Wild-type Littermates Research Articles

Depletion of mitochondrial CypD in endothelial and smooth muscle cells attenuates vascular dysfunction and hypertension.

Hypertension is a major risk factor of cardiovascular disease affecting nearly half of adult population, but only 25% patients have their blood pressure under control. Hypertension is associated with mitochondrial dysfunction; however, its molecular mechanisms and causative role are still elusive. Understanding these mechanisms is important to develop new therapies. Cyclophilin D (CypD) promotes mitochondrial swelling and dysfunction. The objective of this study is to test if CypD depletion attenuates vascular dysfunction and hypertension. To test this hypothesis, we used endothelial-specific and smooth muscle-specific CypD knockout mice in angiotensin II model of vascular dysfunction and hypertension. Our results show that depletion of endothelial CypD prevents angiotensin II-induced impairment of endothelial-dependent vasorelaxation, preserves endothelial nitric oxide and mitochondrial respiration, reduces hypertension, vascular oxidative stress and vascular metabolic glycolytic-switch compared with wild-type littermates. Depletion of smooth muscle CypD slightly reduces angiotensin II-induced hypertension, partially attenuates reduction in vascular nitric oxide and vasorelaxation, abolishes vascular superoxide overproduction, diminishes angiotensin II-induced vascular glycolysis, hypertrophy and fibrosis. Our data showed an intriguing "metabolic" and "redox" crosstalk between endothelial and smooth muscle cells. Depletion of endothelial CypD reduces not only angiotensin II-induced endothelial glycolysis but also attenuates smooth muscle cell glycolytic switch. Interestingly, depletion of smooth muscle CypD was also not limited to the effect on smooth muscle glycolysis, but it also reduced endothelial cell glycolysis. Vascular oxidative stress was inhibited both in EcCypDKO and SmcCypDKO mice, therefore, cell-specific CypD depletion had a "global" antioxidant effect on the entire vasculature. Our results support a novel function of mitochondrial CypD in regulation of superoxide production and metabolism of vascular smooth muscle and endothelial cells which affect endothelial barrier and smooth muscle vascular functions. We suggest that blocking vascular CypD reduces vascular oxidative stress, improves vascular metabolism and vascular function which may be beneficial in cardiovascular disease.

The impact of circadian rhythm disruption on oxaliplatin tolerability and pharmacokinetics in Cry1-/-Cry2-/- mice under constant darkness.

Circadian rhythms, the 24-h oscillations of biological activities guided by the molecular clock, play a pivotal role in regulating various physiological processes in organisms. The intricate relationship between the loss of circadian rhythm and its influence on the tolerability and pharmacokinetic properties of anticancer drugs is poorly understood. In our study, we investigated the effects of oxaliplatin, a commonly used anticancer drug, on Cry1-/- and Cry2-/- mice (Cry DKO mice) under darkness conditions, where they exhibit free-running phenotype. We administered oxaliplatin at a dosage of 12mg/kg/day at two distinct circadian times, CT8 and CT16, under constant darkness conditions to Cry DKO mice and their wild type littermates. Our results revealed a striking disparity in oxaliplatin tolerance between Cry DKO mice and their wild-type counterparts. Oxaliplatin exhibited severe toxicity in Cry DKO mice at both CT8 and CT16, in contrast to the wild type mice. Pharmacokinetic analyses suggested that such toxicity was a result of high concentrations of oxaliplatin in the serum and liver of Cry DKO mice after repeated dose injections. To understand the molecular basis of such intolerance, we performed RNA-seq studies using mouse livers. Our findings from the RNA-seq analysis highlighted the substantial impact of circadian rhythm disruption on gene expression, particularly affecting genes involved in detoxification and xenobiotic metabolism, such as the Gstm gene family. This dysregulation in detoxification pathways in Cry DKO mice likely contributes to the increased toxicity of oxaliplatin. In conclusion, our study highlights the crucial role of an intact molecular clock in dictating the tolerability of oxaliplatin. These findings emphasize the necessity of considering circadian rhythms in the administration of anticancer drugs, providing valuable insights into optimizing treatment strategies for cancer patients.

Cardiac Health, Type I Collagen, and Aging in the oim/oim Mouse Model of Osteogenesis Imperfecta (OI) and a Cohort of Adults with OI.

Osteogenesis imperfecta (OI) is a heritable connective tissue disorder with marked skeletal fragility and increased recognition as a pleiotropic type I collagenopathy. The impact of OI-causing gene variants on cardiac health and lifespan is just beginning to be understood. To begin to investigate cardiac manifestations of OI-causing type I collagen variants, we utilized the osteogenesis imperfecta murine (oim/oim) model to examine survival with increased age, as well as cardiac function and collagen expression at 4 and 18 months of age. We determined male oim/oim mice had 50% decreased survival by 18 months of age compared to wildtype (Wt) littermates. Cardiac magnetic resonance imaging and echocardiography revealed 18-month-old male oim/oim mice had increased left ventricular end-diastolic and end-systolic volumes concomitant with decreased function, as well as the presence of aortic stenosis in a subset of 4- and 18- month-old male oim/oim mice compared to Wt littermates. Female oim/oim survival and cardiac function were equivalent to their Wt counterparts. Cardiac evaluations of an adult OI patient cohort corroborated increased incidences of valvular dysfunction in the OI patient population with much of the male cohort also presenting with altered left ventricular function. Little is known concerning the impact of OI causing variants on patient cardiac health and the influence of sex and age. Using an OI mouse model, we determined that 18-month-old male oim/oim mice have cardiac dysfunction with decreased lifespan, confirming the need for further investigations to understand pleiotropic extra-skeletal manifestations and disease progression in osteogenesis imperfecta.

Pulmonary vascular remodeling in Fra-2 transgenic mice is driven by type 2 inflammation and accompanied by pulmonary vascular hyperresponsiveness.

Lung vessel remodeling leads to increased pulmonary vascular resistance, causing pulmonary arterial hypertension (PAH), and consequently right ventricular hypertrophy and failure. In patients suffering from systemic sclerosis (SSc), PAH can occur and is a life-threatening complication. Dysregulation of immune processes plays a crucial role in pulmonary vascular remodeling, as has previously been shown in Fos-related antigen-2 (Fra-2) transgenic (TG) mice, a model of SSc-PAH. Here, we investigate whether vascular remodeling in the Fra-2 TG model is driven by type 2 inflammation and is associated with vascular hyperresponsiveness, an important feature of PAH pathobiology. Basal pulmonary arterial pressure and pulmonary vascular responsiveness to hypoxic ventilation and serotonin were increased in isolated, perfused and ventilated lungs of Fra-2 TG mice compared to wild-type (WT) littermates. Similarly, contractile responses of isolated intrapulmonary arteries were elevated in Fra-2 TG mice. We also observed increased expression of contractile genes in Fra-2 overexpressing human pulmonary artery smooth muscle cells (PASMCs) with elevated intracellular calcium levels after IL-13 stimulation. These findings were corroborated by transcriptomic data highlighting dysregulation of vascular smooth muscle cell contraction and type 2 inflammation in Fra-2 TG mice. In vivo, type 2-specific anti-inflammatory treatment with IL-13 neutralizing antibodies improved vascular remodeling in Fra-2 TG mice, similar to corticosteroid treatment with budesonide. Our results underscore the importance of type 2 inflammation and its potential therapeutic value in PAH-associated pulmonary vascular remodeling and hyperresponsiveness in SSc-PAH.

Phospholipase C β4 promotes RANKL-dependent osteoclastogenesis by interacting with MKK3 and p38 MAPK.

Phospholipase C β (PLCβ) is involved in diverse biological processes, including inflammatory responses and neurogenesis; however, its role in bone cell function is largely unknown. Among the PLCβ isoforms (β1-β4), we found that PLCβ4 was the most highly upregulated during osteoclastogenesis. Here we used global knockout and osteoclast lineage-specific PLCβ4 conditional knockout (LysM-PLCβ4-/-) mice as subjects and demonstrated that PLCβ4 is a crucial regulator of receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast differentiation. The deletion of PLCβ4, both globally and in the osteoclast lineage, resulted in a significant reduction in osteoclast formation and the downregulation of osteoclast marker genes. Notably, male LysM-PLCβ4-/- mice presented greater bone mass and fewer osteoclasts in vivo than their wild-type littermates, without altered osteoblast function. Mechanistically, we found that PLCβ4 forms a complex with p38 mitogen-activated protein kinase (MAPK) and MAPK kinase 3 (MKK3) in response to RANKL-induced osteoclast differentiation, thereby modulating p38 activation. An immunofluorescence assay further confirmed the colocalization of PLCβ4 with p38 after RANKL exposure. Moreover, p38 activation rescued impaired osteoclast formation and restored the reduction in p38 phosphorylation caused by PLCβ4 deficiency. Thus, our findings reveal that PLCβ4 controls osteoclastogenesis via the RANKL-dependent MKK3-p38 MAPK pathway and that PLCβ4 may be a potential therapeutic candidate for bone diseases such as osteoporosis.

Trans 10, cis 12-conjugated linoleic acid in low concentration reduces while in high concentration enhances adipocyte metabolism but effectively improves hepatic steatosis of obese mice.

Trans 10, cis 12-conjugated linoleic acid (t10c12-CLA)-producing mice were used to investigate the antiobesity of obese males. Compared to wild-type littermates, high concentration t10c12-CLA in biallelic Pai/Pai mice reduced fat by up-regulation lipid metabolism in white adipose tissue (WAT). In contrast, low concentration t10c12-CLA in monoallelic Pai/wt mice could not reduce fat for down-regulation lipid metabolism in WAT. Simultaneously, t10c12-CLA enhanced thermogenesis and beta-oxidation in brown adipose tissue, alleviated steatosis by declining lipid metabolism in the liver, and lowered circulating triglycerides. On the other hand, low concentration t10c12-CLA specifically resulted in decreased circulating fibroblast growth factor 21, elevated glucose and high-density lipoprotein, whereas high concentration t10c12-CLA specifically increased circulating and hepatic cholesterol levels via up-regulation of low-density lipoprotein receptor in the liver. In conclusion, high concentration t10c12-CLA enhances local lipid metabolism in WAT and leads to fat loss, whereas low concentration t10c12-CLA attenuates the enzymic activities in WAT and fails to reduce fat. T10c12-CLA can effectively and concentration independently improve steatosis by attenuating hepatic lipid metabolism. These results suggest that low concentration of t10c12-CLA is beneficial, but high concentration is unfavorable to obese male mammals.

Dose escalation pre-clinical trial of novel DOK7-AAV in mouse model of DOK7 congenital myasthenia

Abstract Congenital myasthenic syndromes are a group of inherited disorders characterised by defective neuromuscular transmission and fatigable muscle weakness. Causative mutations have been identified in over 30 genes, including DOK7, a gene encoding a post-synaptic protein crucial in the formation and stabilisation of the neuromuscular junction. Mutations in this gene is one of the leading three most prevalent causes of congenital myasthenia in diverse populations across the globe. The majority of DOK7 congenital myasthenic patients experience varying degrees of disability despite receiving optimised treatment (usually salbutamol), necessitating the development of improved therapeutic approaches. Here we executed a dose escalation pre-clinical trial using a DOK7 congenital myasthenic syndrome mouse model to assess the efficacy of AMP-101, an innovative recombinant adeno-associated viral gene replacement therapy. This mouse model harbours a duplication in the Dok7 gene which corresponds to the mutation most commonly found in DOK7 congenital myasthenia patients, c.1124-1127dupTGCC. The model has a much more severe phenotype than patients, and lives for only a few days. AMP-101 is based on AAVrh74 and contains human DOK7 cDNA under the control of a muscle-restricted promoter. Three doses of AMP-101 (2x1013vg/kg, 6x1013vg/kg or 1x1014 vg/kg) were administered intra-peritoneally at four days of age. We show that the two higher doses of 6x1013vg/kg and 1x1014 vg/kg generated enlarged neuromuscular junctions and rescued the very severe phenotype of the model. Treated mice became at least as strong as wild type littermates, as demonstrated by using an inverted screen hang test, a rotarod test, and a grip strength test. EMG showed that the treated model mice had decrement of compound muscle action potential on repetitive nerve stimulation, which indicates defective signalling at the neuromuscular junction. However, male models treated with 1x1014 vg/kg showed the least decrement which was not statistically different from wild type littermates. Western blot analysis demonstrated robust expression of DOK7 in the diaphragm and tibialis anterior muscles. This data shows that AMP-101 is an effective treatment in a mouse model for DOK7 congenital myasthenia, and suggests that AMP-101 is a promising candidate to move forward to clinic trials as a gene therapy for patients.

Overexpression of triadin in adipose tissue causes cardiac dysfunction

Abstract Introduction As a member of the protein complex that regulates the ryanodine receptor (RyR) , triadin plays a critical role in intracellular calcium homeostasis. Our previous results demonstrate that the NAD-dependent deacetylase sirtuin-1 (SIRT1) downregulates the expression of triadin isoform Trisk 51 in adipose tissue. A transgenic mouse model with adipose tissue selective overexpression of Trisk 51 (Adipo-TRDN) was established. Aim The present study is designed to investigate the role of SIRT1-triadin signalling in the development of dilated cardiomyopathy/heart failure with reduced ejection fraction. Methods Wild type (WT) littermates, Adipo-TRDN without or with human SIRT1 overexpressed in adipose tissue (Adipo-TRDN/SIRT1) were used for this study. All mice were provided with standard chow diet, normal drinking water and kept in a 12/12 light-dark cycle. Echocardiography was performed in 30 weeks old male mice using Visual Sonics Vevo 2100 Imaging System to measure systolic and diastolic function using B-mode, M-mode, Color Doppler and Tissue Doppler parameters. Results Systolic cardiac impairment was present in the Adipo-TRDN mice compared with their littermate controls. Left ventricular internal diameter during systole (LVID, s) was increased in Adipo-TRDN compared with WT littermates (3.0031 vs. 2.1878 mm, p-value: 0.0059). Changes in the Left ventricular anterior wall thickness (LVAW) during systole and diastole were found to be non-significant (1.6180 vs. 1.3530 mm, 1.1443 vs. 1.0455mm). Although fractional shortening (FS %) differences were non-significant (27.64% vs. 34.83%), there was a significant reduction in ejection fraction (EF %) in Adipo-TRDN mice compared with their WT controls (49.41% vs. 64.73%, p-value: 0.0472). No differences were found in the Left ventricular mass (LV) (106.80 vs.84.10 mg). Left ventricular volume during systole (LV Vol; s) was also significantly enhanced in Adipo-TRDN compared with their WT controls (35.25 vs.16.30 μL, p-value: 0.0047). Overexpression of human SIRT1 lead to partial improvement in the pathogenesis of dilated cardiomyopathy in Adipo-TRDN mice. Ejection fraction and fractional shortening were both increased in the Adipo-TRDN/SIRT1 compared with Adipo-TRDN mice (70.70% vs. 49.41%, p-value: 0.0017, 39.60% vs. 27.64% p-value: 0.0151). LV mass and LV Vol;s were reduced in the Adipo-TRDN/SIRT1 compared with Adipo-TRDN mice (75.83 vs. 106.80 mg, p-value: 0.0543) (14.02 vs. 35.25 μL, p-value: 0.0005). LVID (s), LVAW (d) and LVAW (s) were also reduced in Adipo-TRDN/SIRT1 compared with Adipo- TRDN mice (2.1641 vs. 3.0031mm, p-value: 0.0041, 0.8728 vs. 1.1443, p-value: 0.0319, 1.2369 vs. 1.6180mm, p-value: 0.0018). Conclusion Development of dilated cardiomyopathy induced by adipocyte specific overexpression of Triadin is partially prevented by adipose SIRT1.

Mutations that prevent phosphorylation of the BMP4 prodomain impair proteolytic maturation of homodimers leading to lethality in mice.

Bone morphogenetic protein4 (BMP4) plays numerous roles during embryogenesis and can signal either alone as a homodimer, or together with BMP7 as a more active heterodimer. BMPs are generated as inactive precursor proteins that dimerize and are cleaved to generate the bioactive ligand and inactive prodomain fragments. In humans, heterozygous mutations within the prodomain of BMP4 are associated with birth defects. We studied the effect of two of these mutations (p.S91C and p.E93G), which disrupt a conserved FAM20C phosphorylation motif, on ligand activity. We compared the activity of ligands generated from BMP4, BMP4S91C or BMP4E93G in Xenopus embryos and found that these mutations reduce the activity of BMP4 homodimers but not BMP4/7 heterodimers. We generated Bmp4S91C and Bmp4E93G knock-in mice and found that Bmp4S91C/S91C mice die by E11.5 and display reduced BMP activity in multiple tissues including the heart. Most Bmp4E93G/E93G mice die before weaning and Bmp4-/E93G mutants die prenatally with reduced or absent eyes, heart and ventral body wall closure defects. Mouse embryonic fibroblasts (MEFs) isolated from Bmp4S91C and Bmp4E93G embryos show accumulation of BMP4 precursor protein, reduced levels of cleaved BMP ligand and reduced BMP activity relative to MEFs from wild type littermates. Because Bmp7 is not expressed in MEFs, the accumulation of unprocessed BMP4 precursor protein in mice carrying these mutations most likely reflects an inability to cleave BMP4 homodimers, leading to reduced levels of ligand and BMP activity in vivo. Our results suggest that phosphorylation of the BMP4 prodomain is required for proteolytic activation of BMP4 homodimers, but not heterodimers.

Epigenetic modulation rescues neurodevelopmental deficits in Syngap1+/- mice.

SYNGAP1 is a Ras GTPase-activating protein that plays a crucial role during brain development and in synaptic plasticity. Sporadic heterozygous mutations in SYNGAP1 affect social and emotional behaviour observed in intellectual disability (ID) and autism spectrum disorder (ASD). Although neurophysiological deficits have been extensively studied, the epigenetic landscape of SYNGAP1 mutation-mediated intellectual disability is unexplored. Here, we have found that the p300/CBP specific acetylation marks of histones are significantly repressed in the hippocampus of adolescent Syngap1+/- mice. Additionally, we observed decreased dendritic branching of newly born DCX+ neurons in these mice, suggesting altered adult hippocampal neurogenesis. To establish the causal relationship of Syngap1+/- phenotype and the altered histone acetylation signature we have treated 2-4 months old Syngap1+/- mice with glucose-derived carbon nanosphere (CSP) conjugated potent small molecule activator (TTK21) of p300/CBP lysine acetyltransferase (CSP-TTK21). The enhancement of the p300/CBP specific acetylation marks of histones by CSP-TTK21 restored synaptic functions, increased dendritic branching of DCX+ neurons, enables the capability to reorganise cortical circuits in response to change in the sensory stimuli, and improves behavioural measures in Syngap1+/- mice that are very closely comparable to wild type littermates. Further, hippocampal RNA-Seq analysis of these mice revealed that the expression of many critical genes such as Adcy1, Ntrk3, Egr1, and Foxj1 which are key regulators of synaptic plasticity and neurogenesis and are well associated with ID/ASD reversed upon CSP-TTK21 treatment. This study could be the first demonstration of the reversal of autistic behaviour and neural wiring upon the modulation of altered epigenetic modification(s).

Alterations in non-REM sleep and EEG spectra precede REM-sleep deficits in a model of synucleinopathy

Background Sleep disturbances often precede motor symptoms in neurodegenerative diseases like Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Neuroinflammation is implicated in PD pathophysiology and may contribute to non-motor symptoms such as sleep disturbances. The Thy1-αSyn mouse model, overexpressing human alpha-synuclein (αSyn), mimics key aspects of PD and DLB, making it valuable for studying related sleep disturbances and neuroinflammatory changes. Objective To investigate early-stage alterations in sleep architecture, electroencephalographic (EEG) patterns, and neuroinflammation in Thy1-αSyn mice. Methods We used telemetric EEG/electromyography (EMG) with video surveillance to compare sleep patterns and EEG spectral power between 2.5- and 4.5-month-old male Thy1-αSyn transgenic mice and wild-type littermates. Neuroinflammation was assessed by examining microglial (Iba1) and astrocytic (GFAP) activation in key sleep-regulating brain regions. Results Thy1-αSyn mice showed decreased resting wake time and increased non-REM sleep, with altered sleep bout frequency and length, indicating significant sleep architecture changes. Spectral analysis revealed a shift from higher to lower frequency bands, suggesting early neural circuitry disruptions due to αSyn overexpression. Significant microglial activation was observed at 3 months, with astrogliosis progressing by 5 months in key sleep-regulating regions, indicating that neuroinflammation may contribute to the observed sleep disturbances. Conclusions Early-stage Thy1-αSyn mice exhibit significant sleep architecture changes, EEG spectral shifts, and neuroinflammatory alterations. These findings suggest that neuroinflammation may play a role in the initial pathophysiological changes in PD and related synucleinopathies. Sleep, EEG, and neuroinflammatory changes could serve as early biomarkers for these diseases.

Transcriptomic Profiling Reveals 17β-Estradiol Treatment Represses Ubiquitin-Proteasomal Mediators in Skeletal Muscle of Ovariectomized Mice.

With a decline of 17β-estradiol (E2) at menopause, E2 has been implicated in the accompanied loss of skeletal muscle mass and strength. We aimed at characterizing transcriptomic responses of skeletal muscle to E2 in female mice, testing the hypothesis that genes and pathways related to contraction and maintenance of mass are differentially expressed in ovariectomized mice with and without E2 treatment. Soleus and tibialis anterior (TA) muscles from C57BL/6 ovariectomized mice treated with placebo (OVX) or E2 (OVX + E2) for 60 days, or from skeletal muscle-specific ERα knockout (skmERαKO) mice and wild-type littermates (skmERαWT), were used for genome-wide expression profiling, quantitative real-time PCR and immunoblotting. Computational detection of estrogen response elements (EREs) was performed with EREFINDER. We found 155 significantly regulated probe sets in response to E2 (p ≤ 0.001). Pathway analyses identified proteasome and ubiquitin-mediated proteolysis as two downregulated pathways in the E2 group. We confirmed downregulation (p ≤ 0.05) in levels of Fbxw7, Psmb6, Ube2h and Ubxn1, as well as pro-apoptotic Bnip3 and inflammatory factor Nfkbia. Computational analysis identified ERE in the promoter regions of Psmb6, Ube2h, Bnip3 and Nfkbia. The overall content of ubiquitinated proteins was modestly but significantly lower in TA muscles from OVX + E2 vs. OVX mice (p = 0.039). There were no differences between skmERαKO and skmERαWT mice or between skmERαKO/OVX and skmERαKO/OVX + E2 mice for any genes assessed, indicating that ERα is required for E2 regulation of those genes. These results suggest that a mechanism whereby E2 protects against losses of skeletal muscle mass and strength is regulation of ubiquitin-proteasomal mediators.

The Impact of a Western Diet High in Phosphate on the CKD-MBD in an Alport Syndrome Model.

Chronic kidney disease - mineral bone disorder (CKD-MBD) is a syndrome that begins early in CKD, contributes to CKD-associated mortality, and includes components of FGF23 elevation, αklotho deficiency, CKD-stimulated vascular disease, and renal osteodystrophy. Hyperphosphatemia, occurring in later stages of CKD, is also driven by mechanisms of CKD-MBD, and has been shown to stimulate vascular calcification. In a mouse model of Alport CKD that is resistant to vascular calcification, we examine the effects of a high-phosphate Western-type diet on the CKD-MBD, and test whether the diet promotes induction of vascular calcification. An X-linked Col4a5 deficient murine homolog of Alport Syndrome (CKD) and wild type (WT) littermates were fed an animal protein 1.2% high phosphate diet or a standard vegetable protein diet. At disease progression equivalent to CKD stage 4-5, we examined kidney histology for fibrosis, blood for BUN (marker of CKD), and markers of CKD-MBD disease progression, kidney tissue for klotho production, and aorta histology and tissue mRNA and protein analysis for vascular calcification. The Western high Pi diet produced hyperphosphatemia in the CKD animals compared to WT and increased plasma PTH (1880 from 110 pg / ml), FGF23 c-term (670 from 120 pg / ml), and FGF23 intact (3780 from 280 pg / ml), and reduced kidney klotho mRNA and protein (57-67% reduction) (all p < 0.01). Referenced against the CKD animals fed vegetable-based diet, the Western high phosphate-fed CKD animals showed higher levels of plasma PTH and FGF23s. In the wild-type control mice with normal renal function, Western diet produced increased PTH, intact FGF23, and reduced renal klotho (all p <0.01). Vascular smooth muscle transdifferentiation and vascular calcification was not induced by Western high phosphate diet in this model of CKD. Our results show that a Western-style high-phosphate diet advances elements of the CKD-MBD. Renal klotho, FGF23 and PTH are affected by diet even with normal kidney function, suggesting a need for early intervention in the management of phosphate homeostasis as a component of CKD therapy. Additionally, CKD, klotho, and FGF23 all are associated with early aging. Therefore, our findings suggest that a Western high Pi diet accelerates aging and would contribute to the systemic complications of CKD - cardiac disease, osteodystrophy, and vascular disease.

Targeting hypoxia-induced signaling via HIF-2 inhibition, a promising treatment strategy for intestinal fibrosis?

Abstract Background and aims With this project, we aim to evaluate if targeting hypoxia-induced signalling via hypoxia inducible factor (HIF)-2 inhibition impacts experimental intestinal fibrosis and validate our findings in stenotic fibroblasts from CD patients. Methods Colonic fibroblasts will be sorted from mice with a fibroblast-specific deletion of the oxygen sensitive enzyme prolyl hydroxylase (PHD2) (i.e. PHD2ff- Col1a2-ERT2:Cre) and their wildtype littermate controls, followed by RNA sequencing and pathway analysis. Validation of key fibrotic mediators will be performed via qPCR or immunostainings. Intestinal fibrosis will be induced in PHD2ff Col1a2-ERT2:Cre mice wildtype littermate controls by chronic administration of dextran sodium sulphate in their drinking water, followed by treatment with the oral HIF-2 inhibitor, belzutifan. Translatability of preclinical findings will be confirmed in bio-banked fibrostenotic fibroblasts from CD patients. Anticipated impact Intestinal fibrosis is a debilitating complication of IBD which cannot be fully prevented by current anti-inflammatory therapies, highlighting the unmet clinical need for anti-fibrotic treatment strategies. Our preliminary data demonstrated that genetic deletion of Phd2 in fibroblasts aggravates experimental intestinal fibrosis without affecting inflammation and is most likely mediated via HIF-2. This suggests that signaling induced by this transcriptional modulator plays a crucial role in the fibrotic process. This proposal will identify the molecular mediators driving this fibrosis-promoting effect and validate if HIF-2 targeting could represent a promising novel anti-fibrotic treatment strategy. Data resulting from this project will therefore represent a final preclinical step and provide proof-of-concept data with the perspective to initiate a clinical trial in fibrostenotic IBD with belzutifan (awaiting EMA approval, but recently FDA-approved for advanced renal cell carcinoma).

SERT-Deficient Mice Fed Western Diet Reveal Altered Metabolic and Pro-Inflammatory Responses of the Liver: A Link to Abnormal Behaviors.

The inheritance of the short SLC6A4 allele, encoding the serotonin transporter (SERT) in humans, increases susceptibility to neuropsychiatric and metabolic disorders, with aging and female sex further exacerbating these conditions. Both central and peripheral mechanisms of the compromised serotonin (5-HT) system play crucial roles in this context. Previous studies on SERT-deficient (Sert-/-) mice, which model human SERT deficiency, have demonstrated emotional and metabolic disturbances, exacerbated by exposure to a high-fat Western diet (WD). Growing evidence suggests the significance of hepatic regulatory mechanisms in the neurobiology of central nervous system disorders, supporting the 'liver-brain' concept. However, the relationship between aberrant behavior and hepatic alterations under conditions of SERT deficiency remains poorly investigated. One-year-old female Sert-/- mice and their wild-type (WT) littermates were subjected to a control diet (CD) or the WD for a duration of three weeks. The WD had a higher caloric content and was characterized by an elevated saturated fat content (21%) compared to the CD (4.5%) and contained 0.2% cholesterol. Mice were evaluated for anxiety-like behavior, exploration and locomotor activity in the open field test, as well as glucose tolerance and histological indicators of hepatic steatosis. Hepatic pro-inflammatory and metabolism-related gene expression and markers of nitrosative stress, were analyzed utilizing real-time polymerase chain reaction (RT-PCR) and correlated with behavioral and histological outcomes. In comparison to unchallenged mice, Sert-/-/WD mutants, but not the WT/WD group, had increased locomotion and anxiety-like behavior, increased hepatic steatosis, and elevated expression of insulin receptor B and pro-inflammatory cytokines interleukin-1β (Il-1β) and Tnf, as well as decreased expression of leptin receptor B. The two genotypes displayed distinct gene expression patterns of nitric oxide (NO)-related molecules inducible NO synthase (iNos) and arginase (Arg2), insulin receptor-related signaling factors: cluster of differentiation 36 (Cd36), ecto-nucleotide pyrophosphatase/phosphodiesterase (Enpp), protein tyrosine phosphatase N1 (Ptpn1), cytochrome P450 omega-hydroxylase 4A14 (Cyp4a14), acyl-CoA synthetase 1 (Acsl1) and phosphatase and tensin homolog (Pten). Furthermore, there were profound differences in correlations between molecular, histological, and behavioral measurements across the two genotypes. Our findings suggest that the genetic deficiency of SERT results in abnormal hepatic pro-inflammatory and metabolic adaptations in response to WD. The significant correlations observed between behavioral measures and pro-inflammatory and metabolic alterations in WD-fed mice suggest the importance of liver-brain interactions and their role in the aberrant behaviors exhibited by Sert-/- mutants. This study presents the first evidence that altered liver functions are associated with pathological behaviors arising from genetic SERT deficiency.

The CC motif chemokine ligand 11 contributes to alcoholic liver disease.

Alcoholic liver disease (ALD) is characterized by aberrant lipid metabolism and chronic inflammation that eventually give rise to cirrhosis and hepatocellular carcinoma. In the present study we investigated the contribution of CC motif chemokine ligand 11 (CCL11) to the ALD pathogenesis. ALD was induced in mice by binge ethanol gavage or chronic ethanol feeding. Bioinformatic analysis of sequencing data indicated that CCL11 expression was up-regulated in hepatocytes from mice subjected to ethanol feeding compared to those from the control mice. Exposure to ethanol led to CCL11 up-regulated in primary murine hepatocytes in vitro. Consistently, Oil Red O (ORO) staining detected elevated lipid accumulation whereas quantitative PCR (qPCR) detected augmented expression of pro-inflammatory mediators in primary murine hepatocytes treated with recombinant CCL11. On the contrary, CCL11 knockout mice (KO) developed a less severe form of ALD compared to wild type littermates when subjected to either binge or chronic ethanol feeding. Finally, CCL11 antagonism by administration with an inhibitor to CCL11 receptor (CCR3i) attenuated ALD in mice. Our data support a role for CCL11 in ALD pathogenesis and provide proof-of-concept that targeting CCL11 can be considered as a therapeutic approach for ALD intervention.

Fibrillin-1 deficiency perturbs aortic cholinergic relaxation and adrenergic contraction in a mouse model of early onset progressively severe Marfan syndrome.

The pathogenic role of nitric oxide (NO) signaling during development of thoracic aortic aneurysm (TAA) in Marfan syndrome (MFS) is currently unclear. We characterized vasomotor function and its relationship to the activity of the NO-generating enzymes in mice with early onset progressively severe MFS. Wire myography, immunoblotting, measurements of aortic NO and superoxide levels were used to compare vasomotor function, contractile-protein levels, and the activity of endothelial and inducible NO synthase (eNOS and iNOS, respectively) in ascending thoracic aortas of Fbn1mgR/mgR mice relative to wild type (WT) littermates. Isometric force measurements of aortic rings from 16-day-old male Fbn1mgR/mgR mice revealed a significant reduction in acetylcholine (ACh)-induced relaxation and increased phenylephrine (PE)-promoted contractility, associated with abnormally low eNOSSer1177 phosphorylation, decreased NO production and augmented superoxide levels. Greater aortic contractility was associated with α1-adrenoceptor upregulation and normal levels of contractile proteins. While iNOS inhibition had no effect on vasomotor functions, mutant aortic rings pre-incubated with a non-specific NOS inhibitor yielded a greater PE response, implying a significant contribution of endothelial dysfunction to aortic hypercontractility. Impaired eNOS signaling disrupts aortic cholinergic relaxation and adrenergic contraction in MFS mice with dissecting TAA.

Bone cell differentiation and mineralization in wild-type and osteogenesis imperfecta zebrafish are compromised by per- and poly-fluoroalkyl substances (PFAS)

Perfluorinated compounds (PFAS) are well recognized toxic pollutants for humans, but if their effect is equally harmful for healthy and fragile people is unknown. Addressing this question represents a need for ensuring global health and wellbeing to all individuals in a world facing the progressive increase of aging and aging related diseases. This study aimed to evaluate the impact of perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA) and perfluorohexanoic acid (PFHxA) exposure on development and skeletal phenotype using the osteogenesis imperfecta (OI) zebrafish model Chihuahua (Chi/+), carrying a dominant glycine substitution in the α1 chain of collagen I and their wild-type (WT) littermates. To this purpose Chi/+ and WT zebrafish expressing the green fluorescent protein under the early osteoblast marker osterix were exposed from 1 to 6 days post fertilization to 0.36, 1.5 and 3.0 mg/L PFOS, 0.005 and 0.5 mg/L PFOA and 0.01, 0.48 and 16.0 mg/L PFHxA, and their development and skeletal phenotype investigated. Morphometric measurements, confocal microscopy evaluation of operculum area delimited by the fluorescent preosteoblasts and mineral deposition analysis following alizarin red staining were employed. PFOS and the highest concentration of PFHxA significantly impaired standard length in both genotypes. Osteoblast differentiation was significantly compromised by PFOS and by PFOA only in Chi/+. Limited to WT exposed to PFOA a reduced mineralization was also observed. No effect was detected after PFHxA exposure. Apoptosis was only activated by PFOA, specifically in Chi/+ mutant operculum osteoblasts. Interestingly, an altered lipid distribution in both WT and mutant fish was revealed after exposure to both pollutants. In conclusion, our data demonstrate that PFAS impair operculum development mainly compromising cell differentiation in mutant fish whereas alter lipid hepatic distribution in both genotypes with a more severe effect on Chi/+ preosteoblast survival. These results represent a first warning sign of the negative impact of PFAS exposure in presence of genetically determined skeletal fragility.

Weighted gene co-expression network analysis reveals the hub genes and molecular mechanism of quiescence.

The hematopoietic stem cell (HSC) continues their functional integrity and return to quiescence quickly even after inflammatory and other proliferative stress. The mechanism which is responsible for this highly regulatory process is not understood clearly. Previous results have shown that CD53 is noticeably upregulated in HSCs in response to a variety of stimuli. Gene expression profile using RNASeq data of HSCs from the bone marrow and spleen of CD53 knock out and their wild-type littermate had been deposited by Greenberg and co-authors, in GEO database, "GSE219050". They reported that knockout of CD53 promotes continued cell cycle. To identify key genes and specific processes are affected in absence of CD53, we applied weighted gene co-expression analysis. The results show that cyan module is correlated and dark red and light cyan are anti-correlated with CD53 loss. CDK1 is identified as more connected gene or hub gene in cyan module and it is upregulated in the absence of CD53. Likewise, hub genes from dark-red module are EP300, EGF, MCL1, LPL and IGF1R. The gene enrichment analysis depicts, two biological processes, MAPK cascade and Delta Notch signalling were suppressed. Similarly, the biological processes involved in light-cyan module are chromatin organisation and hub genes are Ehmt2, Ezh2, Kdm1a, Rbbp4, Esr1 and Mysm1. It uncovers the roles of CD53 in chromatin organisation, and MAPK cascade and Delta Notch signalling are the major contributors in quiescence mechanism. These findings might provide a new avenue in quiescence research.

The CD74 inhibitor DRhQ improves short-term memory and mitochondrial function in 5xFAD mouse model of Aβ accumulation.

Neuroinflammation and mitochondrial dysfunction are early events in Alzheimer's disease (AD) and contribute to neurodegeneration and cognitive impairment. Evidence suggests that the inflammatory axis mediated by macrophage migration inhibitory factor (MIF) binding to its receptor, CD74, plays an important role in many central nervous system (CNS) disorders such as AD. Our group has developed DRhQ, a novel CD74 binding construct which competitively inhibits MIF binding, blocks macrophage activation and migration into the CNS, enhances anti-inflammatory microglia cell numbers and reduces pro-inflammatory gene expression. Here, we evaluate its effects in amyloid-β (Aβ) overexpressing mice. 5xFAD mice and their wild type littermates were treated with DRhQ (100µg) or vehicle for 4 weeks. DRhQ improved cognition and cortical mitochondrial function in both male and female 5xFAD mice. Aβ plaque burden in 5xFAD animals was not robustly impacted by DRhQ treatment in either the hippocampus or the cortex. Cortical microglial activation was similarly not apparently affected by DRhQ treatment, although in the hippocampus there was evidence of a reduction in activated microglia for female 5xFAD mice. Future studies are needed to confirm this possible sex-dependent response on microglial activation, as well as to optimize the dose and timing of DRhQ treatment and gain a better understanding of its mechanism of action in AD.