C57BL6 Mice Research Articles

SAT668 Can We Treat Osteoporosis, Obesity And Neurodegeneration With A Single FSH-blocking Drug?

Abstract Disclosure: A.R. Pallapati: None. J. Gimenez-Roig: None. S. Rojekar: None. F. Korkmaz: None. D. Sant: None. O. Barak: None. F. Sultana: None. O. Moldavski: None. A. Gumerova: None. H.S. Kannangara: None. U. Cheliadinova: None. C.J. Rosen: None. J.N. Caminis: None. M. Meseck: None. V.E. Demambro: None. S.L. Sims: None. S. Gera: None. R. Witztum: None. S. Miyashita: None. V. Ryu: None. M. Saxena: None. T. Frolinger: None. A. Macdonald: None. S. Kim: None. G. Pevnev: None. D. Lizneva: None. T. Yuen: None. M. Zaidi: None. Pharmacological and genetic studies suggest that FSH is an actionable target for diseases affecting millions, notably osteoporosis, obesity and Alzheimer’s disease (AD). Blocking FSH action prevents bone loss (1, 2), fat accrual (3) and AD-like features in mice (4). We recently developed a first-in-class, humanized, epitope-specific FSH blocking antibody that binds to a 13-amino-acid-long sequence of FSHβ—MS-Hu6—with a KD of 7.52 nM (5). We showed that MS-Hu6 binds specifically to FSHβ, without binding to LH and TSH. For efficacy studies, we have blocked FSH action using either MS-Hu6 or the parent murine antibody, Hf2, targeted to the same epitope. Using our Good Laboratory Practice platform (Code of Federal Regulations, Title 21, Part 58), we report that FSH blockade prevents obesity, osteoporosis and AD in mice. We injected 20-week-old C57BL/6 male mice on a high-fat diet with a range of doses of Hf2 or vehicle s.c. five-days-a-week for 8 weeks. Hf2 (100 µg/mouse/day) reduced the increase in fat mass by 33% starting week 3, with a 7% reduction in in body weight. In separate studies, MS-Hu6 not only caused beiging of white adipose tissue in UCP1-reporter ThermoMice (IVIS imaging), but also improved bone density and microstructure (micro-CT) by elevating bone formation (dynamic histomorphometry). The increase in bone mass and improved microstructure were replicated in Cliff Rosen’s lab using C57BL6 mice 24 weeks post-ovariectomy. Novel Object Recognition testing of AD-prone, ovariectomized 3xTg mice showed a deficit in recognition memory, which was reversed after 8 weeks of Hf2 (100 µg/mouse/day for 5-days-a-week) exposure. Biodistribution studies using 89Zr-labelled, biotinylated or unconjugated MS-Hu6 in mice and monkeys showed localization to bone, bone marrow, fat depots and brain tissue. MS-Hu6 displayed a β phase t½ of 7.5 days in humanized Tg32 mice. In monkeys, an acute single injection of MS-Hu6 did not affect vitals, and biochemical parameters remained within the normative range. We tested 215 variations of excipients using a range of physicochemical techniques, including protein thermal shift, size exclusion chromatography, dynamic light scattering, Fourier-transform infrared spectroscopy, circular dichroism spectroscopy, and differential scanning calorimetry, to yield a formulation with thermal, colloidal, monomeric and structural stability at an ultra-high concentration (100 mg/mL) with acceptable viscosity, clarity and turbidity parameters. MS-Hu6 showed the same “humanness” as human IgG1 in silico and was non-immunogenic in ELISPOT assays for IL-2 and IFNγ in human PBMC cultures. In conclusion, MS-Hu6 is efficacious, durable and manufacturable, and is therefore poised for human testing as a multipurpose therapeutic for obesity, osteoporosis, and perhaps for AD. 1Cell, 2006; 2PNAS, 2018,; 3Nature, 2017; 4Nature, 2022; 5. PNAS, 2020, eLife, 2022. Presentation: Saturday, June 17, 2023

SAT046 Canagliflozin Leads To Less Weight Gain, Confers Changes In The Gut Microbiota And Extends Life Span In Male C57BL6 Mice

Abstract Disclosure: E.E. Habeos: None. F. Filippopoulou: None. M. Kanakis: None. G.I. Habeos: None. N. Belmokhtar: None. P. Stathopoulou: None. G. Tsiamis: None. S. Taraviras: None. G. Lagoumintzis: None. D.V. Chartoumpekis: None. Introduction: SGLT2 inhibitors (SGLT2i) are widely prescribed drugs for the treatment of diabetes. Their beneficial health effects appear to be pleiotropic and are not limited to the amelioration of glycemic profile. The SGLT2i-induced glycosuria is suggested to provoke a metabolic shift that mimics the fasting response. Since calorie restriction has been shown to enhance longevity in mice, we hypothesized that treating mice with SGLT2i would also increase their life span. Methods: To this end, 4-months old male C57BL6 mice were fed with a standard chow diet (Control, n=83) or a diet supplemented with 200 mg/kg canagliflozin (Cana, n=92) and were observed up to the natural end of their life. Results: The canagliflozin-treated mice gained less weight over time. For instance, at the age of 10 months, the body weight of control mice was higher (means ± SEM: 33.1 ± 0.26 g) than the body weight of their canagliflozin-treated counterparts (means ± SEM: 31.4± 0.21 g; p<0.01). The development and severity of age-related cataracts were also examined at the age of 14 months, but no statistically significant difference was found in the severity score between the 2 groups. The survival curves of the control and the Cana mice diverged significantly, with the median survival of the control being 107.5 weeks versus 112.5 weeks of the Cana group (p=0.011). As the gut microbiome is known to change with age and be associated with the development of age-related diseases, feces were collected from 17-month-old mice (n=10 per group) and analysis of fecal bacterial 16S rRNA sequences was performed. Differences in beta diversity (diversity of bacterial species across samples) and relative abundance of bacterial species were noted between the Control and the Cana groups. Conclusions-Discussion: Our study highlights the effect of SGLT2i on longevity in a widely used mouse model (C57BL6). The underlying mechanisms leading to the enhancement of longevity are not clear yet. Some microbiota changes associated with the SGLT2i treatment, as described in other mouse models with enhanced longevity, warrant further investigation and analysis. It would be useful to extend these studies employing various SGLT2i with different potencies and selectivity against SGLT so as to determine if the observed effect on longevity is independent of the type of the drug. Presentation: Saturday, June 17, 2023

The inhibitory effect of licorice on the hepatotoxicity induced by the metabolic activation of Euodiae Fructus

Ethnopharmacological relevanceEuodiae Fructus (EF), the dried, unripe, scented fruit of Tetradium ruticarpum (A. Juss, T.G.Hartley), is a traditional food and herb with mild toxicity. In Asia, it is processed with licorice (EFP), which has been used for centuries to alleviate pain and suppress cough. Pharmacological studies have reported that this herb could cause liver injury by activating the P450 3A enzyme, thus carrying the risk of clinical application. Processing with licorice is an effective method to reduce EF toxicity. It is urgent to explore the toxic components of EF and the attenuation mechanism of licorice. Aim of the studyThis study aimed to indicate the specific pathway of EF-induced damage and identify the mechanism of action of licorice in reducing P450 activation and resulting in reduced liver damage. Materials and methodsMale C57BL-6 mice were used to investigate the toxicity of EF to the liver and determine the attenuation effect on P450 from licorice ingestion. Glutathione (GSH) was used to capture the metabolic activation intermediates of EF. The key component reducing the EF toxicity of licorice was investigated by comparing the differences in chemical components and inhibition on the EF metabolism of licorice from different habitats. ResultsThe intermediate product of evodiamine (EVO) in EF was found to be activated by the P450 enzyme during metabolism, causing liver injury and inflammation. Isoliquiritigenin and liquiritigenin in licorice produced by intestinal bacterial metabolism and glycyrrhizin inhibited the metabolic activation of EF. Glycosides in licorice are metabolized into aglycones by intestinal bacteria, inhibiting the metabolic activation of EF and alleviating hepatotoxicity. ConclusionsBy combining with GSH, the electrophilic intermediates produced by the P450 enzyme's metabolic activation of the indole ring of EVO might cause hepatotoxicity. Glycyrrhizin from licorice and the liquiritigenin and isoliquiritigenin generated by intestinal bacterial metabolism play an attenuated function by inhibiting the P450 enzyme and preventing the metabolic activation of EF.

Tanshinone IIA Alleviates Atherosclerosis Through Inhibition of NF-κB and PPARα/ABCA1 Signaling Pathways

This study evaluated the role and underlying mechanisms of Tanshinone IIA (Tan IIA) in atherosclerosis. C57BL mice (control group) and ApoE mice (model group) were administered a conventional and high-fat diet for 20 weeks. The Tan IIA group was obtained by administering a high-fat diet plus 8 weeks of Tan IIA to other mice for 20 weeks, followed by oil red O staining and lipid examination. RAW264.7 cells were transfected with PPARα siRNA+Tan IIA to measure their expression. The results showed little change in body weight between the three groups (P < 0.05). Liver index was significantly increased in the model and Tan IIA groups (P <0.05). Atherosclerotic plaques, plaque cross-sectional area, human oxidized low-density lipoprotein (ox-LDL), low-density lipoprotein cholesterol (LDL-C) and triglyceride (TG) levels, p-NF-κB, p-IKKα, P-Ikkα/β, TNF-α and IL-1β levels were significantly increased in the model group and decreased in the Tan IIA group (P < 0.05). We also noted a decrease in PPARα, PGC-1α and ABCA1 in the model group and an increase in the Tan IIA group. NF-κB expression was increased in the nucleus and decreased in the cytoplasm in the model group, which was reversed by Tan IIA treatment. Tan IIA significantly reduced ox-LDL, LDL-C and TG levels, plaque size and plaque cross-sectional area in atherosclerosis. Tan IIA effectively inhibited NF-κB, activated the PPARα/ABCA1 signalling pathway, and reduce inflammatory pathways, thereby improving lipid deposition and acting as an anti-atherosclerotic agent.

Combination of sacubitril/valsartan and blockade of the PI3K pathway enhanced kidney protection in a mouse model of cardiorenal syndrome.

Angiotensin receptor-neprilysin inhibitor (ARNI) is an established treatment for heart failure. However, whether ARNI has renoprotective effects beyond renin-angiotensin system inhibitors alone in cardiorenal syndrome (CRS) has not been fully elucidated. Here, we examined the effects of ARNI on the heart and kidneys of CRS model mice with overt albuminuria and identified the mechanisms underlying ARNI-induced kidney protection. C57BL6 mice were subjected to chronic angiotensin II infusion, nephrectomy, and salt loading (ANS); they developed CRS phenotypes and were divided into the vehicle treatment (ANS-vehicle), sacubitril/valsartan treatment (ANS-ARNI), and two different doses of valsartan treatment (ANS-VAL M, ANS-VAL H) groups. Four weeks after treatment, the hearts and kidneys of each group were evaluated. The ANS-vehicle group showed cardiac fibrosis, cardiac dysfunction, overt albuminuria, and kidney fibrosis. The ANS-ARNI group showed a reduction in cardiac fibrosis and cardiac dysfunction compared with the valsartan treatment groups. However, regarding the renoprotective effects characterized by albuminuria and fibrosis, ARNI was less effective than valsartan. Kidney transcriptomic analysis showed that the ANS-ARNI group exhibited a significant enhancement in the phosphoinositide 3-kinase (PI3K)-AKT signalling pathway compared with the ANS-VAL M group. Adding PI3K inhibitor treatment to ARNI ameliorated kidney injury to levels comparable with those of ANS-VAL M while preserving the superior cardioprotective effect of ARNI. PI3K pathway activation has been identified as a key mechanism affecting remnant kidney injury under ARNI treatment in CRS pathology, and blockading the PI3K pathway with simultaneous ARNI treatment is a potential therapeutic strategy for treating CRS with overt albuminuria.

The Effects of Chronic Unpredictable Mild Stress and Semi-Pure Diets on the Brain, Gut and Adrenal Medulla in C57BL6 Mice.

Chronic stress is known to perturb serotonergic regulation in the brain, leading to mood, learning and memory impairments and increasing the risk of developing mood disorders. The influence of the gut microbiota on serotonergic regulation in the brain has received increased attention recently, justifying the investigation of the role of diet on the gut and the brain in mood disorders. Here, using a 4-week chronic unpredictable mild stress (CUMS) model in mice, we aimed to investigate the effects of a high-fat high-glycaemic index (HFD) and high-fibre fruit & vegetable "superfood" (SUP) modifications of a semi-pure AIN93M diet on behaviour, serotonin synthesis and metabolism pathway regulation in the brain and the gut, as well as the gut microbiota and the peripheral adrenal medullary system. CUMS induced anxiety-like behaviour, dysregulated the tryptophan and serotonin metabolic pathways in the hippocampus, prefrontal cortex, and colon, and altered the composition of the gut microbiota. CUMS reduced the catecholamine synthetic capacity of the adrenal glands. Differential effects were found in these parameters in the HFD and SUP diet. Thus, dietary modifications may profoundly affect the multiple dynamic systems involved in mood disorders.

Prevention of Inflammation, Neovascularization, and Retinal Dysfunction by Kinin B1 Receptor Antagonism in a Mouse Model of Age-Related Macular Degeneration.

The kallikrein-kinin system (KKS) contributes to vascular inflammation and neovascularization in age-related macular degeneration (AMD), particularly via the kinin B1 receptor (B1R). The aim of the present study was to determine the protective effects of the topical administration of the B1R antagonist (R-954) on inflammation, neovascularization, and retinal dysfunction in a murine model of neovascular AMD. Choroidal neovascularization (CNV) was induced in C57BL6 mice using an argon laser. A treatment with ocular drops of R-954 (100 μg/15 μL, twice daily in both eyes), or vehicle, was started immediately on day 0, for 7, 14, or 21 days. CNV, invasive microglia, and B1R immunoreactive glial cells, as well as electroretinography alterations, were observed within the retina and choroid of the CNV group but not in the control group. The staining of B1R was abolished by R-954 treatment as well as the proliferation of microglia. R-954 treatment prevented the CNV development (volume: 20 ± 2 vs. 152 ± 5 × 104 µm3 in R-954 vs. saline treatment). R-954 also significantly decreased photoreceptor and bipolar cell dysfunction (a-wave amplitude: -47 ± 20 vs. -34 ± 14 µV and b-wave amplitude: 101 ± 27 vs. 64 ± 17 µV in R-954 vs. saline treatment, day 7) as well as angiogenesis tufts in the retina. These results suggest that self-administration of R-954 by eye-drop treatment could be a promising therapy in AMD to preserve retinal health and vision.

Generation of Novel Immunocompetent Mouse Cell Lines to Model Experimental Metastasis of High-Risk Neuroblastoma.

NB, being a highly metastatic cancer, is one of the leading causes of cancer-related deaths in children. Increased disease recurrence and clinical resistance in patients with metastatic high-risk NBs (HR-NBs) result in poor outcomes and lower overall survival. However, the paucity of appropriate in vivo models for HR-NB metastasis has limited investigations into the underlying biology of HR-NB metastasis. This study was designed to address this limitation and develop suitable immunocompetent models for HR-NB metastasis. Here, we developed several highly metastatic immunocompetent murine HR-NB cell lines. Our newly developed cell lines show 100% efficiency in modeling experimental metastasis in C57BL6 mice and feature metastasis to the sites frequently observed in humans with HR-NB (liver and bone). In vivo validation demonstrated their specifically gained metastatic phenotype. The in vitro characterization of the cell lines showed increased cell invasion, acquired anchorage-independent growth ability, and resistance to MHC-I induction upon IFN-γ treatment. Furthermore, RNA-seq analysis of the newly developed cells identified a differentially regulated gene signature and an enrichment of processes consistent with their acquired metastatic phenotype, including extracellular matrix remodeling, angiogenesis, cell migration, and chemotaxis. The presented newly developed cell lines are, thus, suitable and promising tools for HR-NB metastasis and microenvironment studies in an immunocompetent system.

Alkylresorcinols as New Modulators of the Metabolic Activity of the Gut Microbiota.

Alkylresorcinols (ARs) are polyphenolic compounds with a wide spectrum of biological activities and are potentially involved in the regulation of host metabolism. The present study aims to establish whether ARs can be produced by the human gut microbiota and to evaluate alterations in content in stool samples as well as metabolic activity of the gut microbiota of C57BL, db/db, and LDLR (-/-) mice according to diet specifications and olivetol (5-n-pentylresorcinol) supplementation to estimate the regulatory potential of ARs. Gas chromatography with mass spectrometric detection was used to quantitatively analyse AR levels in mouse stool samples; faecal microbiota transplantation (FMT) from human donors to germ-free mice was performed to determine whether the intestinal microbiota could produce AR molecules; metagenome sequencing analysis of the mouse gut microbiota followed by reconstruction of its metabolic activity was performed to investigate olivetol's regulatory potential. A significant increase in the amounts of individual members of AR homologues in stool samples was revealed 14 days after FMT. Supplementation of 5-n-Pentylresorcinol to a regular diet influences the amounts of several ARs in the stool of C57BL/6 and LDLR (-/-) but not db/db mice, and caused a significant change in the predicted metabolic activity of the intestinal microbiota of C57BL/6 and LDLR (-/-) but not db/db mice. For the first time, we have shown that several ARs can be produced by the intestinal microbiota. Taking into account the dependence of AR levels in the gut on olivetol supplementation and microbiota metabolic activity, AR can be assumed to be potential quorum-sensing molecules, which also influence gut microbiota composition and host metabolism.

Multiscale Passive Mechanical Characterization of Rodent Skeletal Muscle

PurposeTo experimentally measure selected passive properties of skeletal muscle at three different scales (macroscopic scale: whole muscle, microscopic scale: single skinned fiber, and submicron scale: single myofibril) within the same animal model (mice), and to compare a primarily slow-twitch fiber muscle (soleus) and a primarily fast-twitch fiber muscle (extensor digitorum longus, EDL) for each scale. MethodsHealthy 3 months old wild-type C57BL6 mice were used. To characterize each scale, soleus (N = 11), EDL (N = 9), slow fibers (N = 17), fast fibers (N = 16), and myofibrils from soleus (N = 11) and EDL (N = 11) were harvested. Passive mechanical (ramp, relaxation) tests were applied at each scale to compare the passive properties (Young's modulus, static and dynamic stresses) within a given scale, across scales and between muscle types. ResultsThe soleus and EDL showed significant passive mechanical differences at the macroscopic scale while no variation was observed between both tissues at the microscopic and submicron scales. The results highlight the importance of the scale that is used to mechanically characterize a multiscale tissue. ConclusionThe present work will allow for a better understanding of the multiscale passive mechanical properties for two muscles with vastly differing physiological and metabolic properties. This study provides referent data to the body of literature that can be built upon in future work.

Maternal impacts of pre-conceptional glyphosate exposure

Maternal glyphosate (GLY) impacts remain unclear despite associations between urinary GLY and birth outcomes. Whether maternal pre-conceptional GLY exposure would have phenotypic and molecular impacts in the dam and offspring was tested. Female C57BL6 mice (6 wk) were exposed to saline (CT; n = 20) or GLY (2 mg/kg; n = 20) per os five d per week for 20 wk. Females were housed with males and on gestation day (GD) 14, divided into: CT non-pregnant (CNP), CT pregnant (CP), GLY non-pregnant (GNP), GLY pregnant (GP). Another cohort (CT; n = 10 or GLY; n = 10) completed three pregnancy rounds and pregnancy index (PI), number of pups per litter and pups surviving to postnatal day (PND) 5 calculated. The PI in GLY mice was higher in breeding rounds 1 and 2, but lower in round 3. Pregnancy increased (P ≤ 0.1) GD14 liver and ovary weight. Spleen weight was increased (P < 0.05) in GP relative to GNP mice. No offspring phenotypic impacts were observed. Approximately six months after cessation of exposure, secondary follicle number was reduced (P < 0.05) by pre-conceptional GLY exposure. The ovarian proteome analyzed by LC-MS/MS was altered (P < 0.05) by pregnancy (49 increased, 43 decreased) and GLY exposure (non-pregnant: 75 increased, 22 decreased, pregnant: 27 increased, 29 decreased; aged dams: 60 increased, 98 decreased) with several histone proteins being altered. These findings support ovarian transient and persistent impacts of GLY exposure and identify pathways as potential modes of action.

Adeno-associated virus-9 reverses delayed gastric emptying of solids in diabetic mice.

Gastroparesis is defined by delayed gastric emptying (GE) without obstruction. Studies suggest targeting heme oxygenase-1 (HO1) may ameliorate diabetic gastroparesis. Upregulation of HO1 expression via interleukin-10 (IL-10) in the gastric muscularis propria is associated with reversal of delayed GE in diabetic NOD mice. IL-10 activates the M2 cytoprotective phenotype of macrophages and induces expression of HO1 protein. Here, we assess delivery of HO1 by recombinant adeno-associated viruses (AAVs) in diabetic mice with delayed GE. C57BL6 diabetic delayed GE mice were injected with 1 × 1012 vg scAAV9-cre, scAAV9-GFP, or scAAV9-HO1 particles. Changes to GE were assessed weekly utilizing our [13 C]-octanoic acid breath test. Stomach tissue was collected to assess the effect of scAAV9 treatment on Kit, NOS1, and HO1 expression. Delayed GE returned to normal within 2 weeks of treatment in 7/12 mice receiving scAAV9-cre and in 4/5 mice that received the scAAV9-GFP, whereas mice that received scAAV9-HO1 did not respond in the same manner and had GE that took significantly longer to return to normal (6/7 mice at 4-6 weeks). Kit, NOS1, and HO1 protein expression in scAAV9-GFP-treated mice with normal GE were not significantly different compared with diabetic mice with delayed GE. Injection of scAAV9 into diabetic C57BL6 mice produced a biological response that resulted in acceleration of GE independently of the cargo delivered by the AAV9 vector. Further research is needed to determine whether use of AAV mediated gene transduction in the gastric muscularis propria is beneficial and warranted.

In vitro and in vivo toxicity of carbon dots with different chemical compositions

Carbon dots (CDs) are easy-obtained nanoparticles with wide range of biological activity; however, their toxicity after prolonged exposure is poorly investigated. So, in vitro and in vivo toxicity of CDs with the surfaces enriched with hydroxylated hydrocarbon chains and methylene groups (CD_GE), carboxyl and phenol groups accompanied with nitrogen (CD_3011), trifluoromethyl (CDF19) or toluidine and aniline groups (CDN19) were aimed to be discovered. CDs’ in vitro toxicity was assessed on A549 cells (real-time cell analysis of impedance, fluorescence microscopy) after 24 h of incubation, and we observed no changes in cell viability and morphology. CDs’ in vivo toxicity was assessed on C57Bl6 mice after multiple dosages (5 mg/kg subcutaneously) for 14 days. Lethality (up to 50%) was observed in CDN19 and CD_3011 groups on different days of dosing, accompanied by toxicity signs in case of CD_3011. There were no changes in serum biochemical parameters except Urea (increased in CDF19 and CD_3011 groups), nor substantial kidney, liver, and spleen injuries. The most impactful for all organs were also CD_3011 and CDF19, causing renal tubule injury and liver blood supply violation. Thus, CDs with a surface enriched with oxygen- and nitrogen-containing functional groups might be toxic after multiple everyday dosing, without, however, significant damages of internal organs in survived animals.

Combined effects of valproate and naringin on kidney antioxidative markers and serum parameters of kidney function in C57BL6 mice.

Valproate is known to disturb the kidney function, and high doses or prolonged intake may cause serum ion imbalance, kidney tubular acidosis, proteinuria, hyperuricosuria, polyuria, polydipsia, and dehydration. The aim of this in vivo study was to see whether naringin would counter the adverse effects of high-dose valproate in C57Bl/6 mice and to which extent. As expected, valproate (150 mg/kg bw a day for 10 days) caused serum hyperkalaemia, more in male than female mice. Naringin reversed (25 mg/kg bw a day for 10 days) the hyperkalaemia and activated antioxidative defence mechanisms (mainly catalase and glutathione), again more efficiently in females. In males naringin combined with valproate was not as effective and even showed some prooxidative effects.

Abstract P237: Bainiku-ekisu Attenuates Cardiac Remodeling In Angiotensin Ii-infused Mice

A fruit of the Prunus mume is a traditional food in Japan. Recently, bainiku-ekisu, an infused juice concentrate of Japanse Prunus mume , is attracting attention as a health promoting supplement. Angiotensin II (AngII) plays a central role in development of hypertension. It has been reported that bainiku-ekisu treatment attenuates the growth-promoting signaling induced by AngII in vascular smooth muscle cells. However, whether bainiku-ekisu has any effect on an animal model of hypertension remains unknown. Therefore, this study was designed to explore the potential anti-hypertensive benefit of bainiku-ekisu utilizing a mouse model of hypertension with AngII infusion. 8- to 10-week-old male C57BL6 mice were randomly divided into four groups and infused with Ang II (1 μg/kg/min) for 2 weeks via osmotic mini-pump or sham-operated, and given 1% BE containing water (BEW) or normal water (control) for 2 weeks. Blood pressure was evaluated every other day by tail cuff measurement and at 2 weeks by telemetry. Cardiac hypertrophy was assessed by heart-to-body weight ratio. After 2 weeks, mice were euthanized, and the aorta and heart collected. Extracted aortas and hearts were fixed and used for histological studies to evaluate vascular remodeling. Increase in heart-to-body weight ratios by Ang II were unaltered by BEW. Aortic medial hypertrophy was observed in control mice (n=5) after 2 weeks of Ang II infusion, which was attenuated in BEW group (96 μm vs. 71 μm, respectively, p&lt;0.01). Control mice with angiotensin II infusion showed cardiac matrix deposition, which was attenuated in BEW group. In addition, attenuation of hypertension was observed in Ang II-infused mice with BEW treatment. We also observed attenuation of protein synthesis induced by AngII with BE incubation in cultured aortic VSMC. In conclusion, BEW prevented Ang II-induced vascular remodeling and hypertension. Potential cardiovascular health benefit to taking BE as a functional food should be further studied.

Abstract P320: Novel Alpha-calcitonin Gene Related Peptide Agonist Analogs In The Treatment Of Cardiovascular Disease

Alpha-calcitonin gene related peptide (α-CGRP) is a potent vasodilator and protects against heart failure and hypertension in various animal models; however, rapid clearance of the peptide from the circulation makes it difficult to use as a therapeutic agent. Recently, we have synthesized two α-CGRP agonist analogs linked with two n-methoxyethylglycine (NMEG) peptoid molecules at either end: 1)- α-CGRP with NMEG molecules at the N-terminal end (N-ter NMEG), and 2)- α-CGRP with NMEG molecules at the C-terminal end (C-ter NMEG). Bioactivity and toxicity of modified peptides were evaluated in in vitro and in vivo conditions. Peptide-peptoid hybrids, N-ter NMEG and C-ter NMEG, were synthesized using a solid-phase peptide synthesis method with a &gt;98% purity. To test the in vitro cell viability of the analogs, rat H9C2 cardiac myocyte cells were treated with 1 μM, 3 μM, and 10 μM concentrations of N-ter NMEG and C-ter NMEG peptides for 4 days followed by an MTT assay. Our in vitro results demonstrated that neither α-CGRP analogs were toxic to H9C2 cells. To test the bioactivity of these peptides, blood pressure (BP) was measured in wild-type C57BL6 mice using a tail-cuff BP analysis system. A bolus dose of 1.2, 3.6, and 12 (in mg/kg b.wt./mouse) of either peptide was injected subcutaneously (n= 4 mice/dose) followed by BP measurement at increasing time points. Our results demonstrate that there is a variance in the bioactivity of the two peptides. Subcutaneous delivery of N-ter NMEG reduced BP similar to the native peptide. After ten minutes of N-ter NMEG delivery, the BP (in mmHg ±SD) at a dose of 1.2 mg/kg was 76 ±5 (baseline BP= 118 ±15), at a dose of 3.6 mg/kg was 79 ±7 (baseline BP= 113 ±9), and at a dose of 12 mg/mg was 83 ±12 (baseline BP= 104 ±6). In contrast, C-ter NMEG administration did not reduce BP at any peptide concentration tested. In summary, in this study we determined that the α-CGRP analog N-ter NMEG, but not C-ter NMEG, is bioactive. Furthermore, N-ter NMEG showed no toxic effects on H9C2 cardiac myocytes in an in vitro viability assay. These results demonstrate that the analog N-ter NMEG is an effective α-CGRP agonist and a promising candidate molecule to treat cardiac diseases.

Abstract 083: Neurovascular And Cognitive Dysfunction In Middle-Aged Mice With High Blood Pressure Variability

Blood pressure variability (BPV) is a novel risk factor for cognitive decline. This study tested the hypothesis that BPV disrupts cellular function and communication at the neurovascular complex (vascular, glia, and neuron) contributing to BPV-induced neurovascular dysfunction. A main goal of the study was to establish a murine BPV model and examine how this risk factor impacts key functional outputs of the neurovascular complex. Middle-aged C57BL6 mice were implanted a cranial window and injected GCaMP viral vectors to monitor Ca 2+ activity in cortical neurons or astrocytes. DSI telemetry was used to monitor cardiovascular variables simultaneously ( in vivo ) with arteriole diameter and astrocyte/neuronal Ca 2+ responses via two-photon imaging. In addition, mice were implanted a programmable pump and infused 6-8 pulses/day of saline (Control) or angiotensin II (Ang II, 18 μg/day) for 20-25 days. Ang II infusions evoked transient BP increases (Δ 23±2 mmHg, mean arterial pressure (MAP, P=0.0001, n=14)), and increased BPV (P=0.04, n=12) measured by the average real variability. The 24-hour MAP was not different compared to Controls, allowing for the investigation of BPV-evoked changes in neurovascular function in the absence of hypertension. BPV mice displayed cognitive decline (NOR, P=0.006, n=10). Myogenic responses were lost in parenchymal arterioles of BPV mice but preserved in Controls (P&lt;0.01 n=6). In addition to impaired vascular function, during Ang II-induced BP increases, astrocytes of BPV mice exhibited greater Ca 2+ event frequency (P&lt;0.0001, n=4-6), but of lesser amplitude and reduced area (P=0.02, n=4-6). Furthermore, the neurovascular coupling response, evoked via whisker stimulation, was blunted in mice with high BPV (P&lt;0.0001, n=6-10). Relevant to inflammation, microglial cells from BPV mice had elevated Iba1 (P=0.03, n=4-5) and CD68+ (P=0.003), an inflammatory marker. These findings show that BPV, in the absence of hypertension, result in a loss of cerebral autoregulation, a shift in spontaneous astrocyte Ca 2+ dynamics during blood pressure fluctuations, and increased microglia reactivity. Future mechanistic studies are needed to determine how BPV contributes to cognitive decline.

Abstract P220: Angiotensin II Dependent And Independent Pressor Effect Of Ang-(1-12)

Background: Ang-(1-12) serves as a functional substrate for the formation of Ang II in the blood and tissues. It remains unknown whether the hypertensive effect of Ang-(1-12) is solely caused by Ang II formation or whether the peptide itself exerts a pressor effect by binding to the AT1-receptor or a yet-to-be-identified receptor subtype. Here we examined the possibility of Ang II independent pressor effects of Ang-(1-12). Methods: Anesthetized male c57bl6 mice were injected with Ang II or Ang-(1-12) (0.2 μg/g BW) and the systolic blood pressure (SBP) was measured continuously for 15 minutes. To evaluate whether Ang-(1–12) vasoconstrictor activity is due to its processing into Ang II, mice were preinjected with Telmisartan or Lisinopril before Ang-(1-12) injection. We further assessed the effect of recombinant (r) ACE2 as a way to augment Ang II metabolism and therefore unravel Ang II independent pressor effects of Ang-(1-12). Results: Ang-(1-12) infusion led to a peak increase in SBP almost as high as Ang II (145 ±24 mmHg vs. 160±26 mmHg, respectively). The pressor effect of Ang-(1-12) was completely abolished by either Telmisartan or Lisinopril (103±11 mmHg and 98±12 mmHg, respectively). rACE2 prevented the sustained rise in SBP induced by Ang II (Fig. 1), but it did not affect the pressor effect of Ang-(1-12) (Fig. 1). Conclusions: Ang-(1–12) hypertensive effect is blocked by RAS blockade consistent with an action dependent on Ang II formation and coupling to AT-1 receptors. In contrast, Ang-(1–12) pressor activity is not modified when Ang II is rapidly metabolized by rACE2. These data suggest a vasoactive action of Ang-(1–12) that is, in part, independent of its conversion into Ang II.

P17-22: Phenotypic characterization of macrophages during peritoneal mesothelioma development induced by a multiwalled carbon nanotube in wild type C57BL mice

P17-22: Phenotypic characterization of macrophages during peritoneal mesothelioma development induced by a multiwalled carbon nanotube in wild type C57BL mice

STING promotes ferroptosis through NCOA4-dependent ferritinophagy in acute kidney injury

Ferroptosis in tubules has been implicated in the pathogenesis of acute kidney injury (AKI), whereas the regulatory mechanism remains unclear. The stimulator of interferon genes (STING) is previously recognized as a critical mediator of innate immunity via a DNA-sensing pathway and has been increasingly linked to lipid peroxidation, a hallmark of ferroptosis. Herein we investigated the role and the underlying mechanism of STING in AKI models established by ischemia/reperfusion (IR) in C57BL mice. The expression level of STING was predominantly increased in tubules of kidney after IR treatment. Besides, STING deficiency markedly alleviated IR-induced lipid peroxidation, tissue damage and renal dysfunction. Consistently, in vitro experiments demonstrated that the increase in ferroptotic cell death, lipid ROS production and the decrease in GSH peroxidase 4 (GPX4) expression in renal tubular cells subjected to ferroptosis agonist or hypoxia/reoxygenation intervention were all mitigated by genetic deficiency or pharmacological inhibition of STING, while all exacerbated by STING overexpression. Further, these detrimental effects of STING overexpression relied on the induction of ferritinophagy, i.e. autophagic degradation of ferritin, leading to iron overload. Mechanistically, STING mediated the initiation of ferritinophagy through interacting with nuclear receptor coactivator 4 (NCOA4), a fundamental receptor for the transfer of ferritin into lysosome. Collectively, STING contributes to ferroptosis during ischemic AKI through facilitating NCOA4-mediated ferritinophagy and shows the potential as a promising therapeutic choice for AKI.