Littermate Mice Research Articles

STK25 acts as a regulator of leukocyte migration

Abstract The recruitment of leukocytes to sites of infection is an essential component of the innate immune response to pathogens. Defects in leukocyte migration can render the host susceptible to recurrent infections. In our efforts to identify new mediators of innate immunity, we found that serine/threonine protein kinase 25 (STK25) promotes TLR-induced proinflammatory cytokine production. While STK25 has previously been implicated in the regulation of cell migration, its role in leukocyte trafficking has not been defined. To investigate the involvement of STK25 in leukocyte migration, we evaluated baseline hematological parameters from Stk25-deficient (Stk25−/−) mice via a Hemavet. We found that Stk25−/− mice exhibit reduced circulating lymphocytes, neutrophils, and monocytes at steady-state compared to age- and gender-matched wild-type littermate (WT) mice. Immunophenotyping by flow cytometry confirmed these findings and revealed a significant reduction in the frequency of Ly6Chi inflammatory monocytes in Stk25−/− peripheral blood (PB) relative to WT. In contrast, we observed no apparent change in the frequencies of neutrophils and Ly6Chi inflammatory monocytes in Stk25−/− bone marrow, suggesting a defect in leukocyte migration. Finally, we examined the role of STK25 in neutrophil trafficking via an in vitro chemotaxis assay. We found that PB neutrophils from Stk25−/− mice displayed a defect in migration toward WT serum, suggesting a cell-intrinsic role for STK25 in the control of cell trafficking. Additionally, PB neutrophils from WT mice exhibited reduced migration toward Stk25−/− serum, supporting a cell-extrinsic function for STK25. Thus, our data support a new role for STK25 as a mediator of leukocyte trafficking.

Abstract 358: Smooth Muscle Cell Specific Beclin-1 Deficiency Accelerates Ascending And Abdominal Aortic Aneurysms In Male Hypercholesterolemic Mice Infused With Angiotensin Ii

Background and Objective: Ascending and abdominal aortic aneurysms (AAs) are permanent dilations of the ascending and abdominal aorta with a mortality greater than 80% after rupture. Vascular smooth muscle cells (SMCs) are pivotal in maintaining aortic structural integrity and function, and SMC-rich aortic medial stability is highly disrupted in AAs. Autophagy is a self-regulatory process by which cell digest, and recycles their cytoplasmic materials for energy purposes under stress. Recent clinical studies highlighted that Beclin-1, a gene indispensable for autophagy induction, is highly upregulated in AA tissues from patients. However, the functional contribution of SMC-derived Beclin-1 in the development of AAs is unknown. Using the well-established Angiotensin II infusion model of AAs, we examined the role of SMC-Beclin-1 during the development of AAs in hypercholesterolemic mice. Methods and Results: Mice with inducible deletion of Beclin-1 in SMCs were produced by breeding male mice hemizygous for Acta2-Cre ERT2 (Cre+) to female Beclin-1 floxed mice in an LDLR -/- background. At 8 weeks of age, male Beclin-1 x Acta2-Cre ERT2 (Cre+) and non-Cre littermates (Cre-) mice were injected with tamoxifen (75mg/kg, i.p.) for 5 consecutive days. After 2 weeks, Western blot analyses showed depletion of Beclin-1 protein in the aortic media from Cre+ mice compared to Cre- littermates. To study the role of Beclin-1in SMCs, male Cre+ and Cre- (N=5-6/group) mice were fed a fat-enriched diet (21% fat; 0.15% cholesterol) for 5 weeks. After 1 week of diet feeding, mice were infused subcutaneously with low dose of AngII (500 ng/kg/min) by osmotic mini-pumps for 4 weeks. Interestingly, depletion of Beclin-1 in SMCs significantly accelerated AngII-induced aortic luminal dilation (Cre-: 1.0 ± 0.02 mm, Cre+: 1.5 ± 0.14 mm; P < 0.001), and expansion of ex-vivo maximal diameters of abdominal aortas in mice (Cre-: 1.1 ± 0.1 mm, Cre+: 1.52 ± 0.1 mm; P < 0.05). In addition, SMC- Beclin-1 depletion significantly increased AngII-induced ascending aortic aneurysms (Cre-: 12.6 ± 0.4 mm 2 , Cre+: 20.9 ± 1.2 mm 2 ; P=0.008 ). Conclusion: These findings demonstrate that SMC-Beclin-1 plays a critical role in suppressing AngII-induced ascending and abdominal aortic aneurysm formation in mice.

Loss of Club Cell Creb Mitigates IL‐1B–mediated Muco‐obstructive Phenotypes in Male Murine Airways

Goblet cell metaplasia and/or mucus hypersecretion are hallmark features of multiple lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis and pulmonary fibrosis. Interleukin 1β (IL‐1B) is a proinflammatory mediator that increases expression of the major gel forming mucins in the airways, Muc5b and Muc5ac, in part through activation of the cAMP response element binding protein (Creb) transcription factor. IL‐1B also induces Spdef, a key regulator of goblet cell differentiation from airway club cells. Here we tested the hypothesis that elimination of murine club cell Creb mitigates muco‐obstructive phenotypes induced by IL‐1B. Mice with floxed Creb1 were bred to Scgb1atm1(cre/ERT)Blh to generate mice with conditional loss of Creb1 from club cells. Wild‐type and transgenic male mice (7‐9/group) littermates received intranasal IL‐1B (0.5 ng/inhalation) or saline (control) for four consecutive days, with tamoxifen administration occurring on days 1 and 3 to induce Cre recombinase expression. Periodic acid–Schiff staining in lung samples showed that IL‐1B increased the number of goblet cells in central airways compared to saline controls. This effect was blunted by conditional loss of Creb1. Similarly, flexiVent measurements demonstrated that IL‐1B increased airway resistance (R), Newtonian resistance (Rn), tissue elastance (ERS), tissue damping (G) and tissue elasticity (H). Loss of club cell Creb1 mitigated all effects of IL‐1B on airway mechanics. Additionally, though the percentage of granulocytes in the bronchoalveolar lung lavage fluid was not increased by IL‐1B, loss of club cell Creb1 decreased the percentage of granulocytes in the bronchoalveolar lung lavage fluid independent of treatment (i.e., saline or IL‐1B). These findings suggest that the muco‐obstructive effects of IL‐1B are dependent upon club cell Creb1 and provide novel insight for therapeutic strategies.

Mutation in the 5'SL Region of Collagen I mRNA Attenuates Diastolic Dysfunction in Male, but not Female, Mice Following 28 Weeks of Feeding a High Fat High Sucrose Diet

IntroductionThe La ribonucleoprotein 6 (LARP6), an RNA binding protein, increases type I collagen expression by regulating mRNA stability and translation. This is mediated via its interaction with the 5’ stem loop (5’SL) structure in collagen I mRNA. Disrupting this interaction by mutating the 5’SL region has been shown to reduce fibrotic remodeling in liver. Therefore, we tested the hypothesis that this interaction plays a role in cardiac fibrosis and stiffening in diet induced obesity in 5’SL mutant and wild‐type littermate mice. In fact, obesity and hypertension promote remodeling and cardiac stiffening due to excess collagen deposition, fibrosis, and microvascular dysfunction, that ultimately lead to diastolic dysfunction.MethodsMale and female 5’SL mice and wild‐type (WT) littermates were fed a high fat high sucrose (HFHS) or control diet starting at 6 weeks of age. After 28 weeks of feeding, mice were assessed for cardiac function by echocardiography. Hearts were collected and a transverse section of the left and right ventricle was fixed for histology. RNA was isolated from the left ventricle and submitted for mRNA sequencing and qPCR.ResultsFollowing 28 weeks of a HFHS diet, male and female mice showed increased body weight, cardiac hypertrophy (heart weight normalized to tibia length), and diastolic dysfunction. However, the 5’SL mutation attenuated the impaired diastolic function in male, but not female mice. Surprisingly, cardiac fibrosis was not altered by the HFHS diet. RNA sequencing revealed that HFHS diet altered gene expression associated with glucose and ketone metabolism, as well as inflammation, in male WT littermates. However, the 5’SL mutation enriched gene networks associated with vascular development and angiogenesis.ConclusionsChronic HFHS feeding leads to increased body weight, hypertrophic cardiomyopathy, and diastolic dysfunction in both male and female mice. However, the 5’SL mutation blunts dysfunction only in male mice, demonstrating sex‐dependent cardioprotective effects.

Fragile X Mental Retardation Protein Mediates the Effects of Androgen on Hippocampal PSD95 Expression and Dendritic Spines Density/Morphology and Autism-Like Behaviors Through miR-125a.

Dysregulated synaptic plasticity is a key feature of neurodevelopmental disorders, including autism. This study investigated whether Fragile X mental retardation protein (FMRP), a selective RNA-binding protein that regulates synaptic protein expression by interacting with miRNAs, mediates the effects of androgens that play an important role in regulating the synaptic plasticity in the hippocampus. Experiments using mouse hippocampal neuron HT22 cells demonstrated that dihydrotestosterone (DHT) increased the expression of postsynaptic density protein 95 (PSD95) by inhibiting FMRP expression. Administration of miR-125a inhibitor upregulated the PSD95 expression and significantly increased the DHT-induced upregulation of PSD95. FMRP knockdown in HT22 cells reduced the expression of miR-125a. Moreover, miR-125a inhibitor upregulated the PSD95 expression in the DHT-treated HT22 cells with FMRP knockdown. Subsequently, the effects of androgen-mediated via FMRP in regulating neural behaviors and PSD95 expression and dendritic spines density/morphology were investigated using Fmr1 knockout (KO) and wild-type littermate (WT) mice. The castration of WT mice reduced the androgen levels, aggravated anxiety and depression, and impaired learning and memory and sociability of mice. DHT supplementation post-castration reversed the alterations in density and maturity of dendritic spines of hippocampal neurons and behavioral disorders in WT mice; however, it did not reveal such effects in Fmr1 KO mice. Further, immunohistochemical staining and western blotting analyses after knocking down miR-125a revealed similar effects of castration and post-castration DHT supplementation on PSD95 protein expression. These findings clarified that FMRP mediated the effects of DHT through miR-125a in regulating the expression of hippocampal synaptic protein PSD95. This study provides evidence for the neuroprotective mechanism of androgen in PSD95 expression and dendritic spines density/morphology and suggests that treatment interventions with androgen could be helpful for the management of synaptic plasticity disorders.

Type 2 Diabetic Mellitus Inhibits Skin Renewal through Inhibiting WNT-Dependent Lgr5+ Hair Follicle Stem Cell Activation in C57BL/6 Mice.

Background Hair follicles are important accessory organs of the skin, and it is important for skin renewal and performs variety of important functions. Diabetes can cause several dermatoses; however, its effect on hair follicles is unclear. The purpose of this study was to investigate the effect of type II diabetes (T2DM) on the hair follicles of mice. Methods Seven-week-old male C57BL/6 littermate mice were divided into two groups. The treatment group was injected with streptozotocin (STZ) to induce T2DM, and the control group was parallelly injected with the same dose of buffer. Seven days after injection, the back is depilated to observe the hair follicle regeneration. Hair follicle regeneration was observed by naked eyes and HE staining. The proliferation of the skin cells was observed by PCNA and K14 staining. The altered genes were screened by RNA sequencing and verified by qRT-PCR. In addition, Lgr5 + GFP/mTmG transgenic mice were used to observe the effect of T2DM on Lgr5 hair follicle stem cells (HFSC). And the expression of WNT4 and WNT8A were measured by Western Blot. Results T2DM inhibited hair follicle regeneration. Compared to control mice, T2DM mice had smaller hair follicles, reduced skin thickness, and less expression of PCNA and K14. RNA sequencing showed that the two groups had significant differences in cell cycle and proliferation-related pathways. Compared with the control mice, the mRNA expression of Lgr4, Lgr5, Wnt4, and Wnt8a was decreased in the T2DM group. Moreover, T2DM inhibited the activation of Lgr5 HFSC and the expression of WNT4 and WNT8A. Conclusions T2DM inhibited hair follicle regeneration and skin cells proliferation by inhibiting WNT-dependent Lgr5 HFSC activation. This may be an important reason for the reduction of skin renewal ability and the formation of chronic wounds caused by diabetes. It is important for the treatment of chronic diabetic wounds and the development of tissue engineering.

Interleukin-1 Mediates Ischemic Brain Injury via Induction of IL-17A in γδ T Cells and CXCL1 in Astrocytes

As a prototypical proinflammatory cytokine, interleukin-1 (IL-1) exacerbates the early post-stroke inflammation, whereas its neutralization is protective. To further investigate the underlying cell-type-specific IL-1 effects, we subjected IL-1 (α/β) knockout (Il1−/−) and wildtype (WT) littermate mice to permanent middle cerebral artery occlusion (pMCAO) and assessed immune cell infiltration and cytokine production in the ischemic hemisphere by flow cytometry 24 h and 72 h after stroke. Il1−/− mice showed smaller infarcts and reduced neutrophil infiltration into the ischemic brain. We identified γδ T cells and astrocytes as target cells of IL-1 signaling-mediated neutrophil recruitment. First, IL-1-induced IL-17A production in γδ T cells in vivo, and IL-17A enhanced the expression of the main neutrophil attracting chemokine CXCL1 by astrocytes in the presence of tumor necrosis factor (TNF) in vitro. Second, IL-1 itself was a potent activator of astrocytic CXCL1 production in vitro. By employing a novel FACS sorting strategy for the acute isolation of astrocytes from ischemic brains, we confirmed that IL-1 is pivotal for Cxcl1 upregulation in astrocytes in vivo. Our results underscore the pleiotropic effects of IL-1 on immune and non-immune cells within the CNS to mount and amplify the post-stroke inflammatory response.

Commensal gut bacterium critically regulates alveolar bone homeostasis

AbstractThe alveolar bone is a unique osseous tissue due to the presence of the teeth and the proximity of commensal oral microbes. Commensal microbe effects on alveolar bone homeostasis have been attributed to the oral microbiota, yet the impact of commensal gut microbes is unknown. Study purpose was to elucidate whether commensal gut microbes regulate osteoimmune mechanisms and skeletal homeostasis in alveolar bone. Male C57BL/6T germfree (GF) littermate mice were maintained as GF or monoassociated with segmented filamentous bacteria (SFB), a commensal gut bacterium. SFB has been shown to elicit broad immune response effects, including the induction of TH17/IL17A immunity, which impacts the development and homeostasis of host tissues. SFB colonized the gut, but not oral cavity, and increased IL17A levels in the ileum and serum. SFB had catabolic effects on alveolar bone and non-oral skeletal sites, which was attributed to enhanced osteoclastogenesis. The alveolar bone marrow of SFB vs. GF mice had increased dendritic cells, activated helper T-cells, TH1 cells, TH17 cells, and upregulated Tnf. Primary osteoblast cultures from SFB and GF mice were stimulated with vehicle-control, IL17A, or TNF to elucidate osteoblast-derived signaling factors contributing to the pro-osteoclastic phenotype in SFB mice. Treatment of RAW264.7 osteoclastic cells with supernatants from vehicle-stimulated SFB vs. GF osteoblasts recapitulated the osteoclast phenotype found in vivo. Supernatants from TNF-stimulated osteoblasts normalized RAW264.7 osteoclast endpoints across SFB and GF cultures, which was dependent on the induction of CXCL1 and CCL2. This report reveals that commensal gut microbes have the capacity to regulate osteoimmune processes in alveolar bone. Outcomes from this investigation challenge the current paradigm that alveolar bone health and homeostasis is strictly regulated by oral microbes.Commensal microbe effects on alveolar bone homeostasis have been attributed to the oral microbiota, yet the impact of commensal gut microbes is unknown. Studies performed with segmented filamentous bacteria (SFB) monoassociated mice revealed commensal gut microbes modulate osteoimmune responses and skeletal homeostasis in alveolar bone. This work challenges the current paradigm that alveolar bone health is strictly regulated by oral microbes.

Dietary salt initiates redox signaling between endothelium and vascular smooth muscle through NADPH oxidase 4

Prevention of phenotype switching of vascular smooth muscle cells is an important determinant of normal vascular physiology. Hydrogen peroxide (H2O2) promotes osteogenic differentiation of vascular smooth muscle cells through expression of Runt related transcription factor 2 (Runx2). In this study, an increase in dietary NaCl increased endothelial H2O2 generation through NOX4, a NAD(P)H oxidase. The production of H2O2 was sufficient to increase Runx2, osteopontin and osteocalcin in adjacent vascular smooth muscle cells from control littermate mice but was inhibited in mice lacking endothelial Nox4. A vascular smooth muscle cell culture model confirmed the direct involvement of the activation of protein kinase B (Akt) with inactivation of FoxO1 and FoxO3a observed in the control mice on the high NaCl diet. The present study also showed a reduction of catalase activity in aortas during high NaCl intake. The findings demonstrated an interesting cell-cell communication in the vascular wall that was initiated with H2O2 production by endothelium and was regulated by dietary NaCl intake. A better understanding of how dietary salt intake alters vascular biology may improve treatment of vascular disease that involves activation of Runx2.

Inhibition of endothelial Nox2 activation by LMH001 protects mice from angiotensin II-induced vascular oxidative stress, hypertension and aortic aneurysm

Endothelial oxidative stress and inflammation attributable to the activation of a Nox2-NADPH oxidase are key features of many cardiovascular diseases. Here, we report a novel small chemical compound (LMH001, MW = 290.079), by blocking phosphorylated p47phox interaction with p22phox, inhibited effectively angiotensin II (AngII)-induced endothelial Nox2 activation and superoxide production at a small dose (IC50 = 0.25 μM) without effect on peripheral leucocyte oxidative response to pathogens. The therapeutic potential of LMH001 was tested using a mouse model (C57BL/6J, 7-month-old) of AngII infusion (0.8 mg/kg/d, 14 days)-induced vascular oxidative stress, hypertension and aortic aneurysm. Age-matched littermates of p47phox knockout mice were used as controls of Nox2 inhibition. LMH001 (2.5 mg/kg/d, ip. once) showed no effect on control mice, but inhibited completely AngII infusion-induced excess ROS production in vital organs, hypertension, aortic walls inflammation and reduced incidences of aortic aneurysm. LMH001 effects on reducing vascular oxidative stress was due to its inhibition of Nox2 activation and was abrogated by knockout of p47phox. LMH001 has the potential to be developed as a novel drug candidate to treat oxidative stress-related cardiovascular diseases.

Vitamin D receptor deficiency increases systolic blood pressure by upregulating the renin-angiotensin system and autophagy.

The vitamin D receptor (VDR) may regulate blood pressure via multiple pathways. The present study investigated the underlying mechanism by which VDR deficiency increases blood pressure. A total of 16 8-week-old male littermate mice were randomly divided into the VDR knockout and wild-type groups (VDR-/- and VDR+/+, respectively). Blood pressure was measured using a four-channel PowerLab data acquisition and ADI software analysis system. After euthanasia, vascular smooth muscle cells (VSMCs) were isolated from the VDR-/- and VDR+/+ mice. Oxidative stress, renin-angiotensin system (RAS) activation and autophagy markers were measured in the isolated VSMCs using reverse transcription-quantitative PCR (RT-qPCR), western blotting and transmission electron microscopy (TEM) assays. Mean systolic pressure was significantly higher in the VDR-/- mice compared with the VDR+/+ mice. RT-qPCR and western blotting analyses indicated that RAS markers (angiotensin II and II type 1 receptor) were significantly upregulated, oxidative stress was increased (evidenced by reduced superoxide dismutase and peroxiredoxin-4) and autophagy was activated (upregulation of autophagy related protein 7, Beclin 1 and microtubule-associated proteins 1A/1B light chain 3A) in the VDR-/- VSMCs compared with the VDR+/+ VSMCs. TEM demonstrated that there were more autophagy bodies in the VDR-/- VSMCs compared with the VDR+/+ VSMCs. In conclusion, VDR deficiency was associated with high blood pressure. The mechanism underlying the increase in blood pressure caused by VDR deficiency may involve activation of the RAS, as well as increased oxidative stress and autophagy of VSMCs.

Amyloid Beta Pathology Exacerbates Weight Loss and Brain Cytokine Responses following Low-Dose Lipopolysaccharide in Aged Female Tg2576 Mice.

Systemic inflammation has been implicated in the progression of Alzheimer’s disease (AD); however, less is understood about how existing AD pathology contributes to adverse outcomes following acute inflammatory insults. In the present study, our goal was to determine how AD-associated amyloid beta (Aβ) pathology influences the acute neuroinflammatory and behavioral responses to a moderate systemic inflammatory insult. We treated 16–18-month-old female Tg2576 (Tg) mice, which overproduce human Aβ and develop plaques, and age-matched wild-type (WT) littermate mice with an intraperitoneal injection of 0.33 mg/kg lipopolysaccharide (LPS) or saline. Mice were then evaluated over the next 28 h for sickness/depressive-like behaviors (food intake, weight loss, locomotion, and sucrose preference), systemic inflammation (serum amyloid A, SAA), blood-brain barrier (BBB) disruption, astrogliosis (glial fibrillary acidic protein/GFAP), Aβ, and cytokine levels in the brain. We found that LPS caused a larger reduction in body weight in Tg vs. WT mice, but that other behavioral responses to LPS did not differ by genotype. BBB disruption was not apparent in either genotype following LPS. Concentrations of the systemic inflammatory marker, SAA, in the blood and brain were significantly increased with LPS but did not significantly differ by genotype. GFAP was increased in Tg mice vs. WT but was not significantly affected by LPS in either genotype. Finally, LPS-induced increases of eight cytokines (IL-1β, IL-6, IL-12 (p40), IL-10, IL-17A, MIP-1α/CCL3, MIP-1β/CCL4, and RANTES/CCL5) were found to be significantly higher in Tg mice vs. WT. In summary, our data show that Aβ pathology exacerbates the neuroinflammatory response to LPS and identifies cytokines that are selectively regulated by Aβ. The association of worse neuroinflammation with greater weight loss in Tg mice suggests that Aβ pathology could contribute to poor outcomes following a systemic inflammatory insult.

Abstract TMP110: Impairment Of Efferocytosis Promotes The Rupture Of Intracranial Aneurysm

Background: Efferocytosis is an apoptotic cell clearing process that reduces the tissue-damaging effects of excessive apoptotic cells. Mer receptor tyrosine kinase (Mertk) is present on the surface of phagocyte cells, such as macrophages, and mediates efferocytosis of apoptotic cells. Cleavage of Mertk by metalloproteinases impairs efferocytosis. Several studies suggested that the severity of atherosclerosis, myocardial infarction, and aortic aneurysms were increased in Mertk KO mice due to the impairment of efferocytosis. Higher levels of apoptosis are found in ruptured aneurysms compared to in unruptured aneurysms, suggesting impaired efferocytosis in rupture-prone aneurysms. However, whether impaired efferocytosis plays a role in the pathophysiology of intracranial aneurysms is unclear. In this project, we investigated the effect of impaired efferocytosis in intracranial aneurysms. Hypothesis: We hypothesize that impairment of efferocytosis promotes the rupture of intracranial aneurysms. Methods: We induced intracranial aneurysms by combining systemic hypertension and a single injection of elastase into the cerebrospinal fluid in mice. Aneurysm rupture rate was used as the primary endpoint. We used Mertk knockout (KO) mice. Results: There was no statistical difference in the formation rate of aneurysms between litter-mate control wild-type mice (WT) and Mertk KO mice. Interestingly, the aneurysmal rupture rate was significantly higher in Mertk KO mice (Mertk vs. WT: 94.7% vs. 61.5%, P < 0.05%). Conclusions: Our experiment suggested that impairment of efferocytosis promotes aneurysm rupture. Inhibiting cleavage of Mertk may be a strategy for preventing the rupture of intracranial aneurysms.

The G Protein-Coupled Receptor, VPAC1, Mediates Vasoactive Intestinal Peptide-Dependent Functional Homeostasis of the Gut Microbiota.

Vasoactive intestinal peptide (VIP) is a neuropeptide involved in the regulation of feeding behavior and circadian rhythms, metabolism, and immunity. Previous studies revealed the homeostatic effects of VIP signaling on the gut microbiota. VIP-deficient mice demonstrate a gut microbiota dysbiosis characterized by reduced α-diversity and decreased relative abundance (RA) of Gram-positive Firmicutes. However, the mechanism by which VIP signaling affects changes in the microbiota is unknown. To investigate the role of the 2 cognate G protein-coupled receptors for VIP (VPAC1 and VPAC2) in VIP-mediated homeostasis of the microbiota, fecal samples from VPAC1- and VPAC2-deficient, heterozygous, and wild-type littermate mice were assessed via targeted amplicon sequencing. Their microbiota profiles were additionally compared with microbiota from VIP-deficient, heterozygous, and wild-type littermates, where genotype-dependent changes in the composition and predicted function of each cohort were compared. While wild-type mice in each line differed in α-diversity and β-diversity, consistent changes in both metrics were observed in VIP-deficient and VPAC1-deficient mice. This includes a dramatic reduction in α-diversity, increased RA of Proteobacteria and Bacteroidetes, and decreased RA of Lachnospiraceae, Ruminococcaceae, Muribaculaceae, and Rikenellaceae. Specific amplicon sequence variants and predicted functions found to differ significantly based on VIP or VPAC1 genotype were concordant in their directions of change. Multiplatform predicted functional profiling suggested a defective VIP-VPAC1 axis was associated with reduced amino acid degradation along with reduced quinol and quinone biosynthesis. Furthermore, alterations in predicted functions include increased sugar degradation, nitrate reduction, and fatty acid biosynthetic pathways, among other changes. We conclude that VIP signaling through VPAC1 is critical for the maintenance of normal function of the gut microbiota.

Systemic inflammation elicits distinct brain immune signaling dynamics in female and male mice with AD pathology

Women are disproportionately affected by Alzheimer's disease (AD), even after adjusting for age and socioeconomic status. Sex-based biological differences in immune response, including hormonal and X-linked immune genes, may contribute to differences in disease progression. However, we have limited understanding of the role of sex in modulating immune response and how these differences are affected by AD pathology. Here, we hypothesized that female and male mice would exhibit distinctive neural immune signaling in response to challenge with the pro-inflammatory stimulus lipopolysaccharides (LPS), and that these differences would be exacerbated in model of AD pathology compared to wild-type controls.Female and male six-month-old 5xFAD amyloid beta (Aβ) and wild type (WT) littermate mice were interperitoneally injected with LPS or saline vehicle once daily for 0,1, or 4 days (N=4). Luminex multiplexed ELISAs was used to quantify inflammatory cytokines, MAPK intracellular signaling phospho-proteins in cortical tissues.In the saline group, we observed no differences between females and males in either 5xFAD or WT mice. Additionally, in wild type mice, we observed no significant differences between females and males after either one or four daily injections of LPS. In contrast, in 5xFAD mice injected with LPS, we found significant differences between females and males after either one or four once daily injections. In particular, female 5xFAD mice after a single injection exhibited significantly increased profile (p=0.014, two tailed t-test) of phospho-proteins and cytokines, including MEK, cJUN, IL-13, IL-17, IL-10, and IL-1α compared to males. After four once-daily LPS injections, female 5xFAD mice exhibited a significantly different profile (p=0.016, two tailed t-test) consisting of increased phosphorylation of ATF2, Mek, cJUN, and HSP-27, together with decreased expression of multiple proinflammatory cytokines including IL-9, VEGF, MIP-1β compared to males.Our data reveal that although sex is not a significant determinant of neural immune response in WT mice, it significantly affects neural immune response to LPS challenge in 5xFAD mice. These data suggest distinctive immune processes in female vs male 5xFAD mice that are not present in healthy controls. Future work will consider the effects of Aβ pathology on these results.

L-type calcium channel subtypes mediating hypothalamic dysfunction caused by amyloid-beta.

In Alzheimer's disease (AD), non-cognitive manifestations attributable to hypothalamic dysfunction such as systemic metabolic deficits can occur prior to the cognitive decline (Ishii, M. and Iadecola C., Cell Metabolism 22:761,2015). We previously found in Tg2576 mice overexpressing the Swedish mutation of amyloid precursor protein that amyloid-beta pathology can cause dysfunction in hypothalamic arcuate neuropeptide Y (NPY) neurons, associated with low adiposity and low plasma leptin levels prior to memory decline or plaque formation (Ishii, M. et al., J Neuroscience 34:9096,2014). Furthermore, the hypothalamic arcuate NPY neuronal dysfunction was caused by amyloid-beta modulating low-threshold activated L-type calcium channels (Ishii, M. et al., J Neuroscience 39:8816,2019). However, which of the two major brain L-type calcium channel subtypes Cav 1.2 or Cav 1.3 is involved has not been established. To examine the role of Cav 1.2, we used the Cre-lox system in genetic mouse models to knock-out Cav 1.2 in hypothalamic arcuate NPY neurons (Cav 1.2 cKO). Cav 1.2 cKO mice were bred to NPY-GFP and Tg2576 mice. Two to 3-month-old male or female mice were used for all studies. Voltage-gated L-type calcium currents from hypothalamic slices containing arcuate NPY neurons were recorded using whole-cell voltage-clamp. Cytoplasmic Ca2+ levels were measured ratiometrically by loading Fura-2AM in dissociated arcuate NPY neurons and recording the emitted fluorescence using an inverted fluorescent microscope. Exogenous oligomeric amyloid-β42 caused a significant increase in cytoplasmic Ca2+ levels and L-type calcium currents in arcuate NPY neurons to a similar extent in both Cav 1.2 cKO and wild-type (WT) littermate mice. These effects of amyloid-β42 were reversed by the L-type calcium channel blocker nimodipine. In vivo, Cav 1.2 cKO mice bred to Tg2576 mice had similar body weight deficits compared to Tg2576 mice without deletion of Cav 1.2 in the arcuate NPY neurons. These results suggest that Cav 1.2 does not have a significant role in amyloid-beta mediated dysfunction of hypothalamic arcuate NPY neurons and that Cav 1.3 is likely the major target of amyloid-beta in these neurons. Future studies modulating Cav 1.3 in hypothalamic neurons will help elucidate the mechanisms underlying hypothalamic dysfunction caused by amyloid-beta pathology and its therapeutic potential in AD.

Sex-specific behavioral impairments produced by neonatal exposure to MK-801 are partially reversed by adolescent CDPPB treatment

Psychomimetic behaviors manifest in adult rodents long after neonatal exposure to the noncompetitive NMDA receptor antagonist MK-801. In the present study, we used this neurodevelopmental model of schizophrenia to evaluate the therapeutic potential of positive allosteric modulation of metabotropic glutamate receptor 5 (mGluR5) during adolescence. To this end, we randomly assigned male and female C57BL6 mouse littermates to one of three treatment groups: (i) neonatal and adolescent saline, (ii) neonatal MK-801 (0.25 mg/kg) and adolescent saline, and (iii) neonatal MK-801 and adolescent CDPPB (10 mg/kg), a positive allosteric modulator of mGluR5. When animals reached adulthood, a wide range of behavioral tests were conducted including sucrose preference, anxiety assessment in the elevated plus maze, and a series of food-reinforced operant procedures meant to assess motor activity, motivation, learning, and attention. Neonatal MK-801 exposure produced profound motor hyperactivity in both sexes and attenuated sucrose preference in males, effects that were reversed by CDPPB. MK-801 produced other deficits such as impaired set shifting or response inhibition deficits that were not reversed by CDPPB. Overall, female mice were more susceptible to MK-801's behavioral effects than males. These findings further support the use of neonatal MK-801 exposure as an animal model of schizophrenia and suggest that CDPPB can reverse the neurodevelopmental progression of some schizophrenia-like behaviors.

Abstract 10832: Transient Receptor Potential Melastatin 7 (TRPM7) Contributes to Myocardial Ischemic Injury

Introduction: Ischemic heart disease is the leading cause of death in the USA. Despite the progress of interventional coronary reperfusion strategies, myocardial ischemia reperfusion (I/R) injury remains a substantial challenge for treatment. TRPM7 is a Ca 2+ -permeable nonselective channel which plays an important role in cardiogenesis and atrial fibrillation. However the role of TRPM7 in pathogenesis of myocardial I/R is unknown. Hypothesis: TRPM7 contributes to myocardial I/R injury. Methods: TRPM7 knockout mice (M7KO) and Cre - control littermates (WT) mice were exposed to 45 min ischemia or sham procedure followed by 24 hrs of reperfusion (n=8 to 12/group). Heart function was evaluated by echocardiograph, and in vitro assays were used for mechanistic investigation. Results: We found that TRPM7 expression was upregulated in I/R injury WT mouse hearts. Global M7KO (gM7KO) improved heart performance via increasing the ejection fraction and fraction shortening by 29.1 ± 4.2 % and 10.8 ± 3.0 % respectively (p<0.05). Trpm7 deletion also reduced the infarct size by 31.1 ± 1.2 % in comparison with WT mice (P<0.05). Myocytes specific knockout (mM7KO) exhibited similar protective effect against I/R injury. Moreover, deletion of Trpm7 significantly reduced tunnel positive cells, suggesting that upregulated TRPM7 in WT mice contributed to myocyte apoptosis in I/R injury. Interestingly, deletion of Trpm7 also significantly reduced the protein level of NLRP3, cleaved-caspase-1 and mature IL-1β after I/R injury (p<0.05), indicating that TRPM7 contributed to pyroptosis during I/R injury. Consistent with the increased IL-1β level induced by I/R injury in WT mice, the cleaved N-terminal Gasdermin D (N-GSDMD) level was also increased in WT I/R injury hearts versus sham control hearts. Deletion of Trpm7 significantly attenuated the increase of the cleaved N-GSDMD level induced by I/R injury (p<0.05). Conclusions: Trpm7 deletion in cardiac myocytes protects mice against myocardial ischemic injury. This is the first study to demonstrate the important role of TRPM7 in I/R injury through the mechanisms of activation of both apoptosis and pyroptosis. Our results suggest that TRPM7 may serve as a novel therapeutic target for myocardial ischemia/reperfusion injury.

Abstract 10955: The Effects of Acute and Chronic Selective Phosphodiesterase 1 Inhibition in Associated Smooth Muscle Cell-Specific Aging Features

Introduction: Aging-related vascular smooth muscle cell (VSMC) remodeling plays an essential role in cardiovascular diseases. Reduced NO-cGMP pathway signalling is a major feature and pathogenic mechanism underlying VSMC dysfunction. Recently, we found that phosphodiesterase (PDE) 1, an enzyme that hydrolyzes and inactivates the cyclic nucleotides cAMP and cGMP, is a potential treatment target for restoring age-related deficits in VSMC signalling. We here tested the effects of PDE1 inhibition in SMC-specific Ercc1 DNA repair mutant mice, a model of isolated, accelerated SMC aging. Design and Method: SM22α Cre+ Ercc1 fl/- (SMC-KO) and SM22α Cre+ Ercc1 fl/+ littermate (SMC-LM) mice receive either vehicle or the PDE1 inhibitor lenrispodun (dose 40mg/kg/day) in drinking water for 8 weeks. Reactive hyperemia was measured in-vivo at the age of 14 and 25 weeks. Mice were sacrificed at 26 weeks. The endothelium-dependent (ED) and -independent (EI) relaxation of the thoracic aorta was assessed by wire myography. Moreover, we investigated the effect of PDE1 inhibition on the expression of selected PDEs involved in the vasculature by qPCR. Results: Reactive hyperemia was dramatically reduced in vehicle-treated SMC-KO vs. SMC-LM at the age of 25 weeks (P=0.0002) but was preserved in lenrispodun-treated SMC-KO mice. In vehicle-treated SMC-KO mice, the ED and EI relaxations were greatly diminished compared to SMC-LM (ED: 23.92±4.34 vs. 68.04±4.86, P<0.0001; EI: 57.93±5.31 vs. 87.43±4.01, P=0.0004). Acute administration of lenrispodun in the organ bath improved both ED and EI responses (ED: 38.61±4.04, P=0.001; EI 69.99±4.29 P=0.01) while the relaxations remained unchanged after chronic lenrispodun treatment. In addition, lenrispodun upregulated PDE1a mRNA expression in SMC-KO, while no changes were observed in other PDEs. Conclusions: PDE1 inhibition may therapeutically be useful in restoring age-related vascular dysfunction by preserving microvascular function and potentiating NO-cGMP signaling. Yet, its chronic effects might be counteracted by the upregulation of PDE1a in vasculature. These results warrant further pharmacodynamic profiling of PDE enzyme regulation during chronic PDE1 inhibitor treatment.

Abstract 11418: Fibrosis and Calcium Signaling Regulation Are Critical Mechanisms in a Three-Hit HFpEF Mouse Model

Introduction: There is a lack of animal models of heart failure with preserved ejection fraction (HFpEF). Hypothesis: A mouse model with increased Ca2+ influx receiving a high-fat diet (HFD) and L-NAME (3 hits) could be a model for HFpEF to help reveal the mechanism. Methods: Cardiomyocyte (CM) specific, inducible Cavβ2a low expression transgenic (Cavβ2a LE TG) and their control littermate mice were fed with either chow diet (LC and CC), HFD+L-NAME (LHLn, CHLn), or HFD+L-NAME+SAHA (LHLnS, CHLnS) since the age of 4months for 6 months. Echocardiography had been done monthly. Hemodynamic analysis, histological analyses, and Western blotting (WB) had been used to figure out the possible mechanisms after euthanization. Results: LHL (3 hits) mice had preserved left ventricular ejection fraction (LVEF) but the highest levels of ANP/BNP and the lowest strain rate. 3-Hit led to obvious diastolic dysfunction indicated by a higher ratio of mitral E wave to mitral annulus e’ velocity (E/e’ ratio), ventricular stiffness index, end-diastolic pressure, and left atrial (LA) dilation rate (90% in LHL mice). LA and LV fibrosis were more severe in the LHL group. Fibrosis-related molecules, including lysyl oxidase, Periostin, transforming growth factor-beta and its downstream transcription factor SMAD2/3 were higher in the LHL group. LHL mice also had lower PLB phosphorylation at Thr17, less SERCA, more NCX, and increased TnI phosphorylation. SAHA treatment could significantly reverse the HFpEF phenotype. Conclusions: 3-Hit is a model for HFpEF, which can be reversed by SAHA treatment. Fibrosis and altered Ca2+ signaling are critical underlying mechanisms for HFpEF.