Fraction Of Mice Research Articles

Wound-Healing Activity of Solvent Fractions and Antiinflammatory Activity of Crude Extract and Solvent Fractions of Acokanthera schimperi Schweinf (Apocynaceae) Leaves in Mice Model

BackgroundIn Ethiopia, Acokanthera schimperi is frequently used to treat wounds and other conditions. Nothing has been published to date on the anti-inflammatory properties of A. schimperi leaves or the wound-healing effects of solvent fractions, despite a report on the crude extract's ability to heal wounds being available in the literature. PurposeTherefore, this experiment has been initiated to look into the solvent fractions' ability to heal wounds and the anti-inflammatory properties of the A. schimperi leaf 80% methanol extract and its fractions in mice. Study designThe experimental study design was conducted to evaluate wound healing and the anti-inflammatory properties of the A. schimperi leaf 80% methanol extract and its fractions in mice. MethodsThe plant's leaf crude extract was fractionated using ethyl acetate, chloroform, and distilled water. The resulting fractions were then combined with a simple ointment base to make an ointment at 5% and 10% w/w. Next, using excision and incision wound models, the ointments' ability to promote wound healing was assessed. The excision model was utilised to estimate wound contraction and epithelization time, whereas the incision model was utilised to measure tensile strength. By dissolving 1% carrageenan in 0.9% saline (w/v) and giving oral dosages of the crude extract and solvent fractions (100, 200, and 400 mg/kg), the anti-inflammatory efficacy of the leaves was assessed concurrently with a carrageenan-induced hind paw edema model. ResultsSignificant wound healing activity was seen in both models in wounds treated with 5% and 10% (w/w) solvent fraction ointment, as shown by higher tissue breaking strength (with 51.38% maximum effect by 10% (w/w) ethyl acetate fraction ointment), shorter epithelization times (within 15.92 days by ethyl acetate fraction ointment 10%), and increased wound contraction (99.40% by ethyl acetate fraction ointment 10%). The extract (hydromethanolic) and solvent fractions also showed notable dose-related decreases in inflammation. ConclusionThe study's findings supported A. schimperi's traditional use as a wound healing agent by showing that the solvent fractions of the plant's leaves promoted wound healing, at least in part, through their anti-inflammatory effect.

Geniposide alleviates heart failure with preserved ejection fraction in mice by regulating cardiac oxidative stress via MMP2/SIRT1/GSK3β pathway.

Heart failure with preserved ejection fraction (HFpEF) is a complex clinical syndrome with cardiac dysfunction, fluid retention and reduced exercise tolerance as the main manifestations. Current treatment of HFpEF is using combined medications of related comorbidities, there is an urgent need for a modest drug to treat HFpEF. Geniposide (GE), an iridoid glycoside extracted from Gardenia Jasminoides, has shown significant efficacy in the treatment of cardiovascular, digestive and central nervous system disorders. In this study we investigated the therapeutic effects of GE on HFpEF experimental models in vivo and in vitro. HFpEF was induced in mice by feeding with HFD and L-NAME (0.5 g/L) in drinking water for 8 weeks, meanwhile the mice were treated with GE (25, 50 mg/kg) every other day. Cardiac echocardiography and exhaustive exercise were performed, blood pressure was measured at the end of treatment, and heart tissue specimens were collected after the mice were euthanized. We showed that GE administration significantly ameliorated cardiac oxidative stress, inflammation, apoptosis, fibrosis and metabolic disturbances in the hearts of HFpEF mice. We demonstrated that GE promoted the transcriptional activation of Nrf2 by targeting MMP2 to affect upstream SIRT1 and downstream GSK3β, which in turn alleviated the oxidative stress in the hearts of HFpEF mice. In H9c2 cells and HL-1 cells, we showed that treatment with GE (1 μM) significantly alleviated H2O2-induced oxidative stress through the MMP2/SIRT1/GSK3β pathway. In summary, GE regulates cardiac oxidative stress via MMP2/SIRT1/GSK3β pathway and reduces cardiac inflammation, apoptosis, fibrosis and metabolic disorders as well as cardiac dysfunction in HFpEF. GE exerts anti-oxidative stress properties by binding to MMP2, inhibiting ROS generation in HFpEF through the SIRT1/Nrf2 signaling pathway. In addition, GE can also affect the inhibition of the downstream MMP2 target GSK3β, thereby suppressing the inflammatory and apoptotic responses in HFpEF. Taken together, GE alleviates oxidative stress/apoptosis/fibrosis and metabolic disorders as well as HFpEF through the MMP2/SIRT1/GSK3β signaling pathway.

Long-term impact of postnatal nutritional programming on cardiac sensitivity to ischemia-reperfusion injury in vivo and on glucose metabolism

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Fondation de France Introduction Nutritional environment during intra uterine and postnatal period has a major impact on health and disease in adulthood. The early programming of cardiometabolic risk through a postnatal overfeeding in rodents leads to a permanent increase in body weight, associated with alteration of left ventricular contractility, higher cardiac sensitivty to ischemic or chemo-toxic stresses and alterations of glucose métabolism in adulthood. However, these data have mainly obtained in young rodents and less is known for older animals. Purpose Our aim was to evaluate the impact of postnatal overfeeding (PNOF) on cardiac function, on cardiac sensitivity to ischemia-reperfusion (I-R) injury in vivo, on glucose metabolism and on metabolic profile of pericardial adipose tissue (PAT) in young (4 months), adult (6 months) and old (12 months) male mice. Methods PNOF was induced by the reduction of litter size of C57/BL6 mice immediately after birth: normally-fed group (NF) was composed of 9 male pups/mother and overfed group (PNOF) of 3 pups/mother. Echocardiography and blood sugar tests (glucose tolerance and insulin resistance) were performed at all ages. An in vivo ischemia-reperfusion injury was induced by the ligation of the left anterior descending coronary artery for 45 minutes followed by 24 h of reperfusion. PAT was harvested and weighted, in order to investigate by rt-qPCR its inflammatory status. All experiments were performed in 4-, 6- and 12-months aged mice. Results PNOF induced an early and permanent increase in body weight in PNOF group, independently of mice’s age (+23% at 24 days of life, +14% at 4 months, +23% at 6 and 12 months, compared to NF group). Echocardiographic measurements showed an alteration of left ventricular ejection fraction in mice aged from 4 months up to 12 months (-11 % on average, compared to NF groups). PNOF mice presented glucose intolerance and insulin resistance from 4 to 12 months of age. Moreover, PNOF mice had a higher PAT mass indexed to body weight with increased inflammatory status and decreased activin A mRNA expression at 6 and 12 months. Finally, PNOF induced a significant increase of infarct size at 4 months (+37%, p<0.01, n=8), 6 months (+32%, p<0.05, n=9) and 12 months (+38%, p<0,05, n=9) after cardiac surgery. Conclusion Nutritional programming through short-term PNOF induced an early and permanent overweight, an increase in pericardial fat mass with a greater inflammatory status, associated with alterations of glucose metabolism, alteration of systolic function and a higher sensitivity to ischemia reperfusion injury induced in vivo. The cellular mechanisms of these pathological modifications induced by PNOF need further exploration but could involve alterations in cardiomyocyte pathways and/or modification of global inflammatory status. Therefore, the PNOF mice model corresponds to a relevant experimental model of programmed metabolic syndrome as we known in humans.

Enhancing impact of <i>Prosopis africana</i> fraction on biochemical indices and oxidative balance in <i>Plasmodium berghei</i>-infected mice

This study addresses the ongoing global health challenge of malaria by exploring the therapeutic potential of the Prosopis africana fraction in mice infected with Plasmodium berghei. Using 42 male mice divided into six groups, including a control group, infected groups without treatment, infected treated with 20 mg/kg bw chloroquine, and infected groups treated with different doses of Prosopis africana fraction at 6.25 mg/kg,12.5mg/kg and 25 mg/kg bw respectively. The mice were induced by NK-65 chloroquine sensitive Plasmodium berghei and administered oral treatment for four days. After the treatment period, biochemical analysis of blood samples and tissues was conducted, focusing on liver and kidney function markers, as well as oxidative stress indicators. Mice infected with Plasmodium berghei exhibited significant alterations in biochemical indices and oxidative balance compared to the control group. However, treatment with Prosopis africana fraction mitigated these changes, restoring biochemical parameters and reestablishing oxidative balance. Notably, the dose at 12.5 mg/kg bw of Prosopis africana fraction demonstrated superior efficacy in reversing the alterations induced by malaria infection. These findings suggest that Prosopis africana fraction holds therapeutic potential in mitigating the biochemical disruptions and oxidative stress associated with malaria infection.

Effects of a Global Rab27a Null Mutation on Murine PVAT and Cardiovascular Function.

RAB27A is a member of the RAS oncogene superfamily of GTPases and regulates cell secretory function. It, is expressed within blood vessels and perivascular adipose tissue. We hypothesized that loss of RAB27A would alter cardiovascular function. Body weight of Rab27aash mice was measured from 2 to 18 months of age, along with glucose resorption at 6 and 12 months of age and glucose sensitivity at 18 months of age. Body weight and cellular and molecular features of perivascular adipose tissue and aortic tissue were examined in a novel C57BL/6J Rab27a null strain. Analyses included morphometric quantification and proteomic analyses. Wire myography measured vasoreactivity, and echocardiography measured cardiac function. Comparisons across ages and genotypes were evaluated via 2-way ANOVA with multiple comparison testing. Significance for myography was determined via 4-parameter nonlinear regression testing. Genome-wide association data linked rare human RAB27A variants with body mass index and glucose handling. Changes in glucose tolerance were observed in Rab27aash male mice at 18 months of age. In WT (wild-type) and Rab27a null male mice, body weight, adipocyte lipid area, and aortic area increased with age. In female mice, only body weight increased with age, independent of RAB27A presence. Protein signatures from male Rab27a null mice suggested greater associations with cardiovascular and metabolic phenotypes compared with female tissues. Wire myography results showed Rab27a null males exhibited increased vasoconstriction and reduced vasodilation at 8 weeks of age. Rab27a null females exhibited increased vasoconstriction and vasodilation at 20 weeks of age. Consistent with these vascular changes, male Rab27a null mice experienced age-related cardiomyopathy, with severe differences observed by 21 weeks of age. Global RAB27A loss impacted perivascular adipose tissue and thoracic aorta proteomic signatures, altered vasocontractile responses, and decreased left ventricular ejection fraction in mice.

Anti-anxiety and Hypnotic Effects of Lawsonia inermis Hydroalcoholic Extract

Background: Sleep is a vital biological feature, and insomnia causes maladaptive physical and psychological functions. The main flaws of current insomnia medications are significant side effects. A suitable substitute can be herbal products. We aimed to evaluate the anti-anxiety, sleep-inducing, and - prolonging effects of Lawsonia inermis extract and fractions in mice. Methods: Male albino mice were pretreated intraperitoneally (i.p.) with either three doses (40, 80, and 160 mg/kg) of L. inermis extract or n-butanol fraction (NBF), ethyl acetate fraction (EAF), and water fraction (WF), 30 minutes before i.p. injection of 30 mg/kg pentobarbital. Sleep latency and duration of sleep were recorded. For anxiolytic activity, elevated plus-maze (EPM) tests were used. Moreover, the toxicity of the extract was determined in both in vivo and in vitro experiments. Results: L. inermis extract (160 mg/kg) significantly reduced sleep latency and increased sleep duration. EAF at 160 mg/kg decreased sleep latency and increased sleep duration. Flumazenil reversed the hypnotic effect of L. inermis extract (160 mg/kg). L. inermis extract (80, 160 mg/kg) increased the time spent and the number of entries in the open arms of EPM. The tested extracts and fractions administration found no adverse effects on PC12 cell viability. The LD50 was 2.4 g/kg. Conclusion: L. inermis extracts exhibit anxiolytic and hypnotic effects, probably modulating the GABAergic system.

Targeting HDAC6 to treat heart failure with preserved ejection fraction in mice

Heart failure with preserved ejection fraction (HFpEF) poses therapeutic challenges due to the limited treatment options. Building upon our previous research that demonstrates the efficacy of histone deacetylase 6 (HDAC6) inhibition in a genetic cardiomyopathy model, we investigate HDAC6’s role in HFpEF due to their shared mechanisms of inflammation and metabolism. Here, we show that inhibiting HDAC6 with TYA-018 effectively reverses established heart failure and its associated symptoms in male HFpEF mouse models. Additionally, in male mice lacking Hdac6 gene, HFpEF progression is delayed and they are resistant to TYA-018’s effects. The efficacy of TYA-018 is comparable to a sodium-glucose cotransporter 2 (SGLT2) inhibitor, and the combination shows enhanced effects. Mechanistically, TYA-018 restores gene expression related to hypertrophy, fibrosis, and mitochondrial energy production in HFpEF heart tissues. Furthermore, TYA-018 also inhibits activation of human cardiac fibroblasts and enhances mitochondrial respiratory capacity in cardiomyocytes. In this work, our findings show that HDAC6 impacts on heart pathophysiology and is a promising target for HFpEF treatment.

Inhibition of ferroptosis reverses heart failure with preserved ejection fraction in mice

BackgroundHeart failure with preserved ejection fraction (HFpEF) accounts for approximately 50% of heart failure cases. The molecular mechanisms by which HFpEF leads to impaired diastolic function of the heart have not been clarified, nor have the drugs that target the clinical symptoms of HFpEF patients.MethodsHFpEF chip data (GSE180065) was downloaded from the National Center for Biotechnology Information (NCBI) database. Differentially expressed genes (DEGs) were filtered by the limma package in R and processed for GO and KEGG pathway analyses. Then, ferroptosis-related genes in HFpEF were identified by taking the intersection between DEGs and ferroptosis-related genes. CytoHubba and MCODE were used to screen ferroptosis-related hub DEGs in the protein–protein interaction (PPI) network. Establishment of a mouse HFpEF model to validate the transcript levels of ferroptosis-related hub DEGs and ferroptosis-related phenotypes. Transcript levels of ferroptosis-related hub DEGs and HFpEF phenotypic changes in the hearts of HFpEF mice were further examined after the use of ferroptosis inhibitors.ResultsGO and KEGG enrichment analyses suggested that the DEGs in HFpEF were significantly enriched in ferroptosis-related pathways. A total of 24 ferroptosis-related DEGs were identified between the ferroptosis gene dataset and the DEGs. The established PPI network was further analyzed by CytoHubba and MCODE modules, and 11 ferroptosis-related hub DEGs in HFpEF were obtained. In animal experiments, HFpEF mice showed significant abnormal activation of ferroptosis. The expression trends of the 11 hub DEGs associated with ferroptosis, except for Cdh1, were consistent with the results of the bioinformatics analysis. Inhibition of ferroptosis alters the transcript levels of 11 ferroptosis-related hub DEGs and ameliorates HFpEF phenotypes.ConclusionsThe present study contributes to a deeper understanding of the specific mechanisms by which ferroptosis is involved in the development of HFpEF and suggests that inhibition of ferroptosis may mitigate the progression of HFpEF. In addition, eleven hub genes were recognized as potential drug binding targets.

Formononetin improves heart failure with preserved ejection fraction in mice by activating the PPARα/PGC-1 pathway

Formononetin improves heart failure with preserved ejection fraction in mice by activating the PPARα/PGC-1 pathway

Abstract 13255: Cardiac and Generalized SGLT2 Deficiency Does Not Protect Against Development of Heart Failure With Reduced Ejection Fraction in Mice

Sodium-glucose cotransporter 2 inhibitors (SGLT2i), originally designed for the treatment of diabetes, exert profound cardiovascular benefit and are now the standard-of-care in all forms of heart failure (HF). The mechanism of their benefit in HF is unknown. We hypothesized SGLT2i act via an on-target, extra-cardiac mechanism to promote ketosis and improve myocardial energetics. Generalized- (gKO) and cardiac-specific (csKO) SGLT2 knockout mice were generated by breeding SGLT2 floxed mice with EIIa or αMHC Cre recombinase lines, respectively. While csKO grew and developed normally, gKO mice were slightly smaller compared to WT littermates. There was no baseline cardiac phenotype in either group. The gKO mice exhibited profound glucosuria, decreased fasting serum glucose and augmented fasting ketosis compared to controls, and comparable in magnitude to the metabolic effects of 14 days of treatment with empagliflozin (Empa, 50mg/kg/day orally) in WT mice. Targeted metabolomic analysis of gKO and Empa-treated WT mice identified increased circulating and myocardial levels of the ketone body 3-hydroxybutyrate (1.9-2.3x, p<0.01), along with an increase in a subset of tricarboxylic acid cycle intermediates and C4-OH, a ketone oxidation by-product. Empa treatment in WT mice following trans-aortic constriction with apical myocardial infarction (TAC/MI) improved LV dysfunction and pathological remodeling at 6 weeks (left ventricular ejection fraction (EF) 36±6 vs 28±9, p<0.03; tibia length-normalized biventricular weight 9.6±0.4 vs 11.3±1.6, p<0.001). In contrast, csKO and gKO mice subjected to TAC/MI had no difference in EF, strain, ventricular hypertrophy or dilation compared to WT controls. In conclusion, generalized SGLT2 deficiency phenocopies the metabolic effects of SGLT2i treatment but neither gKO nor csKO afford cardiac protection in an established HF model. These results suggest an off-target mechanism for beneficial effects of SGLT2i in HF.

Hydrogen sulfide alleviates heart failure with preserved ejection fraction in mice by targeting mitochondrial abnormalities via PGC-1α

AimIncreasing evidence has proposed that mitochondrial abnormalities may be an important factor contributing to the development of heart failure with preserved ejection fraction (HFpEF). Hydrogen sulfide (H2S) has been suggested to play a pivotal role in regulating mitochondrial function. Therefore, the present study was designed to explore the protective effect of H2S on mitochondrial dysfunction in a multifactorial mouse model of HFpEF. MethodsWild type, 8-week-old, male C57BL/6J mice or cardiomyocyte specific-Cse (Cystathionine γ-lyase, a major H2S-producing enzyme) knockout mice (CSEcko) were given high-fat diet (HFD) and l-NAME (an inhibitor of constitutive nitric oxide synthases) or standardized chow. After 4 weeks, mice were randomly administered with NaHS (a conventional H2S donor), ZLN005 (a potent transcriptional activator of PGC-1α) or vehicle. After additional 4 weeks, echocardiogram and mitochondrial function were evaluated. Expression of PGC-1α, NRF1 and TFAM in cardiomyocytes was assayed by Western blot. ResultsChallenging with HFD and l-NAME in mice not only caused HFpEF but also inhibited the production of endogenous H2S in a time-dependent manner. Meanwhile the expression of PGC-1α and mitochondrial function in cardiomyocytes were impaired. Supplementation with NaHS not only upregulated the expression of PGC-1α, NRF1 and TFAM in cardiomyocytes but also restored mitochondrial function and ultrastructure, conferring an obvious improvement in cardiac diastolic function. In contrast, cardiac deletion of CSE gene aggravated the inhibition of PGC-1α-NRF1-TFAM pathway, mitochondrial abnormalities and diastolic dysfunction. The deleterious effect observed in CSEcko HFpEF mice was partially counteracted by pre-treatment with ZLN005 or supplementation with NaHS. ConclusionOur findings have demonstrated that H2S ameliorates left ventricular diastolic dysfunction by restoring mitochondrial abnormalities via upregulating PGC-1α and its downstream targets NRF1 and TFAM, suggesting the therapeutic potential of H2S supplementation in multifactorial HFpEF.

B Cell Chronic Lymphocytic Leukemia Development in Mice with Chronic Lung Exposure to Coccidioides Fungal Arthroconidia.

Links between repeated microbial infections and B cell chronic lymphocytic leukemia (B-CLL) have been proposed but not tested directly. This study examines how prolonged exposure to a human fungal pathogen impacts B-CLL development in Eµ-hTCL1-transgenic mice. Monthly lung exposure to inactivated Coccidioides arthroconidia, agents of Valley fever, altered leukemia development in a species-specific manner, with Coccidioides posadasii hastening B-CLL diagnosis/progression in a fraction of mice and Coccidioides immitis delaying aggressive B-CLL development, despite fostering more rapid monoclonal B cell lymphocytosis. Overall survival did not differ significantly between control and C. posadasii-treated cohorts but was significantly extended in C. immitis-exposed mice. In vivo doubling time analyses of pooled B-CLL showed no difference in growth rates of early and late leukemias. However, within C. immitis-treated mice, B-CLL manifests longer doubling times, as compared with B-CLL in control or C. posadasii-treated mice, and/or evidence of clonal contraction over time. Through linear regression, positive relationships were noted between circulating levels of CD5+/B220low B cells and hematopoietic cells previously linked to B-CLL growth, albeit in a cohort-specific manner. Neutrophils were positively linked to accelerated growth in mice exposed to either Coccidioides species, but not in control mice. Conversely, only C. posadasii-exposed and control cohorts displayed positive links between CD5+/B220low B cell frequency and abundance of M2 anti-inflammatory monocytes and T cells. The current study provides evidence that chronic lung exposure to fungal arthroconidia affects B-CLL development in a manner dependent on fungal genotype. Correlative studies suggest that fungal species differences in the modulation of nonleukemic hematopoietic cells are involved.

Multi-omics Analyses Identify Galectin-1 as a Potential Novel Mediator of Heart Failure with Preserved Ejection Fraction in Mice and Humans

Heart failure with preserved ejection fraction (HFpEF) is a clinical syndrome where patients present with symptoms of volume overload despite having a normal systolic function. HFpEF is associated with elevation of inflammatory biomarkers, but the precise pathways leading to cardiac dysfunction is not known. In a deoxycorticosterone acetate (DOCA)-salt model of hypertension and HFpEF, we performed CITE-Seq analysis on total leukocytes isolated from the heart, which revealed significant differential gene expression primarily in the myeloid population. We then took the top 20 genes that were differentially expressed between myeloid cells from DOCA-salt vs sham treated mice and performed a genetic analysis called PrediXcan in Vanderbilt’s DNA databank, BioVU. Genetically predicted expression of two genes were associated with a HFpEF phenotype in humans. Of these, the gene encoding galectin 1, Lgals1, had the lowest p-value (0.02) and highest beta coefficient (0.32) corresponding to an odds ratio for HFpEF of 1.38. The role of galectin-1 in heart failure is unknown, we hypothesized that overexpression of Lgals1 exacerbates HFpEF. We found that siRNA knockdown of Lgals1 in bone marrow derived macrophages stimulated with lipoprotein saccharide and IFNγ, they expressed significantly more inflammatory genes such as IL-1α (p<0.0001), IL-6 (p=0.0219), iNOS (p=0.043), and TNFα (p=0.0031) using a 2-way ANOVA followed by Sidak’s multiple comparison test. Next, we explored the role of Lgals1 in the pathophysiology of HFpEF using Lgals1-/- mice. Surprisingly, despite both in vitro and in vivo data supporting a role of galectin-1 in HFpEF, Lgals1-/- mice did not show a significant difference in HFpEF phenotype (blood pressure, exercise tolerance, echocardiography, heart weight, or pulmonary congestion) after DOCA-salt treatment compared to littermate Lgals1+/+ controls in either males or females (p > 0.05 using Student’s t-test for all parameters measured, n=10-12). One hypothesis for this negative result is that Lgals1 is ubiquitously expressed, and a total body knockout may have compensatory mechanisms. We therefore created Lgals1 floxed mice and future studies will determine the effect of macrophage specific deletion of galectin-1 on HFpEF. In conclusion, while multi-omics approaches in mice and humans identify galectin-1 as a novel mediator of HFpEF, further studies in mice are needed to confirm its roles as a potential therapeutic target. 1R01HL161212-01A1, 19EIA34480023,,19IPLOI34760558, and 1T32HL144446-01 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Chemical characterization, antinociceptive and anti-inflammatory effect of Lippia lacunosa, a species used by the Bandeirantes

Ethnopharmacological relevanceLippia lacunosa Mart. & Schauer is an endemic plant from the Serra do Espinhaço mountain range located on the Atlantic plateau, Brazil. It is known as “chá de pedestre” and “rosmaninho” in folk medicine. This species has a characteristic mango aroma and is widely used by the population for flu, colds, sinus infections, coughing, relaxing baths, and foot baths after long walks. It is often confused with and, therefore, used interchangeably with L. rotundifolia and L. pseudothea. Aim of the studyThis study aimed to increase scientific knowledge on the ethnopharmacological use of Lippia lacunosa through the evaluation of the micromolecular composition and anti-inflammatory and antinociceptive activities of the hexane and ethanolic extracts, essential oil, and fractions in mice. Materials and methodsThe chemical profile of L. lacunosa extracts and fractions were obtained by chromatographic methods such as Ultra-Performance Liquid Chromatography (UPLC), Gas Chromatography (GC), Column Chromatography (CC), and Thin Layer Chromatography (TLC). Carrageenan-induced paw edema was used to investigate the anti-inflammatory activity in mice. Mechanical allodynia induced by carrageenan and hot plate tests were employed to evaluate the antinociceptive activity. ResultsThe main constituents found in the essential oil were the monoterpenes myrcene (13.81%), linalool (6.84%), ipsenone (21.2%), and myrcenone (25.44%); and sesquiterpenes elemol (7.30%) and spathulenol (3.15%). The chromatograph fractionation of essential oil yielded a fraction rich in the main compounds (F33), ipsenone and mircenone. In experimental models of paw edema and mechanical allodynia induced by carrageenan (600 μg, 30 μL, i.pl.), the administration of hexane extract, essential oil (50 or 100 mg/kg, p.o.) or majority fraction (10 mg/kg, p.o.) reduced paw edema. The ethanolic extract (100 mg/kg) reduced mechanical allodynia only in the 2 nd h of evaluation. On the other hand, the hexane extract (50 or 100 mg/kg) and essential oil (100 mg/kg), as well as the majority fraction (10 mg/kg), reduced mechanical allodynia throughout the evaluation period. The hexane extract, essential oil, and majority fraction F33 also reduced the heat-induced nociceptive response. Also, majority fraction F33 did not affect the time mice spent in the rota-rod apparatus. ConclusionsThe elucidation of the composition of the essential oil and the demonstration of the activity of L. lacunosa in experimental models of acute inflammation and also in models of nociceptive and inflammatory pain can help to increase knowledge on the ancient ethnopharmacological use by the Bandeirantes, aiming at the evaluation of the species as a candidate for herbal medicine or phytopharmaceutical in the treatment of patients with inflammatory and painful conditions.

Porcupine inhibitor CGX1321 alleviates heart failure with preserved ejection fraction in mice by blocking WNT signaling.

Heart failure with preserved ejection fraction (HFpEF) is highly prevalent, and lacks effective treatment. The aberration of WNT pathway underlies many pathological processes including cardiac fibrosis and hypertrophy, while porcupine is an acyltransferase essential for the secretion of WNT ligands. In this study we investigated the role of WNT signaling pathway in HFpEF as well as whether blocking WNT signaling by a novel porcupine inhibitor CGX1321 alleviated HFpEF. We established two experimental HFpEF mouse models, namely the UNX/DOCA model and high fat diet/L-NAME ("two-hit") model. The UNX/DOCA and "two-hit" mice were treated with CGX1321 (3 mg·kg-1·d-1) for 4 and 10 weeks, respectively. We showed that CGX1321 treatment significantly alleviated cardiac hypertrophy and fibrosis, thereby improving cardiac diastolic function and exercise performance in both models. Furthermore, both canonical and non-canonical WNT signaling pathways were activated, and most WNT proteins, especially WNT3a and WNT5a, were upregulated during the development of HEpEF in mice. CGX1321 treatment inhibited the secretion of WNT ligands and repressed both canonical and non-canonical WNT pathways, evidenced by the reduced phosphorylation of c-Jun and the nuclear translocation of β-catenin and NFATc3. In an in vitro HFpEF model, MCM and ISO-treated cardiomyocytes, knockdown of porcupine by siRNA leads to a similar inhibitory effect on WNT pathways, cardiomyocyte hypertrophy and cardiac fibroblast activation as CGX1321 did, whereas supplementation of WNT3a and WNT5a reversed the anti-hypertrophy and anti-fibrosis effect of CGX1321. We conclude that WNT signaling activation plays an essential role in the pathogenesis of HFpEF, and porcupine inhibitor CGX1321 exerts a therapeutic effect on HFpEF in mice by attenuating cardiac hypertrophy, alleviating cardiac fibrosis and improving cardiac diastolic function.

High Potency Vasoactive Intestinal Peptide Receptor Antagonists Induce Durable Antileukemia/Tumor T Cell Memory in Mice with Once Daily Dosing

Introduction: Allogeneic bone marrow transplantation (allo-BMT) is curative for patients with myeloid leukemia (AML) through the activity of cytotoxic effector donor T cells, but high morbidity and mortality of allo-BMT limits the use of this therapy. A long-standing goal is the development of strategies that induce an autologous immune response to leukemic blasts by the patient's T cells. Vasoactive intestinal peptide (VIP) is a neuropeptide with pleiotropic functions, including modulation of immune and inflammatory responses. Previous studies demonstrated that VIP hybrid (VIPhyb), a peptide antagonist of VIP-signaling, induced T cell-dependent anti-leukemic activity in murine allotransplant models and promoted activation of mouse and human T cells (Li et al., 2016, Cancer Res). We developed a series of novel peptide VIP antagonists (VIP-ANT) based upon predicted binding affinity in silico to human VIP receptors VPAC1 and VPAC2. We tested the anti-cancer potential of these new VIP-ANT peptides in murine models of myeloid leukemia and mastocytoma, investigating the optimal dose and schedule of administration, and explored mechanisms underlying anti-tumor memory induced by VIP-ANT peptides. Methods: B6 (CD45.1, or albino) mice were injected intravenously with 1 x 106 C1498 cells (AML), and DBA/2j mice were injected subcutaneously with 5 x 104 P815 cells (myeloid mastocytoma). Leukemia-bearing mice were treated with subcutaneous injections of VIP-ANT or control scrambled peptides. Treatment started six days after injection of cells when C1498 leukemia cells were detectable in the blood, and luciferase+ P815 cells as tumors were palpable. A range of VIP-ANT doses was administered s.c. at 10, 30, or 100 μg. Treatment schedules tested included twice daily for 14 doses, once daily, once every other day, once every three days, and once weekly for 7 doses. Some mice received a single dose at day +6 following leukemia cell injection. Lymphocyte kinetics and leukemia cell burden were analyzed weekly through submandibular peripheral blood collections. Mice without evidence of AML/mastocytoma >60 days post-inoculation received a second injection of a two-fold higher dose of C1498 (2 × 106 cells) or P815 (1 × 105 cells). Results: We tested over 13 new VIP-ANT peptides. Daily or every other day (qod) administration of the novel VIP-ANT peptides to leukemia/tumor-bearing mice led to clearance of C1498 cells from blood or complete regression of P815 in 40 % of mice, leading to significantly improved survival over 60 days. Daily administration of 10 μg of novel VIP-ANT peptides for 7 days to leukemic mice prolonged survival(p<0.0001 VIP-ANT vs scramble, Fig 1A), with the fraction of mice that remained leukemia-free proportional to the predicted binding affinity of the VIP-ANT to human VPAC1 and VPAC2. With increased dose intensity and dose-density of ANT308, the most potent peptide, 40%-80% of mice survived to 70 days (median survival days (MSD) of 60 days) after ANT308 treatment compared with the survival of <20% in mice receiving scrambled peptide (MSD 36 days). Increasing the dose and schedule of ANT308 administration to 100 μg twice-daily led to 65% survival (MSD not reached) versus 40% with daily dosing, 30% with every-other-day dosing, and 0% survival with scrambled-sequence control peptide (MSD 33 days) (p<0.001, Fig 1B). Notably, the affinity of different VIP-ANT peptides to VPAC1 and VPAC2 VIP receptors significantly correlated with the survival of C1498-leukemic mice following prior treatment with the corresponding peptide. Prior treatment of leukemic mice with VIP-ANT induced protective immunological memory, rendering mice resistant to re-challenge with C1498 acute myeloid leukemia (80% survival) and P815 mastocytoma (60% survival) cells, compared with 0% survival (MSD 21 days) among immunologically naïve mice. Conclusion: Blocking VIP-signaling with high-affinity VIP-R peptide antagonists represents a novel pharmacological approach to enhance adaptive T cell responses to leukemia/myeloid sarcoma-associated antigens in murine leukemia/tumor models. We are continuing PK and toxicology studies with the lead VIP-ANT peptides to support the development of phase 1 clinical trials for patients with relapsed AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 13146: Cardioprotective Effects of Mtss1 Reduction in Dilated Cardiomyopathy

Introduction: MTSS1 is an I-BAR protein that interacts with the actin cytoskeleton and the cell membrane and is speculated to play a role in cell motility, tumor metastasis, and cardiogenesis. Enhancer variants that reduce the expression of MTSS1 in the human left ventricle have been associated with cardioprotective traits in genome-wide association studies. However, the effect of MTSS1 downregulation on the development of heart disease has not been studied in experimental models. Here we tested the hypothesis that reduction of MTSS1 protects against dilated cardiomyopathy (DCM) in vitro and in vivo. Methods: We used CRISPR-Cas9 to knock out MTSS1 in induced pluripotent stem cells (iPSCs) harboring pathogenic variants from patients with severe DCM. DCM iPSC lines with and without MTSS1 were then differentiated into cardiomyocytes and phenotyped in vitro. A DCM mouse model containing a TPM1 D230N transgene was crossed with a mouse model heterozygous for Mtss1 knockout. Progeny from this F1 cross were assessed for differences in cardiac structure and function via echocardiography. Results: MTSS1 deletion restored the beta-adrenergic response in DCM iPSC cardiomyocytes. In parallel, Mtss1 deletion partially rescued DCM-associated phenotypes including left ventricular dilation and reduced ejection fraction in mice. Conclusions: Our initial results suggest a causal link between MTSS1 reduction and cardioprotection in DCM. Inhibiting cardiac MTSS1 may hold therapeutic potential for DCM, though the cellular mechanisms through which MTSS1 affects cardiac phenotypes remain under investigation.

Abstract 581: bbT369, a dual-targeted and CBLB gene-edited autologous CART product, demonstrates anti-lymphoma activity in preclinical mouse models

Abstract Anti-CD19 CAR T cell therapies have improved outcomes for non-Hodgkin lymphoma (NHL) patients. However, only 30-40% of patients treated with commercially available CART cell therapies obtain long term remission, highlighting the need for more efficacious and durable therapies. Emerging clinical data suggest several failure modes for CD19 CAR T cell therapies: including loss or downregulation of CD19 antigen, loss of co-stimulation pathways on tumor cells, exhaustion of CAR-T cells, and immunosuppressive microenvironments. To overcome these hurdles, we devised the next-generation autologous CAR-T cell therapy bbT369. bbT369 is dual targeted (CD79a/CD20) CAR T cell therapy that uses an OR gate design to limit antigen escape, has split 41BB and CD28 co-stimulatory domain architecture to augment T cell activation, and contains a knock-out of the CBLB gene to enhance potency and reduce T cell exhaustion. Here we report the first results with bbT369, demonstrating anti-lymphoma activity in in vitro assays and in vivo using xenograft mouse models. We demonstrate that CD79a and CD20 expression is B cell lineage restricted in normal human tissue and confirm that these proteins are co-expressed in diffuse large B cell samples. To target these antigens, we show a split dual-targeting CAR configuration is optimal for bbT369-directed tumor cell killing. Using an engineered megaTAL, we demonstrate high on-target activity of greater than 75% insertions and deletions (Indels) at the CBLB target site using clinical-scale manufacturing processes and low off-target activity (all off-targets less than 0.2%). In in vitro tumor co-culture assays, we show that inclusion of the CBLB gene edit in bbT369 increases Interleukin (IL)-2 production relative to an unedited anti-CD79a/CD20 CAR T cell control. Using various xenograft mouse models, we showed that bbT369 has similar or improved efficacy compared to anti-CD19 CAR drug product, including in low tumor-antigen models. In the Toledo subcutaneous xenograft model, bbT369 showed a 3-fold increase in T cell expansion compared with an unedited anti-CD79a/CD20 dual-targeting CAR T cell control. Furthermore, while a fraction of mice (3/5) receiving the unedited anti-CD79a/CD20 dual-targeting CAR T cells experienced late relapses (between 60-80 days following initial tumor clearance), all mice (n=5) receiving bbT369 were fully protected from late relapses (up to day 104 of follow-up). Collectively, the data support a first-in-human trial for bbT369 to evaluate initial safety and efficacy in NHL patients. Citation Format: Michael Certo, Christopher Baldeviano, Sharlene Adams, Martin Asimis, Alexander Astrakhan, Andy Chavkin, Maria L. Cabral, Jimmy Chu, Marie Debrue, Devina Desai, John Evans, Pinky Htun, Amanda Iniguez, Jordan Jarjour, Carl Johnson, Harini Kantamneni, Sema Kurtulus, Michael Magee, Unja Martin, Seamus McKenney, Sara Miller, Prashant Nambiar, Vinh Khang Nguyen, Mauris Nnamani, Jen Obrigewitch, Lisa Pechilis, Molly Perkins, Christopher Petersen, Jason Pinger, Cindy Rogers, Nick Rouillard, Kendal Sanson, Emily Thompson, Collin Walter, Roslyn Yi, Sarah Voytek, Philip Gregory. bbT369, a dual-targeted and CBLB gene-edited autologous CART product, demonstrates anti-lymphoma activity in preclinical mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 581.

Gene-mapping study of extremes of cerebral small vessel disease reveals TRIM47 as a strong candidate.

Cerebral small vessel disease is a leading cause of stroke and a major contributor to cognitive decline and dementia, but our understanding of specific genes underlying the cause of sporadic cerebral small vessel disease is limited. We report a genome-wide association study and a whole-exome association study on a composite extreme phenotype of cerebral small vessel disease derived from its most common MRI features: white matter hyperintensities and lacunes. Seventeen population-based cohorts of older persons with MRI measurements and genome-wide genotyping (n = 41 326), whole-exome sequencing (n = 15 965), or exome chip (n = 5249) data contributed 13 776 and 7079 extreme small vessel disease samples for the genome-wide association study and whole-exome association study, respectively. The genome-wide association study identified significant association of common variants in 11 loci with extreme small vessel disease, of which the chr12q24.11 locus was not previously reported to be associated with any MRI marker of cerebral small vessel disease. The whole-exome association study identified significant associations of extreme small vessel disease with common variants in the 5′ UTR region of EFEMP1 (chr2p16.1) and one probably damaging common missense variant in TRIM47 (chr17q25.1). Mendelian randomization supports the causal association of extensive small vessel disease severity with increased risk of stroke and Alzheimer’s disease. Combined evidence from summary-based Mendelian randomization studies and profiling of human loss-of-function allele carriers showed an inverse relation between TRIM47 expression in the brain and blood vessels and extensive small vessel disease severity. We observed significant enrichment of Trim47 in isolated brain vessel preparations compared to total brain fraction in mice, in line with the literature showing Trim47 enrichment in brain endothelial cells at single cell level. Functional evaluation of TRIM47 by small interfering RNAs-mediated knockdown in human brain endothelial cells showed increased endothelial permeability, an important hallmark of cerebral small vessel disease pathology. Overall, our comprehensive gene-mapping study and preliminary functional evaluation suggests a putative role of TRIM47 in the pathophysiology of cerebral small vessel disease, making it an important candidate for extensive in vivo explorations and future translational work.

Neuropharmacological evaluation of the methanol leaf extract of Phyllanthus muellerianus (Kuntze) Exell and its ethyl acetate fraction in mice

Purpose: To investigate the neuropharmacological effects of the methanol leaf extract (ME) and fractions of Phyllanthus muellerianus (PM) (Phyllanthaceae) (Kuntze) Exell (PM) in mice.&#x0D; Methods: Acute toxicity was carried out on the extract using standard protocol. ME was fractionated into hexane (HF), ethyl acetate (EF), and methanol (MF) fractions. Pentylenetetrazol (PTZ)-induced seizure, open field (OF) and motor coordination (rotarod) tests were models employed. Mice allotted into fourteen groups of six animals each were treated orally with 100, 200, or 400 mg/kg of the extract and fractions in pentylene tetrazole (PTZ) seizure model. Seizure was induced with intraperitoneal (ip) injection of 70 mg/kg of PTZ. The positive and negative controls employed were phenobarbitone (35 mg/kg) and 5 ml/kg of 7 % Tween 80, respectively. In the OF and motor coordination tests, six groups of six mice were treated orally with ME and EF at 200 and 400 mg/kg doses. Control groups received either 5 ml/kg of 7% Tween 80 or diazepam (1 mg/kg ip) as negative and positive controls respectively&#x0D; Results: In the PTZ model, only EF abolished seizures completely (p&lt;0.05), when compared with the negative control, producing 100% protection, even better than the phenobarbitone which gave 83.3% protection. In the OFT, in comparison with the control, ME at 400 mg/kg (p &lt; 0.05) decreased both the number of line crossing and the number of assisted rearing similar to that produced by diazepam. EF increased both the locomotor and exploratory activities significantly (p &lt; 0.05) in mice. ME at 400 mg/kg significantly (p &lt; 0.05) evoked reduction in the time of fall of mice from the rotarod when compared to the control in the same way as diazepam while EF did not elicit any appreciable differences.&#x0D; Conclusion: ME has anticonvulsant, sedative, and anxiolytic activities, while EF possesses anticonvulsant and anxiolytic activities devoid of sedative and cognitive impairment. The observed anticonvulsant effect was better than that produced by phenobarbitone. Thus, it may be a good lead for developing antiepileptic and other central nervous system active agents.