionally, the siRNA-mediated knockdown of MAPK11 (p38β MAPK) reduced the induction of ODZ1 in response to EGF. Overall, we show that EGF may activate an EGFR-mediated signaling pathway through p38β MAPK, to upregulate the invasion factor ODZ1, which may initiate morphological changes for tumor cells to invade the surrounding parenchyma. These data identify a new candidate of the EGF-EGFR pathway for novel therapeutic approaches.

Exploring the potential of pomegranate (Punica granatum) peel extract and punicalagin as novel anti-hypercholesterolemic agents

Background: Hypercholesterolemia, characterized by elevated low-density lipoprotein (LDL) cholesterol levels, is a significant risk factor for cardiovascular diseases. Familial and behavioral factors contribute to its onset, and while lifestyle changes offer some benefits, medical interventions are essential to manage this condition effectively. Statins, the primary medications used to lower LDL cholesterol, have limitations, including the unintended upregulation of proprotein convertase subtilisin/kexin type 9 (PCSK9). Therefore, exploring alternative approaches to enhance LDL receptor expression while targeting PCSK9 is crucial, with herbal medicine showing promise in this endeavor. Objective: This study investigates the potential of pomegranate peel extract and punicalagin, a key pomegranate component, in lowering LDL cholesterol in HepG2 cells. The research explores their effects on LDL receptor (LDLR) expression, LDL uptake, and proprotein convertase subtilisin/kexin type 9 (PCSK9) gene expression. Methods: HepG2 cells were treated with different concentrations of pomegranate peel extract (37.5, 75, 150, and 300 μg/ml) and punicalagin (12.5, 25, 50, and 100 μg/ml). Next, the viability of HepG2 cells, the protein levels of LDL receptors and LDL uptake, and the expression levels of PCSK9 were measured. Results: Pomegranate peel extract (37.5, 75, 150, and 300 μg/ml) and punicalagin (12.5, 25, 50, and 100 μg/ml) did not exhibit significant cytotoxicity and significantly increased LDLR protein levels (P-value= 0.00, after 24 hours) and LDL cholesterol uptake (P-value= 0.00, after 24 hours). Furthermore, they exhibited a dose-dependent reduction in the expression of PCSK9 (pomegranate: P-value= 0.01; punicalagin: P-value= 0.00). Conclusion: These findings suggest that pomegranate peel extract and punicalagin have the potential to serve as effective agents in managing hypercholesterolemia by enhancing LDL receptor expression and reducing PCSK9 expression in HepG2 cells. Further research is required to explore their effects on transcription factors and PCSK9 function. Keywords: Pomegranate; Punicalagin; HepG2 cell; LDL; Cardiovascular; Punica granatum

Peripheral-central network analysis of cancer cachexia status accompanied by the polarization of hypothalamic microglia with low expression of inhibitory immune checkpoint receptors

While the excessive inflammation in cancer cachexia is well-known to be induced by the overproduction of inflammatory mediators in the periphery, microflora disruption and brain dysfunction are also considered to contribute to the induction of cancer cachexia. Hypothalamic microglia play a crucial role in brain inflammation and central-peripheral immune circuits via the production of inflammatory mediators. In the present study, we evaluated possible changes in excessive secretion of gut microbiota-derived endotoxin and the expression timeline of several inflammation-regulatory mediators and their inhibiting modulators in hypothalamic microglia of a mouse model of cancer cachexia following transplantation of pancreatic cancer cells. We demonstrated that the plasma level of lipopolysaccharide (LPS) was significantly increased with an increase in anaerobic bacteria, especially Firmicutes, in the gut at the late stage of tumor-bearing mice that exhibited dramatic appetite loss, sarcopenia and severe peripheral immune suppression. At the early stage, in which tumor-bearing mice had not yet displayed “cachexia symptoms”, the mRNA expression of pro-inflammatory cytokines, but not of the neurodegenerative and severe inflammatory modulator lipocalin-2 (LCN2), was significantly increased, whereas at the late “cachexia stage”, the level of LCN2 mRNA was significantly increased along with significant decreases in levels of inhibitory immune checkpoint receptors programmed death receptor-1 (PD-1) and CD112R in hypothalamic microglia. In addition, a high density of activated neurons in the paraventricular nucleus (PVN) of the hypothalamus region and a significant increase in corticosterone secretion were found in cachexia model mice. Related to the cachexia state, released corticosterone was clearly increased in normal mice with specific activation of PVN neurons. A marked decrease in the natural killer cell population was also observed in the spleen of mice with robust activation of PVN neurons as well as mice with cancer cachexia. On the other hand, in vivo administration of LPS in normal mice induced hypothalamic microglia with low expression of inhibitory immune checkpoint receptors. These findings suggest that the induction of cancer cachexia may parallel exacerbation of the hypothalamic inflammatory status with polarization to microglia expressed with low levels of inhibitory immune checkpoint receptors following LPS release from the gut microflora.

Social-Single Prolonged Stress affects contextual fear conditioning in male and female Wistar rats: Molecular insights in the amygdala

Stress exposure can lead to post-traumatic stress disorder (PTSD) in male and female rats. Social-Single Prolonged Stress (SPS) protocol has been considered a potential PTSD model. This study aimed to pharmacologically validate the Social-SPS as a PTSD model in male and female rats. Male and female Wistar rats (60-day-old) were exposed to Social-SPS protocol and treated with fluoxetine (10 mg/Kg) or saline solution intraperitoneally 24 h before euthanasia. Two cohorts of animals were used; for cohort 1, male and female rats were still undisturbed until day 7 post-Social-SPS exposure, underwent locomotor and conditioned fear behaviors, and were euthanized on day 9. Animals of cohort 2 were subjected to the same protocol but were re-exposed to contextual fear behavior on day 14. Results showed that fluoxetine-treated rats gained less body weight than control and Social-SPS in both sexes. Social-SPS effectively increased the freezing time in male and female rats on day eight but not on day fourteen. Fluoxetine blocked the increase of freezing in male and female rats on day 8. Different mechanisms for fear behavior were observed in males, such as Social-SPS increased levels of glucocorticoid receptors and Beclin-1 in the amygdala. Social-SPS was shown to increase the levels of NMDA2A, GluR-1, PSD-95, and CAMKII in the amygdala of female rats. No alterations were observed in the amygdala of rats on day fourteen. The study revealed that Social-SPS is a potential PTSD protocol applicable to both male and female rats.