Wild-type Control Mice Research Articles

Age-associated increase in anxiety-like behavior in Lpaatδ/Agpat4 knockout mice

• Rearing activity is elevated in 14- and 20-month old Lpaatδ −/− mice. • 20 month-old Lpaatδ −/− mice show increased anxiety-like behavior in the E.P.M. and O.F.T. • NMDA receptor levels are not significantly different in aged Lpaatδ −/− mice. • Brain expression of serotonin regulatory genes 5htr1a and slc6a4 is lower in 20 month-old Lpaatδ −/− mice. We have previously reported deficits in phospholipids and NMDA-receptors in brains of 2–3 month-old mice deficient in the mitochondrial enzyme lysophosphatidic acid acyltransferase delta (LPAATδ)/acylglycerophosphate acyltransferase 4 (AGPAT4), which were associated with deficits in learning and memory. An initial assessment suggested these mice may also be predisposed to an anxiogenic-like behavioral phenotype, but this was not fully evident in young mice. Since some animal and human studies report worsening anxiety from middle-to-older adulthood, we hypothesized that aging may reveal a significant anxiety-like phenotype in Lpaatδ -deficient mice. We analyzed anxiety-like behavior in 14- and 20-month old Lpaatδ deficient (Lpaat δ −/− ) mice and their wildtype age-matched littermate controls. Fourteen month-old Lpaatδ -deficient mice had ∼3-fold greater rearing activity over 24-h than controls. Aging was associated with a significant decrease in rearing activity in Lpaatδ -deficient mice, but not in wildtype control mice. However, rearing activity remained 59% higher in Lpaatδ -deficient mice than controls at 20 months of age. Behavior of Lpaatδ -deficient mice in the open field test and elevated plus maze also suggested a more anxiogenic-like phenotype, although differences between the genotypes were statistically significant only at 20 months. Brain NMDA receptor subunits were not significantly different between different ages or genotypes, which differed from prior results in younger mice. An initial screen of genes related to behavior indicated lower brain expression in Lpaatδ -deficient mice of the serotonin metabolism genes 5htr1a and slc6a4. These studies have implications for understanding associations between LPAATδ/AGPAT4 gene variants and human anxiety.

Essential role of M1 macrophages in blocking cytokine storm and pathology associated with murine HSV-1 infection.

Ocular HSV-1 infection is a major cause of eye disease and innate and adaptive immunity both play a role in protection and pathology associated with ocular infection. Previously we have shown that M1-type macrophages are the major and earliest infiltrates into the cornea of infected mice. We also showed that HSV-1 infectivity in the presence and absence of M2-macrophages was similar to wild-type (WT) control mice. However, it is not clear whether the absence of M1 macrophages plays a role in protection and disease in HSV-1 infected mice. To explore the role of M1 macrophages in HSV-1 infection, we used mice lacking M1 activation (M1-/- mice). Our results showed that macrophages from M1-/- mice were more susceptible to HSV-1 infection in vitro than were macrophages from WT mice. M1-/- mice were highly susceptible to ocular infection with virulent HSV-1 strain McKrae, while WT mice were refractory to infection. In addition, M1-/- mice had higher virus titers in the eyes than did WT mice. Adoptive transfer of M1 macrophages from WT mice to M1-/- mice reduced death and rescued virus replication in the eyes of infected mice. Infection of M1-/- mice with avirulent HSV-1 strain KOS also increased ocular virus replication and eye disease but did not affect latency-reactivation seen in WT control mice. Severity of virus replication and eye disease correlated with significantly higher inflammatory responses leading to a cytokine storm in the eyes of M1-/- infected mice that was not seen in WT mice. Thus, for the first time, our study illustrates the importance of M1 macrophages specifically in primary HSV-1 infection, eye disease, and survival but not in latency-reactivation.

C[sbnd]C chemokine ligand 3 deficiency ameliorates diet-induced steatohepatitis by regulating liver macrophage recruitment and M1/M2 status in mice

Background and aimsThe global prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing. Chemokines and their receptors have potential as therapeutic targets of NAFLD. We investigated the role of CC chemokine ligand 3 (CCL3) in the development of murine and human NAFLD. MethodsCCL3-knockout mice (CCL3−/−) and littermate CCL3 wild-type control mice (WT) were fed a high-cholesterol and high-fat (CL) diet for 16 weeks to induce NAFLD. We investigated the impact of CCL3 gene deletion in bone marrow cells and leptin-deficient ob/ob mice on CL diet-induced steatohepatitis. We assayed the serum CCL3 levels in 36 patients with biopsy-proven NAFLD and nine healthy control subjects. ResultsCompared with normal chow (NC), the CL diet induced steatohepatitis and hepatic fibrosis and elevated the plasma CCL3 level. In the liver, CCL3 protein colocalized with F4/80+ macrophages, especially CD11c+ M1-like macrophages, rather than other cell types. CCL3−/− attenuated CL diet-induced steatohepatitis and fibrosis associated with M2-dominant liver macrophages compared with the WT. The reconstitution of bone marrow (BM) cells from CCL3−/− attenuated steatohepatitis in WT mice fed a CL diet. Furthermore, crossing CCL3−/− onto the ob/ob background prevented CL diet-induced NAFLD in ob/ob mice, which was associated with a lesser inflammatory phenotype of liver macrophages. Also, the serum and hepatic levels of CCL3 were significantly increased in patients with non-alcoholic steatohepatitis (NASH) compared to those with simple fatty liver (NAFL) and healthy subjects. ConclusionOur data indicate that CCL3 facilitates macrophage infiltration into the liver and M1 polarization in the progression of steatohepatitis and highlight the need for further studies to determine the effect of CCL3-CCR1 and -CCR5 signaling blockade on the treatment of NAFLD.

Increased CaMKII-dependent pro-arrhythmic activity in a novel mouse model of obstructive sleep apnoea

Abstract Background Obstructive sleep apnoea (OSA) is frequently associated with atrial arrhythmias, but detailed mechanisms remain elusive. Most recently, we found an increased CaMKII-dependent pro-arrhythmic activity in patients with sleep apnoea. Since patients suffer from various confounding comorbidities, we have developed a novel mouse model of OSA by tongue enlargement. Purpose We tested if mice with OSA exhibit increased atrial CaMKII-dependent pro-arrhythmic activity. Methods Polytetrafluorethylene (PTFE) was injected into the tongue of 12 wild-type (WT) and 10 CaMKII knock-out (CKO) mice. 9 WT and 9 CKO mice were used as control without PTFE injection. Inspiratory flow limitations and apnoeas were monitored during murine sleep phases by whole-body plethysmography (Buxco). After eight weeks, isolated atrial cardiomyocytes were incubated with the Ca-sensitive dye FURA-2 AM for 15 min. Regular Ca transients were elicited by electrical field stimulation (1 Hz, 20 V for 4 ms) using epifluorescence microscopy. Pro-arrhythmic non-stimulated events were defined as deviations from diastolic Ca baseline between two stimulated Ca transients. Results Sonographic measurements revealed a significant increase in mean tongue diameter from (in mm) 3.7±0.1 to 5.1±0.1 after PTFE injection (n=23, p<0.0001). There was a significant correlation between magnitude of tongue diameter and frequency of apnoeas in OSA mice (p=0.046, r2=0.19, Fig. 1A). Interestingly, we observed a significantly increased frequency of pro-arrhythmic events of (in s–1) 0.06±0.01 in WT OSA mice compared to 0.02±0.01 in WT control mice (p=0.047, Fig. 1B). Similar results were observed at higher stimulation frequencies (2 and 4 Hz). There was a significant correlation of pro-arrhythmic events with inspiratory flow limitations (p=0.03, r2=0.24, Fig. 1C) and with the frequency of apnoeas by strong trend (p=0.06, r2=0.18). In contrast, no increase in atrial pro-arrhythmic events was observed in CKO mice after PTFE injection (for CKO mice after PTFE vs. CKO mice without PTFE, 0.03±0.01 s–1 vs. 0.03±0.01 s–1, p=0.89, Fig. 1B). Accordingly, the correlations between pro-arrhythmic events and both inspiratory flow limitations (p=0.36, r2=0.05, Fig. 1C) and apnoeas (p=0.82, r2=0.004) were completely abolished in CKO mice. Conclusion In a novel mouse model of obstructive sleep apnoea, atrial pro-arrhythmic activity was increased in a CaMKII-dependent fashion, which may have therapeutic implications. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Benedikt Schaner / Mr., this work is supported by a research grant of the German Cardiac Society (DGK); Stefan Wagner / Professor, was funded by DFG grants Figure 1

Crucial role of extra-domain A containing fibronectin for the development of pulmonary hypertension and associated right heart failure

Abstract Background Pulmonary vascular and right ventricular myocardial remodelling are unique phenomena in PH progression. Both processes are accompanied by an abundant re-expression of the extra-domain A of fibronectin (ED-A+ Fn) therefore qualifying as promising biomarker or even therapeutic target. Nevertheless, its functional role in PH pathogenesis remains unclear until now. Objective: The purpose of our study was to analyse the development of PH and RHF in a mouse model of monocrotaline (MCT)-induced PH comparing C57BL/6 ED-A+ Fn knockout (KO) and wild-type (WT) mice. Methods PH was induced by subcutaneous injection of a single dose of MCT (60 mg/kg body weight). Subgroups were additionally treated with the dual endothelin receptor antagonist Macitentan (MAC, 15mg/kg body weight per day from day 14 to 28). There were 6 experimental groups: sham-treated control WT mice (WTco, n=4); MCT induced PH WT mice (WTPH, n=6); MCT induced PH WT mice treated with MAC (WTPH_MAC, n=6); sham-treated control KO mice (KOco, n=4); MCT induced PH KO mice (KOPH, n=6); MCT induced PH KO mice treated with MAC (KOPH_MAC, n=6). Between day 26 and 28, transthoracic echocardiography and right heart catheterization were performed. Both, lung and cardiac tissue samples were subjected to histological analyses. Results Right heart catheterization revealed significantly increased RVPsys values in WTPH (87.0±16.4mmHg) compared to WTco (36.1±9.4mmHg; p=0.034) animals, which showed, at least in trend, a diminution in the WTPH_MAC group (67.1±20.9mmHg; p=n.s.). There was a non-significant increase in RVPsys in the KOPH (55.6±14.9mmHg) compared to KOco mice (37.2±5.6mmHg; p=n.s.) without any differences compared to the KOPH_MAC group (60.9±14.0mmHg; p=n.s.). When comparing the WTPH and the KOPH group, RVPsys was significantly lower in the KO animals (p=0.014), while there were no differences between the WTPH_MAC and the KOPH_MAC group (p=n.s.). Echocardiographic evaluation including surrogate parameters of right ventricular (RV) overload and failure were significantly altered in WTPH compared to WTco animals (p<0.05) and could not be shown to be relevantly improved in the WTPH_MAC group (p=n.s.). The majority of echocardiographic parameters did not significantly differ between the KOPH and the KOco group (p=n.s.). Lung tissue analysis revealed significant alterations in both, the WTPH and the KOPH group, each compared to the corresponding control (p<0.05). The level of lung tissue damage was significantly decreased in KOPH compared to WTPH mice (p<0.05). In RV, the amount of interstitial fibrosis was increased in the WTPH (p=0.009) but not in the KOPH group (p=n.s.), each compared to the corresponding controls. Conclusions The findings of the current study underline the hypothesis that ED-A+ Fn is a key player in the pathogenesis of PH and associated RHF. Thus, it might represent a promising therapeutic target, e.g., by the administration of neutralizing antibodies. Funding Acknowledgement Type of funding sources: Public hospital(s). Main funding source(s): University Hospital Jena

Lack of lymphocytes exacerbate heat stroke severity in male mice through enhanced inflammatory response

Though it has long been thought that the immune system is implicated in the pathophysiology of heat stroke, the underlying mechanisms are still poorly understood. As it has been reported in the literature that lymphocyte disturbance occurs in heat stroke patients or animals, we attempted to seek experimental evidence to define the role of lymphocytes in the pathophysiology of heat stroke. In our study, we used male Balb/c mice to establish a passive heat stroke model. We found that lymphocyte-deficient Severe combined immunodeficient (SCID) mice exposed to heat stress exhibited exacerbated heat stroke severity, which could be indicated by increased rates of mortality and serum levels of inflammatory cytokines compared to wildtype control mice. We further showed, through the depletion of T lymphocytes in wildtype mice and the transfer of wildtype lymphocytes into SCID mice, respectively, that T lymphocytes were both necessary and sufficient to alleviate the severity of heat stroke by inhibiting the early inflammatory response. Moreover, we found that the severity of heat injuries in heat-stressed wildtype mice showed great inter-individual variability, and the early number of T lymphocytes could be negatively associated with the severity of heat stroke. Our results suggest that lack of T lymphocytes could exacerbate the severity of heat stroke by augmenting inflammatory response, and the early circulating T lymphocytes may serve as a potential biomarker for the diagnosis of heat stroke.

Comparison of the Amyloid Load in the Brains of Two Transgenic Alzheimer's Disease Mouse Models Quantified by Florbetaben Positron Emission Tomography.

Alzheimer’s disease (AD) is characterized by formation of amyloid plaques and neurofibrillary tangles in the brain, which can be mimicked by transgenic mouse models. Here, we report on the characterization of amyloid load in the brains of two transgenic amyloidosis models using positron emission tomography (PET) with florbetaben (FBB), an 18F-labeled amyloid PET tracer routinely used in AD patients. Young, middle-aged, and old homozygous APP/PS1 mice (ARTE10), old hemizygous APPswe/PS1ΔE9, and old wild-type control mice were subjected to FBB PET using a small animal PET/computed tomography scanner. After PET, brains were excised, and ex vivo autoradiography was performed. Plaque pathology was verified on brain sections with histological methods. Amyloid plaque load increased progressively with age in the cortex and hippocampus of ARTE10 mice, which could be detected with both in vivo FBB PET and ex vivo autoradiography. FBB retention showed significant differences to wild-type controls already at 9 months of age by both in vivo and ex vivo analyses. An excellent correlation between data derived from PET and autoradiography could be obtained (rPearson = 0.947, p < 0.0001). Although amyloid load detected by FBB in the brains of old APPswe/PS1ΔE9 mice was as low as values obtained with young ARTE10 mice, statistically significant discrimination to wild-type animals was reached (p < 0.01). In comparison to amyloid burden quantified by histological analysis, FBB retention correlated best with total plaque load and number of congophilic plaques in the brains of both mouse models. In conclusion, the homozygous ARTE10 mouse model showed superior properties over APPswe/PS1ΔE9 mice for FBB small animal amyloid PET imaging. The absolute amount of congophilic dense-cored plaques seems to be the decisive factor for feasibility of amyloidosis models for amyloid PET analysis.

BRCA1/Trp53 heterozygosity and replication stress drive esophageal cancer development in a mouse model

BRCA1 germline mutations are associated with an increased risk of breast and ovarian cancer. Recent findings of others suggest that BRCA1 mutation carriers also bear an increased risk of esophageal and gastric cancer. Here, we employ a Brca1/Trp53 mouse model to show that unresolved replication stress (RS) in BRCA1 heterozygous cells drives esophageal tumorigenesis in a model of the human equivalent. This model employs 4-nitroquinoline-1-oxide (4NQO) as an RS-inducing agent. Upon drinking 4NQO-containing water, Brca1 heterozygous mice formed squamous cell carcinomas of the distal esophagus and forestomach at a much higher frequency and speed (∼90 to 120 d) than did wild-type (WT) mice, which remained largely tumor free. Their esophageal tissue, but not that of WT control mice, revealed evidence of overt RS as reflected by intracellular CHK1 phosphorylation and 53BP1 staining. These Brca1 mutant tumors also revealed higher genome mutation rates than those of control animals; the mutational signature SBS4, which is associated with tobacco-induced tumorigenesis; and a loss of Brca1 heterozygosity (LOH). This uniquely accelerated Brca1 tumor model is also relevant to human esophageal squamous cell carcinoma, an often lethal tumor.

Knee Osteoarthritis Progression Is Delayed in Silent Information Regulator 2 Ortholog 1 Knock-in Mice.

Overexpression of silent information regulator 2 ortholog 1 (SIRT1) is associated with beneficial roles in aging-related diseases; however, the effects of SIRT1 overexpression on osteoarthritis (OA) progression have not yet been studied. The aim of this study was to investigate OA progression in SIRT1-KI mice using a mouse OA model. OA was induced via destabilization of the medial meniscus using 12-week-old SIRT1-KI and wild type (control) mice. OA progression was evaluated histologically based on the Osteoarthritis Research Society International (OARSI) score at 4, 8, 12, and 16 weeks after surgery. The production of SIRT1, type II collagen, MMP-13, ADAMTS-5, cleaved caspase 3, Poly (ADP-ribose) polymerase (PARP) p85, acetylated NF-κB p65, interleukin 1 beta (IL-1β), and IL-6 was examined via immunostaining. The OARSI scores were significantly lower in SIRT1-KI mice than those in control mice at 8, 12, and 16 weeks after surgery. The proportion of SIRT1 and type II collagen-positive-chondrocytes was significantly higher in SIRT1-KI mice than that in control mice. Moreover, the proportion of MMP-13-, ADAMTS-5-, cleaved caspase 3-, PARP p85-, acetylated NF-κB p65-, IL-1β-, and IL-6-positive chondrocytes was significantly lower in SIRT1-KI mice than that in control mice. The mechanically induced OA progression was delayed in SIRT1-KI mice compared to that in control mice. Therefore, overexpression of SIRT1 may represent a mechanism for delaying OA progression.

B-Cell Deficiency Exacerbates Inflammation and Bone Loss in Ligature-Induced Experimental Periodontitis in Mice.

ObjectivePeriodontitis, one of the most prevalent chronic oral infectious diseases in humans, is induced by the breakdown in the balance between the biofilm and host immune system. Previous studies have shown the presence of large numbers of B cells in periodontitis lesions, implicating that B lymphocytes play a predominant role during the pathogenesis of periodontitis. This study aimed to investigate the role of all B cells in the initiation of periodontitis.MethodsExperimental periodontitis was induced in B cell-deficient (CD19Cre) mice and wild-type (WT) control mice by 5-0 silk ligation around the maxillary second molar. Four weeks after ligation, alveolar bone loss was determined by micro-computed tomography. The levels of inflammatory cytokines and receptor activator of NF-κB ligand (RANKL)/osteoprotegerin in periodontal lesions were analyzed using real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry. Lymphocyte populations in the cervical lymph nodes and spleen and among the peripheral blood mononuclear cells were detected by flow cytometry.ResultsB-cell deficiency resulted in increased severity of alveolar bone loss in mouse experimental periodontitis, which was associated with increased osteoclast activity and upregulated RANKL expression in the periodontal lesions. In addition, gingiva cytokine expression profiles were shifted to T helper type 1 (Th1) and Th17 in the CD19Cre mice with ligature-induced periodontitis compared with WT mice. In addition, a reduced CD4+/CD8+ T cell ratio was observed in the CD19Cre mice.ConclusionB-cell deficiency exacerbates the inflammation and alveolar bone loss in ligature-induced experimental periodontitis in mice, implicating that B cells may overall play a protective role in the initiation of periodontitis.

Inner hair cell dysfunction in Klhl18 mutant mice leads to low frequency progressive hearing loss.

Age-related hearing loss in humans (presbycusis) typically involves impairment of high frequency sensitivity before becoming progressively more severe at lower frequencies. Pathologies initially affecting lower frequency regions of hearing are less common. Here we describe a progressive, predominantly low-frequency recessive hearing impairment in two mutant mouse lines carrying different mutant alleles of the Klhl18 gene: a spontaneous missense mutation (Klhl18lowf) and a targeted mutation (Klhl18tm1a(KOMP)Wtsi). Both males and females were studied, and the two mutant lines showed similar phenotypes. Threshold for auditory brainstem responses (ABR; a measure of auditory nerve and brainstem neural activity) were normal at 3 weeks old but showed progressive increases from 4 weeks onwards. In contrast, distortion product otoacoustic emission (DPOAE) sensitivity and amplitudes (a reflection of cochlear outer hair cell function) remained normal in mutants. Electrophysiological recordings from the round window of Klhl18lowf mutants at 6 weeks old revealed 1) raised compound action potential thresholds that were similar to ABR thresholds, 2) cochlear microphonic potentials that were normal compared with wildtype and heterozygous control mice and 3) summating potentials that were reduced in amplitude compared to control mice. Scanning electron microscopy showed that Klhl18lowf mutant mice had abnormally tapering of the tips of inner hair cell stereocilia in the apical half of the cochlea while their synapses appeared normal. These results suggest that Klhl18 is necessary to maintain inner hair cell stereocilia and normal inner hair cell function at low frequencies.

DJ-1 attenuates the glycation of mitochondrial complex I and complex III in the post-ischemic heart

DJ-1 is a ubiquitously expressed protein that protects cells from stress through its conversion into an active protease. Recent work found that the active form of DJ-1 was induced in the ischemic heart as an endogenous mechanism to attenuate glycative stress—the non-enzymatic glycosylation of proteins. However, specific proteins protected from glycative stress by DJ-1 are not known. Given that mitochondrial electron transport proteins have a propensity for being targets of glycative stress, we investigated if DJ-1 regulates the glycation of Complex I and Complex III after myocardial ischemia–reperfusion (I/R) injury. Initial studies found that DJ-1 localized to the mitochondria and increased its interaction with Complex I and Complex III 3 days after the onset of myocardial I/R injury. Next, we investigated the role DJ-1 plays in modulating glycative stress in the mitochondria. Analysis revealed that compared to wild-type control mice, mitochondria from DJ-1 deficient (DJ-1 KO) hearts showed increased levels of glycative stress following I/R. Additionally, Complex I and Complex III glycation were found to be at higher levels in DJ-1 KO hearts. This corresponded with reduced complex activities, as well as reduced mitochondrial oxygen consumption ant ATP synthesis in the presence of pyruvate and malate. To further determine if DJ-1 influenced the glycation of the complexes, an adenoviral approach was used to over-express the active form of DJ-1(AAV9-DJ1ΔC). Under I/R conditions, the glycation of Complex I and Complex III were attenuated in hearts treated with AAV9-DJ1ΔC. This was accompanied by improvements in complex activities, oxygen consumption, and ATP production. Together, this data suggests that cardiac DJ-1 maintains Complex I and Complex III efficiency and mitochondrial function during the recovery from I/R injury. In elucidating a specific mechanism for DJ-1’s role in the post-ischemic heart, these data break new ground for potential therapeutic strategies using DJ-1 as a target.

Transgenic Mice Overexpressing Human Alpha-1 Antitrypsin Exhibit Low Blood Pressure and Altered Epithelial Transport Mechanisms in the Inactive and Active Cycles.

Human alpha-1 antitrypsin (hAAT) is a versatile protease inhibitor, but little is known about its targets in the aldosterone-sensitive distal nephron and its role in electrolyte balance and blood pressure control. We analyzed urinary electrolytes, osmolality, and blood pressure from hAAT transgenic (hAAT-Tg) mice and C57B/6 wild-type control mice maintained on either a normal salt or high salt diet. Urinary sodium, potassium, and chloride concentrations as well as urinary osmolality were lower in hAAT-Tg mice maintained on a high salt diet during both the active and inactive cycles. hAAT-Tg mice showed a lower systolic blood pressure compared to C57B6 mice when maintained on a normal salt diet but this was not observed when they were maintained on a high salt diet. Cathepsin B protein activity was less in hAAT-Tg mice compared to wild-type controls. Protein expression of the alpha subunit of the sodium epithelial channel (ENaC) alpha was also reduced in the hAAT-Tg mice. Natriuretic peptide receptor C (NPRC) protein expression in membrane fractions of the kidney cortex was reduced while circulating levels of atrial natriuretic peptide (ANP) were greater in hAAT-Tg mice compared to wild-type controls. This study characterizes the electrolyte and blood pressure phenotype of hAAT-Tg mice during the inactive and active cycles and investigates the mechanism by which ENaC activation is inhibited in part by a mechanism involving decreased cathepsin B activity and increased ANP levels in the systemic circulation.

Cardiac Effects of Novel Histamine H2 Receptor Agonists.

In an integrative approach, we studied cardiac effects of recently published novel H2 receptor agonists in the heart of mice that overexpress the human H2 receptor (H2-TG mice) and littermate wild type (WT) control mice and in isolated electrically driven muscle preparations from patients undergoing cardiac surgery. Under our experimental conditions, the H2 receptor agonists UR-Po563, UR-MB-158, and UR-MB-159 increased force of contraction in left atrium from H2-TG mice with pEC50 values of 8.27, 9.38, and 8.28, respectively, but not in WT mice. Likewise, UR-Po563, UR-MB-158, and UR-MB-159 increased the beating rate in right atrium from H2-TG mice with pEC50 values of 9.01, 9.24, and 7.91, respectively, but not from WT mice. These effects could be antagonized by famotidine, a H2 receptor antagonist. UR-Po563 (1 µM) increased force of contraction in Langendorff-perfused hearts from H2-TG but not WT mice. Similarly, UR-Po563, UR-MB-158, or UR-MB-159 increased the left ventricular ejection fraction in echocardiography of H2-TG mice. Finally, UR-Po563 increased force of contraction in isolated human right atrial muscle strips. The contractile effects of UR-Po563 in H2-TG mice were accompanied by an increase in the phosphorylation state of phospholamban. In summary, we report here three recently developed agonists functionally stimulating human cardiac H2 receptors in vitro and in vivo. We speculate that these compounds might be of some merit to treat neurologic disorders if their cardiac effects are blocked by concomitantly applied receptor antagonists that cannot pass through the blood-brain barrier or might be useful to treat congestive heart failure in patients. SIGNIFICANCE STATEMENT: Recently, a new generation of histamine H2 receptor (H2R) agonists has been developed as possible treatment option for Alzheimer's disease. Here, possible cardiac (side) effects of these novel H2R agonists have been evaluated.

Loss of CX3CR1 augments neutrophil infiltration into cochlear tissues after acoustic overstimulation.

The cochlea, the sensory organ for hearing, has a protected immune environment, segregated from the systemic immune system by the blood-labyrinth barrier. Previous studies have revealed that acute acoustic injury causes the infiltration of circulating leukocytes into the cochlea. However, the molecular mechanisms controlling immune cell trafficking are poorly understood. Here, we report the role of CX3CR1 in regulating the entry of neutrophils into the cochlea after acoustic trauma. We employed B6.129P-Cx3cr1tm1Litt /J mice, a transgenic strain that lacks the gene, Cx3cr1, for coding the fractalkine receptor. Our results demonstrate that lack of Cx3cr1 results in the augmentation of neutrophil infiltration into cochlear tissues after exposure to an intense noise of 120dB SPL for 1hr. Neutrophil distribution in the cochlea is site specific, and the infiltration level is positively associated with noise intensity. Moreover, neutrophils are short lived and macrophage phagocytosis plays a role in neutrophil clearance, consistent with typical neutrophil dynamics in inflamed non-cochlear tissues. Importantly, our study reveals the potentiation of noise-induced hearing loss and sensory cell loss in Cx3cr1-/- mice. In wild-type control mice (Cx3cr1+/+ ) exposed to the same noise, we also found neutrophils. However, neutrophils were present primarily inside the microvessels of the cochlea, with only a few in the cochlear tissues. Collectively, our data implicate CX3CR1-mediated signaling in controlling neutrophil migration from the circulation into cochlear tissues and provide a better understanding of the impacts of neutrophils on cochlear responses to acoustic injury.

MicroRNA-7 overexpression positively regulates the CD8+ SP cell development via targeting PIK3R1

microRNA-7 (miR-7), a distinct miRNA family member, has been reported to be involved in the biological functions of immune cells. However, the potential role of miR-7 in the CD8+ T cell development remains to be elucidated. In this study, we estimated the potential effects of miR-7 overexpression in the thymic CD8+ SP cell development using miR-7 overexpression mice. Our results showed that compared with those in control wild type (WT) mice, the volume, weight and total cell numbers of thymus in miR-7 overexpression (OE) mice increased significantly. The absolute cell number of CD8+ SP cells in miR-7 OE mice increased and its ability of activation and proliferation enhanced. Futhermore, we clarified that miR-7 overexpression had an intrinsic promote role in CD8+ SP cell development by adoptive cell transfer assay. Mechanistically, the expression level of PIK3R1, a target of miR-7, decreased significantly in CD8+ SP cells of miR-7 OE mice. Moreover, the expression level of phosphorylated (p)-AKT and p-ERK changed inversely and indicating that miR-7 overexpression impaired the balance of AKE and ERK pathways. In summary, our work reveals an essential role of miR-7 in promoting CD8+ SP cell development through the regulation of PIK3R1 and balance of AKT and ERK pathways.

Collagen XII Deficiency Increases the Risk of Anterior Cruciate Ligament Injury in Mice.

Anterior cruciate ligament (ACL) rupture is a common knee injury for athletes. Although surgical reconstruction is recommended for the treatment of ACL ruptures, 100% functional recovery is unlikely. Therefore, the discovery of risk factors for ACL ruptures may prevent injury. Several studies have reported an association between polymorphisms of the collagen XII gene COL12A1 and ACL rupture. Collagen XII is highly expressed in tendons and ligaments and regulates tissue structure and mechanical property. Therefore, we hypothesized that collagen XII deficiency may cause ACL injury. To elucidate the influence of collagen XII deficiency on ACL, we analyzed a mouse model deficient for Col12a1. Four- to 19-week-old male Col12a1-/- and wild-type control mice were used for gait analysis; histological and immunofluorescent analysis of collagen XII, and real-time RT-PCR evaluation of Col12a1 mRNA expression. The Col12a1-/- mice showed an abnormal gait with an approximately 2.7-fold increase in step angle, suggesting altered step alignment. Col12a1-/- mice displayed 20–60% ACL discontinuities, but 0% discontinuity in the posterior cruciate ligament. No discontinuities in knee ligaments were found in wild-type mice. Collagen XII mRNA expression in the ACL tended to decrease with aging. Our study demonstrates for the first time that collagen XII deficiency increases the risk of ACL injury.

Abstract MP45: The Roles Of Telomeric Repeat Binding Factor 2-interacting Protein (TERF2IP) K240 Sumoylation In Endothelial Cells On Atherogenesis

Background and Objectives: It is not yet clear how the pro-atherogenic signaling events in endothelial cells (ECs) such as those that lead to EC senescence, apoptosis and activation are interconnected and promote atherosclerotic plaque formation in the area exposed to disturbed blood flow (d-flow). TERF2IP, a member of the shelterin complex of the telomere, regulates all three pathological events. We investigated the role of TERF2IP K240 SUMOylation in the process of d-flow-induced atherosclerotic plaque formation. Methods and Results: We found that d-flow increased TERF2IP K240 SUMOylation in ECs and that it was suppressed by a p90RSK specific inhibitor, FMK-MEA. This SUMOylation was independent of TERF2IP S205 phosphorylation. The d-flow-induced senescence, DNA damage, and apoptosis were inhibited in ECs with TERF2IP depletion or point-mutated phosphorylation (S205A) and SUMOylation (K240R) sites. NF-κB activation induced by d-flow or overexpression of p90RSK was also significantly inhibited in ECs overexpressing the TERF2IP S205A phosphorylation mutant. However, cells overexpressing the TERF2IP K240R SUMOylation mutant showed no effect on the d-flow or p90RSK-medaited NF-κB activation. To determine the biological function of TERF2IP K240 SUMOylation, we generated TERF2IP K240R knock-in (KI) mice and examined d-flow-induced atherosclerotic plaque formation using partial left carotid ligation model mice fed a high-fat diet after AAV8-PCSK9 injection. We found no differences in body weights and cholesterol levels between TERF2IP K240R KI and wild type control (WT) mice, but plaque formation was significantly inhibited in the KI mice compared to WT animals (Oil Red O positive area (%): 19.0 +/- 12.5 (KI mice, n=8) vs 61.2 +/- 24.3 (WT mice, n=7), p = 0.0008). Bone marrow from WT mice were transplanted into KI and WT mice, which were then injected with AAV8-PCSK9 virus and fed a high-fat diet for 16 weeks, but we still found that plaque formation was inhibited in the KI mice. Conclusion: TERF2IP SUMOylation plays a role in in EC senescence but not in activation. The significant inhibition of plaque formation in the TERF2IP K240R KI mice is due to downregulation of TERF2IP SUMOylation in ECs not in myeloid cells.

Abstract MP46: Mutagenesis Of PRR Cleavage Site In Mice Attenuates Angiotensin II-induced Hypertension And Suppresses Renin Expression

It is well demonstrated that activation of renal (pro)renin receptor (PRR) contributes to AngII-induced hypertension. Relatively, less is known about involvement of soluble PRR (sPRR), the extracellular domain of PRR, primarily generated by site-1 protease(S1P) and furin. Moreover, the relationship between PRR/sPRR and the renin-angiotensin system (RAS) has been debated. In the present study, we used CRISPR/Cas9 strategy to generate mice with mutagenesis of the overlapping cleavage site for both proteases in PRR (termed as PRR R279V/L282V ) to examine the phenotype during angiotensin II (AngII) infusion with particular emphasis on circulating and intrarenal renin status. PRR R279V/L282V mice exhibited a reduction of sPRR level in plasma by ~53% and in the kidney by ~82%, were fertile, and had no gross developmental abnormalities. At basal condition, PRR R279V/L282V mice had drastically suppressed renin levels from plasma (plasma total prorenin/renin content [ng/ml]: 9.3 ± 0.7 in PRR R279V/L282V mice vs. 12.8 ± 0.9 in WT mice, n = 5, p &lt; 0.05), urine (urinary total prorenin/renin excretion [ng/24h]: 0.54 ± 0.11 in PRR R279V/L282V mice vs. 1.05 ± 0.15 in WT mice, n = 5, p &lt; 0.05), and the kidney as compared to wild-type controls (WT). By telemetry, the hypertensive response of PRR R279V/L282V to AngII infusion (300 ng/min/kg) was blunted (MAP [mmHg] in Day 10: 125.9 ± 4.6 in PRR R279V/L282V mice vs. 138.5 ± 2.8 in WT mice, n = 8 for each group, p &lt; 0.05) in parallel with attenuated response of intrarenal renin and renal medullary α-ENaC expression. We further examined the direct role of sPRR in renin regulation in As4.1 cells, a renin-expressing cell line isolated from mouse renal tumor and M-1 cells, a mouse cell line derived from the collecting duct. The exposure to sPRR-His in both cell types consistently elevated renin activity, and renin expression at both protein and mRNA levels, all of which were sensitive to inhibition by ICG-001, a β-catenin signaling inhibitor. Together, these results represent strong evidence favoring sPRR as a mediator of AngII-induced hypertension and a master regulator of renin expression at systemic and local levels. Therefore, PRR should be considered as an integrative member of the RAS.

Exposure of Mice during Spermatogenesis: A Role of Inhibitor Kinase 2 in Pro-Opiomelanocortin Neurons.

Background:Epidemiological studies have shown that exposure to ambient fine particulate matter with aerodynamic diameter less than or equal to 2.5 μm () correlates with a decrease in sperm count, but the biological mechanism remains elusive.Objectives:This study tested whether hypothalamic inflammation, an emerging pathophysiological mediator, mediates the development of lower epididymal sperm count due to exposure.Methods:Inhibitor kinase 2 (IKK2) was conditionally knocked out either in all neurons or subtypes of hypothalamic neurons of mice. Effects of concentrated ambient (CAP) exposure on hypothalamic inflammation, the hypothalamic–pituitary–gonadal (HPG) axis, and epididymal sperm count of these mouse models were then assessed. Furthermore, to test whether hypothalamic inflammation is sufficient to decrease sperm production, we overexpressed constitutively active IKK2 (IKK2ca) either in all neurons or subtypes of hypothalamic neurons and assessed hypothalamic inflammation, the HPG axis, and sperm production of these overexpression mouse models.Results:CAP-exposed wild-type control mice vs. filtered air (FA)-exposed wild-type control mice had a higher expression of hypothalamic inflammatory markers, lower functional indexes of the HPG axis, and a lower epididymal sperm count. In contrast, all these measurements for CAP- vs. FA-exposed mice deficient of IKK2 in all neurons were comparable. We also found that overexpression of IKK2ca in either all neurons or pro-opiomelanocortin (POMC) neurons only, but not in Agouti-related protein (AgRP) neurons only, resulted in lower functional indexes of the HPG axis and a lower epididymal sperm count. Moreover, we showed that CAP- vs. FA-exposed mice deficient of IKK2 in POMC neurons had a comparable expression of hypothalamic inflammatory markers, comparable functional indexes of the HPG axis, and a comparable epididymal sperm count.Discussion:This mouse model study shows a causal role of IKK2 of POMC neurons in the development of lower epididymal sperm count due to exposure, providing a mechanistic insight into this emerging pathogenesis. https://doi.org/10.1289/EHP8868