Littermate Controls Research Articles

The potassium channel subunit KV1.8 (Kcna10) is essential for the distinctive outwardly rectifying conductances of type I and II vestibular hair cells

In amniotes, head motions and tilt are detected by two types of vestibular hair cells (HCs) with strikingly different morphology and physiology. Mature type I HCs express a large and very unusual potassium conductance, gK,L, which activates negative to resting potential, confers very negative resting potentials and low input resistances, and enhances an unusual non-quantal transmission from type I cells onto their calyceal afferent terminals. Following clues pointing to KV1.8 (Kcna10) in the Shaker K channel family as a candidate gK,L subunit, we compared whole-cell voltage-dependent currents from utricular HCs of KV1.8-null mice and littermate controls. We found that KV1.8 is necessary not just for gK,L but also for fast-inactivating and delayed rectifier currents in type II HCs, which activate positive to resting potential. The distinct properties of the three KV1.8-dependent conductances may reflect different mixing with other KV subunits that are reported to be differentially expressed in type I and II HCs. In KV1.8-null HCs of both types, residual outwardly rectifying conductances include KV7 (Knq) channels. Current clamp records show that in both HC types, KV1.8-dependent conductances increase the speed and damping of voltage responses. Features that speed up vestibular receptor potentials and non-quantal afferent transmission may have helped stabilize locomotion as tetrapods moved from water to land.

The potassium channel subunit KV1.8 (Kcna10) is essential for the distinctive outwardly rectifying conductances of type I and II vestibular hair cells.

In amniotes, head motions and tilt are detected by two types of vestibular hair cells (HCs) with strikingly different morphology and physiology. Mature type I HCs express a large and very unusual potassium conductance, gK,L, which activates negative to resting potential, confers very negative resting potentials and low input resistances, and enhances an unusual non-quantal transmission from type I cells onto their calyceal afferent terminals. Following clues pointing to KV1.8 (Kcna10) in the Shaker K channel family as a candidate gK,L subunit, we compared whole-cell voltage-dependent currents from utricular HCs of KV1.8-null mice and littermate controls. We found that KV1.8 is necessary not just for gK,L but also for fast-inactivating and delayed rectifier currents in type II HCs, which activate positive to resting potential. The distinct properties of the three KV1.8-dependent conductances may reflect different mixing with other KV subunits that are reported to be differentially expressed in type I and II HCs. In KV1.8-null HCs of both types, residual outwardly rectifying conductances include KV7 (Knq) channels. Current clamp records show that in both HC types, KV1.8-dependent conductances increase the speed and damping of voltage responses. Features that speed up vestibular receptor potentials and non-quantal afferent transmission may have helped stabilize locomotion as tetrapods moved from water to land.

Complement factor B, not the membrane attack complex component C9, promotes neointima formation after arterial wire injury

Background and aimsVascular restenosis due to neointima hyperplasia limits the long-term patency of stented arteries, resulting in angioplasty failure. The complement system has been implicated in restenosis. This study aimed to investigate the role of complement factor B (fB), an essential component of the alternative pathway of complement activation, in neointima formation. MethodsAngioplasty wire injury was conducted using 12-week-old mice deficient in fB or C9 (the main component of the membrane attacking complex, C5b-9) and littermate controls and neointima formation were assessed. Vascular smooth muscle cell (SMC) and endothelial cell (EC) proliferation and migration were examined in vitro. ResultsfB was mainly detected in SMCs of stenotic arteries from humans and mice. Deletion of fB substantially reduced the neointima area and intima-to-media area ratio without affecting the media area at 28 days after injury. At 7 days after injury, fB deficiency decreased SMC proliferation, unaltering neointimal macrophage infiltration and EC reendothelialization. Vascular SMC-expressed fB, not the circulation-sourced fB, played an essential role in SMC proliferation and migration in vitro. fB deficient mice exhibited lower levels of the soluble form of C5b-9, however, deletion of C9 did not alter neointima formation after wire injury, consistent with the null impact of C9 deficiency on SMC proliferation in vitro. ConclusionfB promotes neointima formation following wire-induced artery injury independent of forming the membrane-attacking complex. This is attributable to fB-dependent SMC proliferation and migration without affecting EC function. Targeting fB might protect against restenosis after percutaneous coronary intervention.

Effect of metabolic disorders on reactive gliosis and glial scarring at the early subacute phase of stroke in a mouse model of diabetes and obesity

It is well recognized that type II Diabetes (T2D) and overweight/obesity are established risk factors for stroke, worsening also their consequences. However, the underlying mechanisms by which these disorders aggravate outcomes are not yet clear limiting the therapeutic opportunities. To fill this gap, we characterized, for the first time, the effects of T2D and obesity on the brain repair mechanisms occurring 7 days after stroke, notably glial scarring. In the present study, by performing a 30-minute middle cerebral artery occlusion (MCAO) on db/db (obese diabetics mice) and db/+ (controls) mice, we demonstrated that obese and diabetic mice displayed larger lesions (i.e. increased infarct volume, ischemic core, apoptotic cell number) and worsened neurological outcomes compared to their control littermates. We then investigated the formation of the glial scar in control and db/db mice 7 days post-stroke. Our observations argue in favor of a stronger and more persistent activation of astrocytes and microglia in db/db mice. Furthermore, an increased deposition of extracellular matrix (ECM) was observed in db/db vs control mice (i.e. chondroitin sulfate proteoglycan and collagen type IV). Consequently, we demonstrated for the first time that the db/db status is associated with increased astrocytic and microglial activation 7 days after stroke and resulted in higher deposition of ECM within the damaged area. Interestingly, the injury-induced neurogenesis appeared stronger in db/db as shown by the labeling of migrating neuroblast. This increase appeared correlated to the larger size of lesion. It nevertheless raises the question of the functional integration of the new neurons in db/db mice given the observed dense ECM, known to be repulsive for neuronal migration. Carefully limiting glial scar formation after stroke represents a promising area of research for reducing neuronal loss and limiting disability in diabetic/obese patients.

Assessing the Benefit of Dietary Choline Supplementation Throughout Adulthood in the Ts65Dn Mouse Model of Down Syndrome

Background/Objectives: Down syndrome (DS) is the most common cause of early-onset Alzheimer’s disease (AD). Dietary choline has been proposed as a modifiable factor to improve the cognitive and pathological outcomes of AD and DS, especially as many do not reach adequate daily intake levels of choline. While lower circulating choline levels correlate with worse pathological measures in AD patients, choline status and intake in DS is widely understudied. Perinatal choline supplementation (Ch+) in the Ts65Dn mouse model of DS protects offspring against AD-relevant pathology and improves cognition. Further, dietary Ch+ in adult AD models also ameliorates pathology and improves cognition. However, dietary Ch+ in adult Ts65Dn mice has not yet been explored; thus, this study aimed to supply Ch+ throughout adulthood to determine the effects on cognition and DS co-morbidities. Methods: We fed trisomic Ts65Dn mice and disomic littermate controls either a choline normal (ChN; 1.1 g/kg) or a Ch+ (5 g/kg) diet from 4.5 to 14 months of age. Results: We found that Ch+ in adulthood failed to improve genotype-specific deficits in spatial learning. However, in both genotypes of female mice, Ch+ significantly improved cognitive flexibility in a reverse place preference task in the IntelliCage behavioral phenotyping system. Further, Ch+ significantly reduced weight gain and peripheral inflammation in female mice of both genotypes, and significantly improved glucose metabolism in male mice of both genotypes. Conclusions: Our findings suggest that adulthood choline supplementation benefits behavioral and biological factors important for general well-being in DS and related to AD risk.

Longevity, enhanced memory, and altered density of dendritic spines in hippocampal CA3 and dentate gyrus after hemizygous deletion of Pde2a in mice.

Studies using acute or subchronic pharmacological inhibition of phosphodiesterase 2 A (PDE2A) have led to its proposal as a target for treatment of cognitive deficits associated with neuropsychiatric and neurodegenerative disease. However, the impact of continuous inhibition of PDE2A on memory is unknown. Moreover, the neuroanatomical regions mediating memory enhancement have not been categorically identified. To address these open questions, we studied knockout mice and hippocampus restricted manipulations. Pde2a heterozygous knockout mice are viable with no gross histological abnormalities. The mice exhibit enhanced spatial and object recognition memory that is independent of anxiolytic effects and is paralleled by increased density of dendritic mushroom and thin spines in hippocampal CA3 and dentate gyrus in adult mice. In CA1, subtle alterations in spine density were seen, while theta-burst LTP and paired-pulse facilitation were normal. Spatial memory enhancement persists in aged Pde2a heterozygous knockout mice, and to our surprise these mice live significantly longer than wild-type littermate controls. In summary, we provide evidence that life-long reduction of PDE2A expression promotes spine formation and maturation, exerts beneficial effects on memory, and increases lifespan.

Sensory neuron LKB1 mediates ovarian and reproductive function

Treatments for reproductive disorders in women consist of hormone replacement therapy, which have negative side effects that impact health, spurring the need to understand new mechanisms to employ new therapeutic strategies. Bidirectional communication between sensory neurons and the organs they innervate is an emerging area of interest in tissue physiology with a relevance in reproductive disorders. We hypothesized that the metabolic activity of sensory neurons has a profound effect on reproductive phenotypes. To investigate this phenomenon, we utilized a murine model with conditional deletion of liver kinase B1 (LKB1), a serine/threonine kinase that regulates cellular metabolism in sensory neurons (Nav1.8cre; LKB1fl/fl). LKB1 deletion in sensory neurons resulted in reduced ovarian innervation from dorsal root ganglia neurons and increased follicular turnover compared to littermate controls. Female mice with this LKB1 deletion had significantly more pups per litter compared to wild-type females. Interestingly, the LKB1 genotype of male breeders had no effect on fertility outcomes, thus indicating a female-specific role of sensory neuron metabolism in fertility. In summary, LKB1 expression in peripheral sensory neurons plays an important role in modulating fertility of female mice via ovarian sensory innervation.

Immune cell-enriched single-cell RNA sequencing unveils the interplay between infiltrated CD8+ T resident memory cells and choroid plexus epithelial cells in Alzheimer's disease

Alzheimer's disease (AD) is a progressive neurological disorder and the leading cause of dementia. Despite significant efforts, treatment strategies targeting amyloid-β have been less successful than anticipated. Recently, the role of neuroinflammation and adaptive immune response in AD pathogenesis has gained attention. Here, we performed immune cell-enriched single-cell RNA sequencing of brain parenchymal cells from 12-month-old 5xFAD, an AD mouse model. We analyzed 11,587 single cells and found distinct differences in T cell and choroid plexus cell populations between 5xFAD mouse and littermate control. Subsequent sub-clustering of T cells in the 5xFAD mouse revealed distinct subtypes, with CD8+ resident memory T cells (TRM) being the most prevalent T cell type. In addition, we observed an increase in T cell exhaustion markers, including Pdcd1, Ctla4, and Havcr2, with a particularly significant elevation of PD-1 and TIM-3 in CD8+ TRM in 5xFAD mouse. Furthermore, choroid plexus (ChP) epithelial cells showed altered gene expression patterns, with higher expression of MHC class I and Type I IFN-stimulated genes in 5xFAD mouse compared to the control mouse, suggesting an association with clonal expansion of AD-specific T cells in the brain. Through single-cell RNA sequencing (scRNA-seq) analysis, our study highlights the potential role of resident memory CD8+ T cell and their possible interactions with ChP epithelial cells. This study provides an exploration of the brain microenvironment landscape in AD, revealing critical insights into its underlying mechanisms.

Abstract C015: IL-27R signaling modulates protective versus pathogenic T cell responses in hepatocellular carcinoma

Abstract Liver cancer is the 3rd leading cause of cancer death. Hepatocellular carcinoma (HCC) is the major form of primary liver cancer, with a growing incidence rate estimated to surge by 50% in the coming twenty years. Although some success has been achieved with immune checkpoint blockade inhibitors, many HCC patients do not respond well to the treatments. Therefore, it is critical to better understand immune mechanisms regulating HCC to identify new biomarkers and develop novel immunotherapies for HCC patients. HCC is driven by chronic liver diseases and is frequently associated with chronic inflammation in the liver. Cytokines are small mediators of inflammation that play an essential role in inflammatory diseases and tumorigenesis. IL27 is a member of the IL6/IL12 superfamily with context-dependent roles in various inflammatory disorders and cancer. IL27 receptor (R) is expressed by most immune cells and several non-immune cells and plays pivotal roles in the regulation of immune responses. Recent work from our lab shows that IL27R signaling suppresses anti-cancer immune response in HCC, acting via the control of innate cytotoxic cells, and Il27ra -/- mice develop significantly less HCC. However, how IL27R signaling regulates T cell subsets in HCC in vivo remains elusive. Here, using diethylnitrosamine (DEN)-induced mouse model of HCC we found a reduction of regulatory T cells (Tregs) in tumor-bearing Il27ra-/-mice along with lowered HCC burden. ScRNA sequencing of CD45+ cells isolated from HCC tumors indicated a less proliferative phenotype of Tregs and more cytotoxic and less exhausted CD8 T cells in mice with Il27ra deficiency. Pathway analysis suggested that IL27R-deficient Tregs were more quiescent, as characterized by the downregulation of various metabolic pathways. Our newly generated Foxp3 cre Il27ra flox mice show a significant reduction of DEN-driven HCC accompanied by an increase of tumor-infiltrating activated CD8 T cells compared to IL27R sufficient controls. Treg-specific deletion of IL27R caused the upregulation of TCF1 in Tregs which has been recently demonstrated to control Treg immune-suppressive functions in colorectal cancer. Meanwhile, tumor-infiltrating effector CD8 T cells were increased in Foxp3 cre+ Il27ra flox mice relative to their Foxp3 cre- Il27ra flox littermate controls. Interestingly, CD8 T cell specific deletion of IL27R did not impact HCC growth in CD8 cre Il27ra flox mice. Overall, our data suggest that IL27R signaling suppresses anti-HCC immunity by enhancing the pro-tumorigenic Tregs and suppressing anti-tumor effector CD8 T cell functions. Citation Format: Zhengzheng Shi, Jiani Zhu, Aleksandra Mazitova, Anastasiia Marchenko, Sergei Grivennikov, Ekaterina Koltsova. IL-27R signaling modulates protective versus pathogenic T cell responses in hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C015.

VEGFC Overexpression in Kidney Progenitor Cells Is a Model of Renal Lymphangiectasia.

Lymphangiogenesis is believed to be a protective response in the setting of multiple forms of kidney injury and mitigates the progression of interstitial fibrosis. To augment this protective response, promoting kidney lymphangiogenesis is being investigated as a potential treatment to slow the progression of kidney disease. As injury-related lymphangiogenesis is driven by signaling from the receptor VEGFR3 (vascular endothelial growth factor receptor 3) in response to the cognate growth factor VEGF (vascular endothelial growth factor)-C released by tubular epithelial cells, this signaling pathway is a candidate for future kidney therapeutics. However, the consequences to kidney development and function to targeting this signaling pathway remain poorly defined. We generated a new mouse model expressing Vegfc under regulation of the nephron progenitor Six2Cre driver strain (Six2Vegfc). Mice underwent a detailed phenotypic evaluation. Whole kidneys were processed for histology and 3-dimensional imaging. Six2Vegfc mice had reduced body weight and kidney function compared with littermate controls. Six2Vegfc kidneys demonstrated large peripelvic fluid-filled lesions with distortion of the pelvicalcyceal system which progressed in severity with age. Three-dimensional imaging showed a 3-fold increase in total cortical vascular density. Histology confirmed a substantial increase in LYVE1+ (lymphatic vessel endothelial hyaluronan receptor-1)/PDPN+ (podoplanin)/VEGFR3+ lymphatic capillaries extending alongside EMCN+ (endomucin) peritubular capillaries. There was no change in EMCN+ peritubular capillary density. Kidney lymphatic density was robustly increased in the Six2Vegfc mice. There were no changes in peritubular blood capillary density despite these endothelial cells also expressing VEGFR3. The model resulted in malformation of the lymphatic hilar plexus, resulting in severe hydronephrosis that resembled a human condition termed renal lymphangiectasia. This study defines the vascular consequences of augmenting VEGFC signaling during kidney development and provides new insight into human renal lymphatic malformations.

Abstract 4117657: Targeting GPR39 for Hypoxia-induced Pulmonary Hypertension in Mice

Background: Primary pulmonary arterial hypertension (PAH) is a disease affecting young subjects. It has very poor prognosis and optimal treatment is not available. 15-hydroxyeicosatetraenoic acid (15-HETE), a potent vasoconstrictor, has been implicated in the pathogenesis of PAH. Elevated levels of 15-HETE have been shown to be associated with worse prognosis in patients with PAH. Oral ingestion of 15-HETE leads to development of PAH in rodents. We recently showed that 15-HETE is the endogenous agonist for the G-protein coupled receptor 39 (GPR39) present in vascular smooth muscle cells. Hypothesis: We, therefore, hypothesized that global deletion of GPR39 (knock-out [KO] mice) would protect from PAH by removing the receptor through which 15-HETE causes vasoconstriction. Methods: Accordingly, 13 wild-type (WT) and 16 KO mice were placed in a hypoxia chamber (10% O 2 ). Litter-mate controls (7 WT and 13 KO) were subjected to normoxia (room air, 21% O 2 ). At the end of 4 weeks heart rate as well as aortic and right ventricular (RV) pressures were measured (latter using micromanometer-tipped catheter), and the animals were then euthanized for measurement of RV wall thickness and RV size from which RV wall stress was calculated. Results: Heart rate and systolic blood pressure were not different between the 4 groups . WT hypoxic animals developed PAH with peak RV systolic pressures (RVSP) being significantly higher than normoxic WT and hypoxic WT and hypoxic KO mice. (Figure 1) Similar results were noted for RV end-diastolic pressure (Figure 2) and RV wall stress (Figure 3). Conclusions: By deleting GPR39, the target for 15-HETE, we were able to prevent hypoxia induced PAH and RV dysfunction. Pharmacological inhibition of GPR39 may open new frontiers in the treatment of PAH.

Abstract 4131128: Cardiomyocyte-specific Deletion of Creg1 Impairs Autophagy and Leads to Age-dependent Dilated Cardiomyopathy

CREG1 is a small glycoprotein predominantly localized in the endolysosomal system. In mouse heart, CREG1 expression increases with age. We previously observed cardiac hypertrophy and ventricular non-compaction in a small percentage of constitutive Creg1 knockout mice during the neonatal stage. To elucidate the physiological function of CREG1 in the heart, we generated cardiomyocyte-specific Creg1 knockout (cm Creg1 KO) and knock-in (cm CREG1 KI) mice by crossing Creg1 fl/fl and Rosa hCREG1/hCREG1 with Myh6-Cre mice, respectively. By 6 months of age, the cm Creg1 KO mice developed severe myocardial interstitial fibrosis. Echocardiography at 10 months revealed markedly dilated left ventricles and significantly reduced ejection fractions. All the cm Creg1 KO mice succumbed to heart failure between 10-12 months, while Creg1 fl/fl control littermates exhibited no obvious cardiac abnormalities. Necropsy of deceased cm Creg1 KO mice showed increased heart-to-body weight ratios, enlarged cardiac chambers, and left atrial thromboses. Electron microscopy revealed sparse and elongated myofibers, scant and bizarre-shaped mitochondria, empty cytoplasm, and increased interstitial collagen accumulation. The cm CREG1 KI mice exhibited better endurance to nutritional deprivation than Rosa hCREG1/hCREG1 littermate controls. In sharp contrast to cm Creg1 KO hearts, cm CREG1 KI and Rosa hCREG1/hCREG1 control hearts displayed very mild fibrosis at 8 months, suggesting that CREG1 overexpression may confer cardioprotective effects. In cultured cardiomyocytes, CREG1 colocalizes with the autophagosome marker LC3B following serum deprivation. Immunofluorescence microscopy demonstrated a marked reduction of LC3B puncta in cm Creg1 KO hearts. Analysis of autophagic flux using CAG-RFP-EGFP-LC3 transgenic mice and immunoblotting indicated that the absence of CREG1 impairs both autophagosome formation and degradation, whereas knock-in of CREG1 enhances these autophagic processes. These findings suggest that CREG1 promotes autophagy and protects the heart from age-dependent dilated cardiomyopathy.

Abstract 4134456: Hybrid Cardiac Targeting Peptide Ameliorates HFpEF Phenotype in Mice

Introduction: Heart failure with preserved ejection fraction (HFpEF) is a form of heart failure which is rapidly rising in incidence in the US today. Despite this, no therapies with mortality benefit exist for this syndrome. Previous work with our novel hybrid cardiac targeting peptide (hCTP), a cardiomyocyte-specific cell penetrating peptide, has shown multiple beneficial biological effects (improved calcium handling, anti-inflammatory properties) beyond its transduction abilities. Therefore, we hypothesized that treatment with hCTP in a mouse model of HFpEF would ameliorate the pathophysiology of this disease. Methods: To establish a HFpEF model, 6-week-old, C57BL/6NJ mice were placed on a high-fat diet (HFD), 60% kCal from lard, and water with 0.5 g/L of an iNOS inhibitor (N-Nitro-L-arginine methylester) to induce hypertension. After 5 weeks of the diet, HFD mice and their littermate controls underwent baseline exercise testing and echocardiograms. Exercise capacity was measured after 2 days of exercise training with 2 days of rest in between; mice were placed on a treadmill at a 10-degree angle and made to run at increasingly fast speeds (5-30 m/min) until exhaustion. Echocardiograms were performed under 1.5% inhalational isoflurane. After baseline testing, the mice were treated with vehicle, or either 2.5 or 5 mg/Kg hCTP, either 3 or 5 days a week (five groups, n = 3 per group). After four weeks of treatment, the mice were again subjected to the same battery of tests and euthanized. Data was compared using two way ANOVA with Sidak’s test to compare measurements pre- and post-treatment. Results: Treatment with hCTP at either concentration for five days/week led to an increase in running distance from 39.8 m to 116.3 m (p = 0.03, n = 6), compared to changes in control distance from 208.4 m to 157.8 m (p = 0.399). Running distance did not correlate with weight (R 2 < .001). Treatment with 5 mg/Kg hCTP for 5 days/week led to a normalization of the E/e’ ratio, from 43.3 to 26.9 (p = 0.004, n = 3) compared to a change in the control of 34.7 to 26.1 (p = 0.312). Ejection fractions remained consistent (>55%) across all groups. Conclusions: Our data suggests that hCTP appears to be a promising treatment in ameliorating some of the physiological effects in a murine model of HFpEF. Gene and protein expression studies in heart tissue are underway. Further work is warranted with a larger sample size.

Abstract 4120741: Estrogen Regulates Abdominal Aortic Aneurysm Tregs via CD45 + Fibroblast-derived IL-33

Background: Sexual dimorphisms manifest during the progression of abdominal aortic aneurysm (AAA). Regulatory T cells (Tregs) are a subset of T cells characterized by their immunosuppressive and tissue-repairing functions, therefore playing a protective role in AAA. Aims: We aim to elucidate the mechanism underlying sexual dimorphism in Tregs presented in AAA aortas, which is significant for unraveling the pathogenesis of AAA and developing gender-specific therapeutic approaches. Methods and Results: AAA mice were induced by receiving porcine pancreatic elastase. By flow cytometry, we observed that ovariectomy (OVX) increased the ratio of Tregs in female mice, while estrogen led to a decrease in aortic Tregs. Additionally, compared to littermate controls, estrogen receptor α (ERα) KO mice exhibited an increase in Treg ratio. Similar results were observed for the ST2 + Treg ratio. IL-33, as an alarming molecule, can promote the maintenance of ST2 + Tregs in tissues. Western blot revealed lower IL-33 expression levels in female mice than in male mice. By analyzing the IL-33 expression profiles of various cells in AAA tissue, we found that IL-33 mainly originates from fibroblasts. We found two subpopulations of fibroblasts, CD45 + and CD45 - fibroblasts. Immunofluorescence on the aortas verified the presence of cells co-expressing CD45 and PDGFRα in AAA fragments. Furthermore, IL-33 expression only showed gender differences in CD45 + fibroblasts. Adoptive transfer of CD45.1 + mouse bone marrow macrophages into CD45.2 + mice revealed that aortic CD45.1 + cells could regain the PDGFRα phenotype, with a lower proportion of female PDGFRα + cells in CD45.2 - CD45.1 + cells than in male mice. Lyz2(cre/+); Rosa26(tdtomato/+) mice showed the presence of lyz2-tdtomato + cells expressing PDGFRα in the aortic adventitia after AAA induction. This suggests that CD45 + fibroblasts are partially derived from bone marrow macrophages, which can migrate to aneurysms and acquire a PDGFRα phenotype. Conclusions: Female sex hormones negatively regulate CD45 + fibroblast formation and IL-33 secretion between female and male mice during AAA. This resulted in reduced Treg function in female mice compared to male mice.

TMOD-21. MOUSE MODEL TO CONDITIONALLY EXPRESS A DIFFUSE MIDLINE GLIOMA DERIVED MUTATION IN THE ONCOGENIC PHOSPHATASE PPM1D

Abstract Diffuse Midline Gliomas (DMGs) are incurable brain tumors of children and adults, and there is an urgent need for new therapeutic approaches. Their location within critical areas of the brain precludes complete surgical removal. While current treatment mainly relies on radiation therapy, its efficacy remains palliative. DMGs commonly harbor truncating mutations in the Mn2+/Mg2+-dependent protein phosphatase 1D (PPM1D), leading to a gain-of-function in PPM1D by enhancing protein stability and activity. Thus, investigating PPM1D protein expression and its regulatory mechanisms is crucial for understanding its oncogenic role and identifying therapeutic targets. To address this, we developed a Ppm1d-loxP-exon6-loxP-exon6-E518X-tag mouse allele (Ppm1d-FL). This allele allows conditional expression of DMG-derived truncating mutations from the endogenous Ppm1d locus when the loxP sites are recombined in the presence of Cre recombinase. Tosimulate primary gliomas, we utilized the RCAS/tv-a retrovirus system and Cre/loxP recombination platforms. Mouse pups were injected with RCAS retroviruses bearing oncogenic platelet-derived growth factor B, Cre recombinase, and luciferase reporter genes into the brainstem. We confirmed that Cre leads to expected recombination of Ppm1d-FL, leading to expression of DMG-derived truncated Ppm1d-E518X-Tag mRNA. We examined tumor-free survival data, which indicates accelerated tumor formation with Ppm1d-FL compared to littermate controls with wildtype Ppm1d. The tumors demonstrated expression of molecular biomarkers commonly found in DMGs. Single-cell RNA sequencing has identified pathways activated or inactivated upon Ppm1d mutation. These data shed light on the complex interplay between PPM1D mutations and glioma pathogenesis and may guide the design of therapies that target PPM1D molecular pathways.

CSIG-19. IDENTIFICATION AND TARGETING OF TUMOR DRIVER SCHWANN(NF2LOSS) CELLS IN SPORADIC AND NF2-RELATED VESTIBULAR SCHWANNOMAS

Abstract Vestibular schwannomas (VS) arise from myelinating Schwann cells of CN VIII and are associated with severe morbidity due to local tumor growth. Sporadic and neurofibromatosis type 2 (NF2) associated VS are both characterized by NF2 (merlin) loss but the mechanisms remain unexplored. Using a multiomic approach, we dissected the role of NF2 loss in VS tumor formation and proliferation. We obtained surgical samples from 15 patients undergoing surgery for VS (sporadic, n=4; NF2, n=9), and performed single-cell or single-nucleus RNA sequencing (scRNAseq, n=4; snRNAseq, n=15), transposase-accessible chromatin (snATAC-seq, n=9), whole exome (n=12), and bulk DNA methylation assays (n=15). Transcription factor binding was assayed with ChIPseq. We validated our key findings with immunoblot, ELISA, RNAscope (n=8), and multiplex immunocytochemistry (mIHC; n=8). NF2fl/fl;Periostin-Cre+mice versus their Cre- control littermates treated with novel inhibitors of TEAD auto-palmitoylation (VTs). We mapped the VS transciptome and identified canonical Schwann cells, macrophages, T-cells and endothelial cells. Using the cells that had Chr22 loss (second hit deletions at the NF2 locus), we generated a NF2loss gene signature. We then identified Schwann(NF2loss) cells in all tumors, and found these enriched for YAP1/TAZ-TEAD, NRG1, and HIF-1α signaling pathways. Schwann(NF2loss) cells showed increased TEAD1 expression and chromatin accessibility for TEAD family transcription factor motifs. TEAD1 binding was observed at promoters of genes including VEGFA, EGFR, with strong enrichment for mitogenic pathways. We also found that VEFGA expression was restricted to Schwann(NF2loss) cells, and confirmed this with RNAscope and mIHC. Schwann(NF2loss) cells were key regulators of macrophages (M2-subtype, tissue resident) through M-CSF/IL-34 signaling. NF2fl/fl;Periostin-Cre+ mice showed increased VEGFA expression, and that was reversible by TEAD inhibition in-vivo with VTs. We report that Schwann(NF2loss) cells are key tumor drivers in VS. These cells are highly proliferative (YAP/TAZ-TEAD axis), drive angiogenesis (VEGFA expression), and promote tumor mass accretion (macrophage influx via M-CSF/IL-34 pathway) that can all be targeted via TEAD inhibition.

Dendritic cell mineralocorticoid receptor controls blood pressure by regulating T helper 17 differentiation: role of the Plcβ1/4-Stat5-NF-κB pathway.

Dendritic cells (DCs) are closely related to blood pressure (BP) regulation. Mineralocorticoid receptor (MR) is an important drug target for antihypertensive treatment. However, the role of DC MR in the pathogenesis of hypertension has not been fully elucidated. This study aimed to determine the role of DC MR in BP regulation and to explore the mechanism. Renal biopsy and peripheral blood samples were collected from hypertensive patients (HTN) for immunostaining and flow cytometry. Dendritic cell MR knockout (DCMRKO) mice, DC MR overexpressing (DCMROV) mice, DCMROV/IL-17A knockout (DCMROV/IL-17AKO) mice and finerenone-treated C57BL/6 mice were infused with angiotensin II (Ang II) to establish hypertensive models. Western blotting, chromatin immunoprecipitation, co-immunoprecipitation, and in vivo DC depletion or adoptive transfer were used to delineate the functional importance of DC MR in hypertension development. Mineralocorticoid receptor antagonists (spironolactone and finerenone) suppressed DC aggregation and activation, as well as hypertension in HTN and mice. Compared with littermate control (LC) mice, dendritic cell MR knockout mice had strikingly decreased BPs and attenuated target organ damage after Ang II infusion. Flow cytometry showed that DC MR deficiency mitigated Ang II-induced DC activation and T helper 17 (Th17) cell differentiation. RNA sequencing revealed that MR-deficient DCs had elevated expression of Plcβ1 and Plcβ4, knockdown of which reversed the inhibitory effect of MR deficiency on DC activation and Th17 differentiation. Adoptive transfer of MR-deficient DCs protected Ang II-induced hypertension, whereas knockdown of Plcβ1/4 eliminated the protective effects. At the molecular level, MR negatively regulated Plcβ1/4, which recruited SHP-1 to inactivate of Stat5 activity, resulting in enhanced NF-κB activation and Th17 polarization. Furthermore, DCMROV mice manifested more elevated BPs and target organ damage than control mice after Ang II infusion, and these differences were abolished in DCMROV/IL-17AKO mice. Finally, MR antagonists decreased the aggregation of Th17 in HTN and mice. Dendritic cell MR plays important roles in the pathogenesis of hypertension by regulating Th17 through Plcβ1/4-Stat5-NF-κB signalling, and blockade of DC MR is beneficial for treating hypertension.

Reduced microvascular flow-mediated dilation in Syrian hamsters lacking d-sarcoglycan is caused by increased oxidative stress.

d-sarcoglycan mutation reduces mechanotransduction and induces dilated cardiomyopathy with aging. We hypothesized that in young hamsters with d-sarcoglycan mutation, which do not show cardiomyopathy, flow mechanotransduction might be affected in resistance arteries as the control of local blood flow. Flow-mediated-dilation (FMD) was measured in isolated mesenteric resistance arteries, using 3-months old hamsters carrying a mutation in the d-sarcoglycan gene (CH-147) and their control littermates. The FMD was significantly reduced in the CHF-147 group. Nevertheless, passive arterial diameter, vascular structure and endothelium-independent dilation to sodium nitroprusside were not modified. Contraction induced by KCl was not modified, whereas contraction due to phenylephrine was increased. The basal NO production and total eNOS expression levels were not altered. Nevertheless, eNOS phosphorylation, FAKs and RhoA expression were reduced in CH-147. In contrast, p47phox, COX2, iNOS and reactive oxygen species levels were higher in the endothelium of CHF-147 hamsters. Reducing ROS levels using the superoxide dismutase analog Tempol significantly restored the flow-mediated dilation (FMD) levels in CHF-147 hamsters. However, treatment with the COX-2 inhibitor NS-398 showed a non-significant improvement in FMD.

PHLDA1 protects intestinal barrier function via restricting intestinal epithelial cells apoptosis in inflammatory bowel disease

The current approach to treating inflammatory bowel disease (IBD) primarily focuses on managing inflammation rather than maintaining the integrity of the intestinal barrier. In our study, we sought to investigate the potential role of PHLDA1 in preserving intestinal barrier function as a promising strategy for treating IBD. We observed a significant decrease in PHLDA1 expression in intestinal epithelial cells (IECs) of both IBD patients and mice with chemically induced colitis. This deficiency of PHLDA1 led to increased apoptosis of IECs, resulting in a compromised epithelial barrier and the invasion of commensal bacteria into the mucosa. Consequently, this microbial invasion substantially exacerbated colonic inflammation in mice with the specific knockout of PHLDA1 in IECs (Phlda1IEC−KO) compared to their control littermates. Mechanistically, we found evidence of PHLDA1 interacting with MCL1 to protect against K48-linked polyubiquitylation at the K40 lysine residue, thus preventing ubiquitin-proteasome degradation through the MCL1 ubiquitin ligase E3 (Mule). We further confirmed that the PHLDA1-MCL1-Mule signaling pathway plays a critical role in the development of IBD. Notably, our study demonstrated that enhancing MCL1 levels or reducing Mule expression using adeno-associated virus (AAV) attenuated experimental colitis in Phlda1IEC−KO mice. Collectively, our findings emphasize the significance of PHLDA1 in the pathogenesis of IBD and propose that targeting the PHLDA1-MCL1-Mule signaling pathway could be a viable approach for combating IBD.

Single cell analysis of Idh mutant growth plates identifies cell populations responsible for longitudinal bone growth and enchondroma formation

Enchondromas are a common tumor in bone that can occur as multiple lesions in enchondromatosis, which is associated with deformity of the affected bone. These lesions harbor somatic mutations in IDH and driving expression of a mutant Idh1 in Col2 expressing cells in mice causes an enchondromatosis phenotype. Here we compared growth plates from E18.5 mice expressing a mutant Idh1 with control littermates using single cell RNA sequencing. Data from Col2 expressing cells were analysed using UMAP and RNA pseudo-time analyses. A unique cluster of cells was identified in the mutant growth plates that expressed genes known to be upregulated in enchondromas. There was also a cluster of cells that was underrepresented in the mutant growth plates that expressed genes known to be important in longitudinal bone growth. Immunofluorescence showed that the genes from the unique cluster identified in the mutant growth plates were expressed in multiple growth plate anatomic zones, and pseudo-time analysis also suggested these cells could arise from multiple growth plate chondrocyte subpopulations. This data supports the notion that a subpopulation of chondrocytes become enchondromas at the expense of contributing to longitudinal growth.