Week-old Wild Type Research Articles

Supraphysiological Levels of Testosterone Induce Cardiac Dysfunction via NLRP3 Inflammasome Activation in Macrophages

IntroductionTestosterone (Testo) modulates vascular tone and cardiac performance. Athletes who use Testo at supraphysiological doses exhibit increased blood pressure, higher inflammatory marker levels, vascular dysfunction, and cardiac hypertrophy. NLRP3 inflammasome activation, as part of the innate immune system response, contributes to proinflammatory cytokines production, leading e.g to cardiac dysfunction.HypothesisWe hypothesized that supraphysiological levels of Testo promotes cardiac dysfunction via generation of mitochondrial reactive oxygen species (mROS) and activation of the NLRP3 inflammasome.MethodsMale, 12 week‐old wild type (WT) and NLRP3 knockout (NLRP3‐/‐) mice were used. Mice were treated with testosterone propionate [Testo‐P (10 mg/kg)] or vehicle for 30 days. Cardiac function was evaluated using echocardiography. After in vivo experiments, western blot and ELISA were performed to evaluate NLRP3 inflammasome components. ROS generation was evaluated by lucigenin. Bone marrow‐derived macrophages (BMDMs) were isolated, primed with lipopolysaccharide (LPS [500 ng/mL 4 h]) and stimulated with Testo [10‐7 M], for 4, 6, 12 and 24 h. ROS generation was evaluated by DHE fluorescence and MitoSOX.ResultsEchocardiography showed cardiac dysfunction in WT mice treated with Testo‐P, characterized by reduced ejection fraction, shortening fraction, cardiac output and systolic volume. Testo‐P also increased interventricular septum, left ventricle posterior wall and decreased left ventricle internal diameter. All these effects were observed in NLRP3‐/‐mice. Furthermore, WT mice treated with Testo‐P showed increased cardiac expression of NLRP3 receptor, active Caspase‐1 and IL‐1β levels. These effects were prevented in NLRP3‐/‐. In addition, increased ROS generation was observed in the left ventricle of WT mice, but not NLRP3‐/‐ mice, treated with TP. Testo‐P‐treated WT mice showed increased macrophage infiltrate in the left ventricle, and this effect was not seen in the WT vehicle mice. In in vitro experiments, BMDMs primed with LPS and stimulated with Testo showed increased expression of the NLRP3 receptor and IL‐1β levels after 12 and 24 h. In addition, Testo‐stimulated BMDMs exhibited increased mROS generation.ConclusionSupraphysiological levels of testosterone induce cardiac dysfunction via mROS generation and NLRP3 inflammasome activation. This study was approved by the Ethics Committee on Animal Experimentation of the Ribeirao Preto Medical School (020/2021).

Abstract 285: Thrombospondin-5 Is Necessary For Males But Not Females In Hindlimb Ischemia Recovery

Introduction: Ameliorating critical limb ischemia (CLI), in part, relies on arteriogenesis and angiogenesis. We have shown thrombospondin-5 (TSP5) is proangiogenic in vitro. The role of TSP5 in CLI and the differences between males and females are unknown. Hypotheses: blood flow recovery, arteriogenesis and angiogenesis after limb ischemia is diminished (1) in TSP5 null mice compared to controls, and (2) in female compared to male TSP5 null mice. Methods: 14-16 week-old wild type (WT; C57Bl6) or TSP5 null male and female mice underwent left femoral artery ligation and transection (n=6-9/group). Laser Doppler data was collected preoperative, post ligation and at sacrifice (day 14). Blood flow recovery was expressed as a ratio of ischemic/non-ischemic limb. Immunohistochemistry was performed using anti-α-smooth-muscle-actin on the vastus lateralis to quantify arteriogenesis and anti-CD31 on the gastrocnemius to quantify angiogenesis. Arteriole and capillary density were determined by vessel counts/5HPF. Data was analyzed by ANOVA with post hoc testing, with p<0.05 being significant. Results: TSP5 null mice had decreased blood flow recovery in males (WT 0.85 ± 0.04 vs TSP5 0.48 ± 0.06, p<0.001) and females (WT 0.63 ± 0.07 vs TSP5 0.36 ± 0.04, p<0.01). No difference in flow recovery was seen between male and female TSP5 nulls (male 0.48 ± 0.06 vs female 0.36 ± 0.04 p<0.14). Arteriogenesis was impaired in male TSP5 null mice (WT 3.91 ± 0.24 vs TSP5 2.6 ± 0.42, p<0.02), but increased in females (WT 2.5 ± 0.26 vs TSP5 4.35 ± 0.73, p<0.01). Angiogenesis was decreased in TSP5 null males (WT 10.9 ± 0.93 vs TSP5 7.03 ± 0.84, p<0.01), but not females (WT 6.1 ± 0.50 vs TSP5 5.6 ± 0.80 p<0.9). In contrast, flow recovery, angiogenesis and arteriogenesis was decreased in WT females compared to WT males (p< 0.05). Conclusions: Our findings suggest TSP5 is relevant to vascular remodeling following hindlimb ischemia with a divergence in mechanism between males and females. TSP5 is necessary in males for arteriogenesis and angiogenesis. TSP5 in females may be supressive of arteriogenesis and has no effect on angiogenesis. Further investigation into these sexually dimorphic adaptive processes may lead to specific targeted CLI treatments.

Distinct Alteration of Gene Expression Programs in the Small Intestine of Male and Female Mice in Response to Ablation of Intestinal Fabp Genes.

Fatty acid-binding proteins (Fabps) make up a family of widely distributed cytoplasmic lipid-binding proteins. The small intestine contains three predominant Fabp species, Fabp1, Fabp2, and Fabp6. Our previous studies showed that Fabp2 and Fabp6 gene-disrupted mice exhibited sexually dimorphic phenotypes. In this study, we carried out a systematic comparative analysis of the small intestinal transcriptomes of 10 week-old wild-type (WT) and Fabp gene-disrupted male and female mice. We found that the small intestinal transcriptome of male and female mice showed key differences in the gene expression profiles that affect major biological processes. The deletion of specific Fabp genes induced unique and sex-specific changes in the gene expression program, although some differentially expressed genes in certain genotypes were common to both sexes. Functional annotation and interaction network analyses revealed that the number and type of affected pathways, as well as the sets of interacting nodes in each of the Fabp genotypes, are partitioned by sex. To our knowledge, this is the first time that sex differences were identified and categorized at the transcriptome level in mice lacking different intestinal Fabps. The distinctive transcriptome profiles of WT male and female small intestine may predetermine the nature of transcriptional reprogramming that manifests as sexually dimorphic responses to the ablation of intestinal Fabp genes.

Deficiency of PDK4 Sensitizes Mouse Liver to Diethylnitrosamine and Arsenic Toxicity Through Inducing Apoptosis

BackgroundPyruvate dehydrogenase kinase 4 (PDK4) is a metabolic switch that regulates glucose oxidation and the TCA cycle in the mitochondria. The liver detoxifies xenobiotics and is constantly challenged by various injuries. How the loss of this metabolic regulator, PDK4, contributes to liver injuries remains elusive. Previous studies from our lab have revealed that knockout of Pdk4 in mice resulted in increased cellular proliferation via E2F1 transcription factor‐mediated increase in cyclins.ObjectiveThe objective of this study was to elucidate a novel function of PDK4 in toxin‐induced liver injury.MethodsTwo week‐old wild‐type (WT) (C57BL/6J, male) and Pdk4−/− mice were i.p. injected with a single dose of diethylnitrosamine (DEN) (25 mg/kg body weight) or two month‐old mice were fed with arsenic at 50 ppm (one month) via distilled drinking water. DEN treated mouse samples were collected at day 6 after DEN injection due to unexpected death of Pdk4−/− mice. Liver tissues were collected after both treatments for western blot (WB), IHC, and qPCR. Pdk4 shRNA was used to generate Pdk4 knockdown cells for mitochondrial respiration analysis to measure cellular oxygen consumption rate and extracellular acidification rate (Seahorse Bioscience). Electron microscopy was applied in order to visualize mitochondria.ResultsTUNEL analysis revealed a significant level of spontaneous apoptotic cells in Pdk4−/− mouse livers, which increased with age in the absence of treatment indicating an age‐dependent progression. Microscopy and staining revealed extensive hepatic damage in DEN treated Pdk4−/− mouse livers while WT mouse livers showed no sign of damage which suggests loss of Pdk4 increases hepatic sensitivity to chemical toxicant DEN administration. TUNEL analysis of Arsenic treated mouse liver sections revealed increased apoptosis in Pdk4−/− mice compared to WT mice. Caspase‐3 activity was analyzed via western blot and activity assays revealing a caspase‐3 dependent induction of apoptosis in Pdk4−/− mouse livers. Mitochondrial respiration analysis revealed shPDK4 knockdown cells had significantly higher oxygen consumption rate and a higher respiratory capacity than control cells. Mitochondrial respiration analysis also revealed that shPDK4 knockdown cells had higher extracellular acidification rates suggesting increased glycolytic capacity.ConclusionsIn the present study, we showed that age‐dependent spontaneous hepatic apoptosis happened in systematic Pdk4 knockout (Pdk4−/−) mice and that PDK4‐deficiency increased the sensitivity of adult mice to arsenic insult, as well as juvenile mice to toxicity of DEN, which correlated with a lethal consequence and massive hepatic apoptosis and abnormal mitochondrial respiration.Support or Funding InformationNIH R01ES025909, R01DK104656, R01DK080440, R21AA022482, R21AA024935, VA Merit Award 1I01BX002634, P30 DK34989 (Yale Liver Center).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lentinan Exerts its Anti-Inflammatory Activity by Suppressing TNFR1 Transfer to the Surface of Intestinal Epithelial Cells through Dectin-1 in an in vitro and mice model

It has been reported that lentinan, β-1,3; 1,6-glucan derived from Lentinula edodes, suppresses intestinal inflammation and ameliorates symptoms of colitis. However, the mechanism of intestinal anti-inflammatory activity of lentinan and how it is recognized by intestinal epithelial cells remains largely unclear. The receptor for lentinan on intestinal epithelial cells was identified using an in vitro gut inflammation model consisting of Caco-2 and RAW264.7 cells. Colitis was induced in 7 to 8 week-old wild-type (WT) or knockout (KO) mice by the free intake of water containing 2% dextran sulfate sodium (DSS) for 7 days. Lentinan was administered daily via intragastric administration. Tumor necrosis factor receptor 1 (TNFR1)-GFP complex was constructed to monitor its movement in Caco-2 cells using confocal and total internal fluorescence microscopy. The results indicated that lentinan suppressed DSS-induced body weight loss, shortening of colon length, histological score, and inflammatory cytokine mRNA expression in the inflamed tissues of WT mice, whereas these suppressive effects of lentinan were not observed in Dectin-1 KO mice. Furthermore, lentinan reduced TNFR1 expression in intestinal epithelial cells of WT mice but not those from Dectin-1 KO mice. Using TNFR1-GFP constructs, it was confirmed that lentinan reduced TNFR1 expression on Caco-2 cell membranes through Dectin-1 ligation. Our study revealed that lentinan suppressed intestinal inflammation by Dectin-1-mediated regulation of TNFR1 transfer to the surface of intestinal epithelial cells.

Abstract 1928: Comprehensive identification of miR-122 targetome in the liver and HCC by HITS-CLIP analysis reveals conserved regulation of AMPK signaling in hepatocarcinogenesis

Abstract miR-122, a conserved liver-specific miRNA, is critical for maintaining liver homeostasis. Loss of miR-122 is associated with loss of hepatic phenotype, metastasis and poor prognosis. Recent in vivo work has established miR-122 as a bona fide tumor suppressor, with poorly differentiated, Afp-positive, high grade, spontaneous HCC observed in liver-specific (LKO) and germ-line (KO) miR-122 knockout mice. We hypothesized that the pathophysiology exhibited by KO mice would be mediated through de-repressed targets in KO livers and therefore, dysfunctional regulation of specific pathways involved in liver homeostasis and carcinogenesis. Although the role of miR-122 in the maintenance of liver homeostasis is well established, systematic and direct biochemical elucidation of the miR-122 target network remains incomplete. To this end, we performed Ago-dHITS-CLIP analysis in livers of 6 week-old wild type (WT) and KO mice to identify miRNA targetome in vivo, which revealed ∼1800 miR-122 binding sites, with over 30 fold enrichment for the miR-122 seed sequence sites in WT compared to KO livers. Surprisingly, a majority of miR-122 binding sites were observed on coding exons (40%) followed by 3’-UTRs, introns and 5’-UTRs. Motif analysis of miR-122 dependent binding sites lacking the seed sequence revealed a novel G-bulged motif in addition to the canonical miR-122 seed sequences. Moreover, the hepatic gene expression profile of these mice measured by RNA-seq showed up regulation of ∼2500 genes and down regulation of ∼1200 genes in KO livers compared to WT livers. A comparison of HITS-CLIP and RNA-seq data revealed that majority of miR-122 targets harboring canonical binding sites at their 3’UTRs as well as some coding regions, are significantly upregulated in miR-122 KO livers without notable alteration in the expression of or G-bulged targets at the RNA level. HITS-CLIP analysis of several human HCC and matching benign liver tissues also identified both canonical and G-bulged targets; where the majority of both are located in the coding regions followed by 3’UTRs. Association of miR-122 targets with Argonaute was precipitously reduced in HCCs expressing lower miR-122 levels than respective benign livers. Notably, though miR-122 is completely conserved and highly expressed in human and mouse livers, a large number of identified targets seem to be species-specific. Ingenuity Pathway Analysis of enriched targets revealed conserved pathway specific regulation in mouse and human livers. Pathways commonly associated with critical roles in liver carcinogenesis and liver metabolism, such as AMPK signaling and PI3/AKT pathways, were the major targets of miR-122-mediated regulation. This study suggests that miR-122 functionality is conserved across species. Citation Format: Juan Barajas. Comprehensive identification of miR-122 targetome in the liver and HCC by HITS-CLIP analysis reveals conserved regulation of AMPK signaling in hepatocarcinogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1928.

Abstract 150: A Population of ApoB-100 Peptide-specific CD8+ T cells Tracks Atherosclerosis in ApoE-/- Mice

Background: Growing evidence supports a potential role for CD8+ T cells in atherogenesis, yet CD8+T cell antigens relevant to the disease remain to be defined. The current study was undertaken to identify and characterize endogenous, apoB-100 antigen-specific CD8+ T cells in atherosclerosis using fluorescently tagged, synthetic soluble MHC-I/peptide complexes called Pentamers (Pent). Methods and Results: Self-reactive CD8+ T cells were tested for recall response to a 20-mer peptide derived from apoB-100 under investigation as a potential vaccine candidate. CD8+IFN-γ+ T cells in splenocytes of apoE-/- mice were significantly increased after 48 hours of peptide stimulation, which was enhanced by a 6-week high fat diet. We then tested the apoB-100 peptide for potential binding to mouse MHC-I alleles H2Db and H2Kb. Binding assays identified 13 sequential fragments of 8-mer lengths with potential H2Kb binding. Pents corresponding to the 13 peptide fragments were then synthesized and were screened for reactivity to apoB-100-specific CD8+ T cells from 13 week-old wild type and apoE-/- mice fed normal chow (NC). Flow cytometric analysis detected higher binding to CD8+ T cells from apoE-/- mice for 3 Pents. The first of the 3, called Pent 2, was then selected as prototype for further analysis. The OVA Pentamer SIINFEKL confirmed Pent 2 specificity. Pent 2+CD8+ T cells were significantly increased in 13 week-old apoE-/- mice fed a high fat diet (HC) for 6 weeks compared to NC fed mice (0.99±0.15% vs. 0.51±0.02%, respectively, N=5 each; P<0.05). Pent 2+CD8+ T cells correlated with plaque size of apoE-/- mice fed NC or HC for 6 weeks (R 2 =0.645, P=0.005, N=10) or for 15-22 weeks (R 2 =0.623, P=0.001, N=13). Pent 2 blocked CD8+ cytolytic activity against oxLDL-primed macrophages by 50% (N=6; P<0.05). There was a shift in the Pent 2+CD8+ Vβ repertoire after HC feeding for 6 weeks compared to NC, with significantly increased Pent 2+CD8+ Effector Memory cells (65.0±5.7% vs. 48.8±6.0%, respectively; P<0.01). Flow cytometry of digested aortic sinus plaques confirmed presence of Pent 2+CD8+ T cells in atherosclerotic plaques. Conclusion: Our study provides evidence of a self-reactive, apoB-100-specific CD8+ T cell population that tracks atherosclerotic disease in apoE-/- mice.

Survival of irradiated recipient mice after transplantation of bone marrow from young, old and "early aging" mice.

Bone marrow transplantation is used to examine survival, hematopoietic stem cell function and pathology in recipients of young and old wild type bone marrow derived stem cells (BMDSCs) as well as cells from p53-based models of premature aging. There is no difference in the long term survival of recipients of 8 week-old p53+/m donor cells compared to recipients of 8 week-old wild-type (WT) donor cells (70 weeks) or of recipients of 16-18 weeks-old donor cells from either p53+/m or WT mice. There is shorter survival in recipients of older versus younger WT donor bone marrow, but the difference is only significant when comparing 8 and 18 week-old donors. In the p44-based model, short term survival/engraftment is significantly reduced in recipients of 11 month-old p44 donor cells compared to 4 week-old p44 or wild type donor cells of either age; mid-life survival at 40 weeks is also significantly less in recipients of p44 cells. BMDSCs are readily detectable within recipient bone marrow, lymph node, intestinal villi and liver sinusoids, but not in epithelial derived cells. These results indicate that recipients of young BMDSCs may survive longer than recipients of old bone marrow, but the difference is marginal at best.

Increased ADAM17 Expression in ACE2 Knockout Mice is Associated with Increased Excitability of Paraventricular Nucleus Pre‐sympathetic Neurons

We previously reported that ACE2 knockout (KO) mice, like humans, develop age‐dependent hypertension. In addition, a disintegrin and metalloprotease 17 (ADAM17) was recently shown to contribute to neurogenic hypertension. Accordingly, we hypothesized that ADAM17 in the paraventricular nucleus (PVN) of hypothalamus contributes to enhanced sympathetic tone in mice lacking ACE2. Adult 10‐12 week‐old wildtype (WT) and ACE2 KO male mice (n=2‐4/group) were injected in the kidney with a pseudorabies virus encoding a green fluorescent protein, for retrograde labeling of sympathetic neurons. After 4 days, whole‐cell patch‐clamp recordings were conducted from labeled pre‐sympathetic PVN neurons. The resting membrane potential of these PVN neurons in WT mice was ‐59.7 ±1.06 mV, while ‐52.7 ±1.6 mV in ACE2 KO mice, which may suggest an increase threshold for excitability in pre‐sympathetic PVN neurons. In another set of mice, the hypothalamus was dissected from WT and KO animals (n=4/group) for western blot analysis. ADAM17 expression showed a 2‐fold increase in KO compared to WT (P<0.05). Finally, using immunohistochemistry, ADAM17 expression was confirmed in parvocellular neurons. Taken together, our data suggest that enhanced expression of ADAM17 in the parvocellular PVN could be associated with increased excitability of pre‐sympathetic neurons in ACE2 KO mice. This dysregulation in neuronal activity might alter the intricate pressor homeostasis leading to increased blood pressure. These results suggest that ADAM17 could be a potential new target for the reduction of enhanced sympathetic activity in neurogenic hypertension.Support: NIH HL093178

Mo1692 Vascular Endothelial Transient Receptor Potential Vanilloid 4 Links Colonic Inflammation in Dextran Sulfate Sodium-Induced Colitis Model Mice

BACKGROUND&AIMS: Transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel, and involved in physical sensing in various types of tissues. TRPV4 is expressed in sensory neuron and vascular endothelial cells in various organs. In gastrointestinal tracts, TRPV4 activation in epithelial cells increases in intracellular calcium concentrations and induces colitis. However, the localization and the role of the TRPV4 channel expressed in vascular endothelial cells in colitis are still poorly defined. In the present study, we investigated the alteration of TRPV4 expression in the colonic vascular endothelial cells during dextran sulfate sodium (DSS)-induced colitis. METHODS: Eight week-old wild type (WT) and TRPV4 knock-out (TRPV4 KO) mice were used. Colitis was induced by 2% DSS solution given as drinking water for 7 days. The disease activity index (DAI) was evaluated during the treatment, while colon length, histological damage, and myeloperoxidase (MPO) activity were examined post-DSS treatment. Immunohistochemical analysis was performed in distal colon. TRPV4-immunoreactivity was detected by using immunohistochemical staining with fluorescein-conjugated tyramide amplification. CD31, CD105, CD11b, CD68, Ly6B.2, and VEGF receptor-2 were detected by indirect staining with their specific antibodies. Vascular leakage was investigated using Evans blue dye extrusion assay. RESULTS: TRPV4 KO mice protected against DSS-induced weight loss, neutrophil (MPO), colon shortening, diarrhea, occult-fecal blood, and histological damage. TRPV4 immunoreactivities were detected mostly in epithelial cells of normal distal colon. DSS treatment drastically increased TRPV4 expression in vascular endothelial cells of mucosa and submucosal layer which colocalized with CD31, CD105, and VEGF receptor-2, but not epithelial cells. Repeated intrarectal and intravenous administration of TRPV4 agonist GSK1016790A exacerbated the severity of DSS colitis. Increased vascular permeability was observed following single intravenous administration of GSK1016790A in DSS colitis and this effect was abolished with TRPV4 antagonist RN1734. CD11b, CD68, Ly6B.2 immunopositive cells were detected around TRPV4 positive vascular endothelial cells in mucosa and submucosal layer in DSS colitis. CONCLUSION: These findings indicate that the alteration of TRPV4 in vascular endothelial cells may contribute to progression of colonic inflammation via increasing vascular permeability.

Mo1693 Activation of Bone Marrow-Derived TRPV4-Expressing M2 Macrophages Promotes Intestinal Contraction

BACKGROUND&AIMS: Transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel, and involved in physical sensing in various types of tissues. TRPV4 is expressed in sensory neuron and vascular endothelial cells in various organs. In gastrointestinal tracts, TRPV4 activation in epithelial cells increases in intracellular calcium concentrations and induces colitis. However, the localization and the role of the TRPV4 channel expressed in vascular endothelial cells in colitis are still poorly defined. In the present study, we investigated the alteration of TRPV4 expression in the colonic vascular endothelial cells during dextran sulfate sodium (DSS)-induced colitis. METHODS: Eight week-old wild type (WT) and TRPV4 knock-out (TRPV4 KO) mice were used. Colitis was induced by 2% DSS solution given as drinking water for 7 days. The disease activity index (DAI) was evaluated during the treatment, while colon length, histological damage, and myeloperoxidase (MPO) activity were examined post-DSS treatment. Immunohistochemical analysis was performed in distal colon. TRPV4-immunoreactivity was detected by using immunohistochemical staining with fluorescein-conjugated tyramide amplification. CD31, CD105, CD11b, CD68, Ly6B.2, and VEGF receptor-2 were detected by indirect staining with their specific antibodies. Vascular leakage was investigated using Evans blue dye extrusion assay. RESULTS: TRPV4 KO mice protected against DSS-induced weight loss, neutrophil (MPO), colon shortening, diarrhea, occult-fecal blood, and histological damage. TRPV4 immunoreactivities were detected mostly in epithelial cells of normal distal colon. DSS treatment drastically increased TRPV4 expression in vascular endothelial cells of mucosa and submucosal layer which colocalized with CD31, CD105, and VEGF receptor-2, but not epithelial cells. Repeated intrarectal and intravenous administration of TRPV4 agonist GSK1016790A exacerbated the severity of DSS colitis. Increased vascular permeability was observed following single intravenous administration of GSK1016790A in DSS colitis and this effect was abolished with TRPV4 antagonist RN1734. CD11b, CD68, Ly6B.2 immunopositive cells were detected around TRPV4 positive vascular endothelial cells in mucosa and submucosal layer in DSS colitis. CONCLUSION: These findings indicate that the alteration of TRPV4 in vascular endothelial cells may contribute to progression of colonic inflammation via increasing vascular permeability.

Gravin Scaffolding Protein Mediates Signaling Involved in theRegulation of Hyperlipidemia and Atherosclerosis

Atherosclerosis is a leading health problem and contributes to cardiovascular related mortality. Furthermore, hyperlipidemia is a major atherogenic factor.Understanding the cellular mechanisms leading to atherosclerosis is still lacking. Gravin (AKAP12), an A‐kinase anchoring protein (AKAP), scaffolds and targets various signaling proteins to specific intracellular locations and has been implicated in mediating lipid metabolism. Thus, the purpose of this study was to determine whether the absence of gravin‐mediated signaling would prevent the initiation and progression of atherosclerosis.5 week‐old wild‐type (WT) and gravin‐truncated (gravin‐t/t) mice were subjected to high fat diet (HFD) or normal diet (ND) for 16‐weeks. Cholesterol, triglyceride and VLDL level in serum were significantly decreased in gravin‐t/t HFD compared to WT HFD mice. In addition, systolic and diastolic blood pressure was also lower in gravin‐t/t HFD mice.Furthermore, gravin‐t/t HFD mice showed lower liver‐to‐body weight ratio as well as decreased lipid accumulation and liver damage.These observations are consistent with the lower serum alanine aminotransferase(ALT) and aspartate aminotransferase (AST) levels identified in gravin‐t/t HFD mice. In addition, gravin‐t/t HFD mice had decreased expression of the active form of sterol‐regulatory‐element‐binding protein 2 (SREBP2), which resulted in lower HMG‐CoA reductase (HMGCR) and low‐density lipoprotein receptor (LDLR) expression. Finally, we observed less aortic lipid accumulation in gravin‐t/t HFD mice. Our data indicates that the absence of gravin mediated signaling were resistance to hyperlipidemia and prevented the progression of atherosclerosis.

Spatially Localized Disruptions of Voltage Activated Calcium Release in Mtm1-Deficient Muscle Fibers

Mutations in the gene encoding the phosphoinositide phosphatase myotubularin (Mtm1) are responsible for myotubular myopathy. We previously showed that muscle fibers from Mtm1-deficient mouse suffer from defective excitation-contraction coupling. Here we measured voltage-clamp activated Ca2+ signals in fibers from the flexor digitorum brevis muscles of 4 week-old wild type (WT) and Mtm1-ko mice under line-scan confocal microscopy using the dye rhod-2. Ca2+ release in the diseased fibers was deficient over the full range of activation: fitting the voltage dependence of the peak rate of rise of the line-averaged rhod-2 F/F0 signals with a Boltzmann function gave mean values for maximum rate, midpoint voltage and slope of 0.29 ± 0.02 and 0.14 ± 0.02 F/F0.ms−1, −8.8 ± 1.5 and 0.95 ± 2.3 mV, 7.0 ± 0.4 and 9.2 ± 0.7 mV in WT (n=22) and Mtm1-ko fibers (n=15), respectively. Furthermore, the mean time to peak rate was significantly delayed by 5-10 ms in Mtm1-ko as compared to WT fibers. These global alterations were associated with severe spatial inhomogeneity of Ca2+ release in the diseased fibers with rhod-2 transients yielding localized disruptions along the scanned line including reduced peak amplitude but also delayed or slower rate of onset, suggestive of specific alteration of the early peak component of the rate of Ca2+ release. In fibers treated with wortmannin and LY294002, the properties of the line-averaged rhod-2 F/F0 signals were unchanged in WT fibers but the mean maximum rate of rise of the rhod-2 signal was enhanced by 60% in Mtm1-deficient fibers. Results show that Mtm1-deficiency provokes EC coupling failure through accumulation of spatially localized disruptions of Ca2+ release. They also suggest that pharmacological inhibition of PtdIns-3-kinase activity has the potency to alleviate defective EC coupling in the diseased fibers.

3D ELECTRON MICROSCOPY TOMOGRAPHY TO ASSESS MITOCHONDRIAL MORPHOLOGY IN THE ADULT HEART

<h3>Rationale</h3> Modulating mitochondrial morphology may alleviate cardiac disease by preventing mitochondrial dysfunction. Mitofusin-2 (Mfn2) tethers junctional sarcoplasmic reticulum (jSR) to mitochondria. Standard 2D electron microscopy (2D-EM) does not allow the accurate assessment of mitochondrial morphology and jSR in the adult heart. Here, we investigate whether 3D-EM tomography, which allows one to image the mitochondria and jSR in 3-dimensions, can provide a more objective and accurate assessment of these features. <h3>Methodology</h3> Electron micrographs of serially sectioned samples, obtained from left ventricular tissue harvested from either 8 week-old wild-type (WT) and Mfn2−/− mice, were imaged using either standard 2D-EM or 3D-EM tomography. Specific regions containing juxtaposed mitochondria and jSR were aligned, segmented, and reconstructed in 3D using Amira V.5. <h3>Results</h3> In 2D-EM the Mfn2−/− mitochondria appeared to be larger, whereas in 3D-EM tomography the mitochondria appeared to be either similar or smaller in size, when compared to WT. Measuring the distance between mitochondria and jSR using 2D-EM gave highly variable results. However, 3D-EM allowed one to reconstruct the space between the juxtaposed organelles in 3D allowing a more accurate measurement of this distance. <h3>Conclusions</h3> Here, we show that 3D-EM tomography is superior to 2D-EM in the assessment of mitochondrial morphology and can be used to more accurately measure the distance between mitochondrial and jSR in the adult heart. 3D-EM tomography will enable one to accurately assess mitochondrial morphology in different cardiac diseases (acute IRI, heart failure, LVH) and in the presence of co-morbidities (diabetes, metabolic syndrome, age).

BMT Doubles The Lifespan Of A Mouse Model Of Farber Disease and Corrects Hematopoietic Defects

Farber disease (FD) is a rare and severe inherited metabolic disorder with childhood lethality and no known cure. Farber patients have mutations in ASAH1, which codes for the protein acid ceramidase (ACDase). ACDase degrades ceramide; it is an essential and ubiquitous hydrolase. Clinical signs of FD include hepatosplenomegaly, granulomas, and histiocytosis. Some FD patients have been reported to have monocytosis and leukocytosis; some also present with neurological impairments. Allogeneic bone marrow transplantation (BMT) has had modest success in treating Farber patients, however it fails in patients with neurological manifestations of the disease. To better understand and improve on BMT for Farber disease, we performed syngeneic BMTs in a mouse model of Farber disease that we have recently generated. In our model a mutation from a patient with dramatically reduced ACDase activity was introduced into the analogous loci in the mouse genome. Our mouse model faithfully recapitulates the clinical signs of FD: mice homozygous for the Asah1 P361R mutation (Hom) show robust and systemic ceramide accumulation, lifespans reduced to 7-13 weeks, growth retardation, hepatosplenomegaly, thymomegaly, and wide-spread histiocytosis. Peripheral blood monocytosis and leukocytosis are evident as are neurological manifestations. Hematopoietic organs are particularly affected, with the spleen, thymus, and lymph nodes of Hom Farber mice all being enlarged compared to wild-type (WT) and heterozygous (Het) littermates. These organs are enlarged because they, along with the bone marrow, are massively infiltrated by foamy histiocytes (manuscript in preparation). In this present study we transplanted bone marrow from 4-5 week-old WT donor mice (pre-treated with 5-fluorouracil to enhance hematopoietic stem cell recovery) into 4-5 week-old Hom mice that were myeloablated with radiation. BMT doubled the lifespan of Hom mice to 21 weeks. Weight loss was also reduced, with Hom mice more closely approaching the weight of WT and Het mice. Hematopoietic organ enlargement was minimalized: spleen and lymph node sizes were significantly reduced and thymii were no longer visible. The prevention of hematopoietic organ enlargement was likely due to a drastic reduction in histiocysosis: much fewer foamy macrophages were found in the organs. Additionally, peripheral blood monocytosis and leukocytosis were prevented. This study demonstrates that BMT is efficacious in the treatment of the peripheral complications of Farber disease, yet it is not a permanent cure especially when the disease has neurological implications. It reveals the specific beneficial effects of BMT on hematopoietic organs in this novel mouse model of FD. Future studies will look at enhancing BMT outcomes in FD by transducing donor hematopoietic stem/progenitor cells with recombinant lentiviral vectors encoding WT ACDase. This combination of BMT and gene therapy in lysosomal storage disorders is clinically relevant as it could be translated for use in FD patients, following future clinical implementation for other disorders of this class, such as Fabry disease, for example. Disclosures:No relevant conflicts of interest to declare.

Gravin Scaffolding Protein Mediates Signaling in High Fat Diet Model

Atherosclerosis is an inflammatory response principally in the walls of arteries contributing to cardiovascular related mortality, which can be induced by diets rich in fats. Gravin (AKAP12), an A‐kinase anchoring protein (AKAP), is targeted to specific intracellular locations with protein kinase A (PKA) as well as other signaling molecules. It has been shown that gravin can regulate cholesterol efflux and LDL receptor expression via SREBPS activation. The purpose of this study was to determine whether the absence of gravin mediated signaling would prevent atherosclerosis induced by a high fat diet. Five week‐old wild‐type (WT) and gravin‐knockout (KO) mice were subjected to high fat diet (HFD) or normal diet (ND) for 16 weeks. Lipid profile was measured via HPLC and arterial blood pressure was measured via a Millar catheter. Cholesterol, triglyceride and VLDL levels as well as systolic and diastolic pressure were significantly decreased in KO compared to WT treated with HFD (p&lt;0.0001); no differences were observed between KO and WT with ND. This data indicates that the absence of gravin mediated signaling is able to decrease lipid metabolism as well as to lower the blood pressure in response to HFD.

Abstract 1196: Identification of lung cancer associated oncoantigens as targets for active immunotherapy

Abstract Non small cell lung cancer (NSCLC) is the leading cause of cancer death in men and women. Therefore, the development of new therapeutic strategies is essential for improving the prognosis and treatment of patients. An interesting experimental model to study human lung adenocarcinoma is represented by p53R172HΔg/K-rasG12D mice. They develop aggressive NSCLC that metastasize to multiple sites and display a stepwise, directly age-related progression, mimicking several features observed in lung cancer patients. Lung cancer progression in p53R172HΔg/K-rasG12D mice was characterized by non-invasive magnetic resonance imaging (MRI), histopathological and immunohistochemical analysis. p53R172HΔg/K-rasG12D mice develop lung adenocarcinomas already evident in 10 week-old mice, with a variable degree of differentiation. A significant gender difference in tumor progression was observed, with females developing more tumors than males. Moreover, by using qRT-PCR, some of the antigens deregulated in human lung cancer turned out to be up-regulated also during cancer progression in our mouse model, suggesting that p53R172HΔg/K-rasG12D mice recapitulate the metastatic nature of human NSCLC. Identification of oncoantigens expressed during tumor development could provide an unprecedented opportunity to address the immune system against these molecules. RNA was extracted from lungs of 10, 20 and 30 week-old wild type (wt) and p53R172HΔg/K-rasG12D mice. Transcription profiling was done using Mouse Exon 1.0 ST arrays: we detected 282 genes significantly differentially expressed during different stages of cancer progression. Interestingly, some of the genes that were already deregulated in the early phases of the tumor development remained deregulated also in advanced stages, suggesting their possible application as early biomarkers or as vaccination targets. These genes were ranked to full-fill the minimal requirement for an oncoantigen; within the plasma membrane proteins we identified potential candidates: CXCR1, SLC16A6, SLC26A9 and ROS1. On the basis of the literature data and mouse data we consider ROS1 an interesting putative oncoantigen to be further investigated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1196. doi:1538-7445.AM2012-1196

Genetic ablation of caveolin-1 increases neural stem cell proliferation in the subventricular zone (SVZ) of the adult mouse brain

Adult neural stem cells are self-renewing multipotent cells that have the potential to replace dysfunctional and/or dying neuronal cells at the site of brain injury or degeneration. Caveolins are well-known tumor-suppressor genes that were recently found to be involved in the regulation of stem cell proliferation. For instance, ablation of the caveolin-1 (Cav-1) gene in mice markedly increases the proliferation of intestinal and mammary stem cells. However, the roles of caveolins in the proliferation of adult neural stem cells still remain unknown. In this study, dual-label immunofluorescence analysis of the proliferation marker, Ki67, and the stem cell markers, nestin and Sox2, was performed on brains of 8 week-old wild-type (WT) and Cav-1 knockout (KO) mice. Our results demonstrate an increased number of Ki67-positive nuclei in the subventricular zone (SVZ) of Cav-1 KO brains. Importantly, our dual-label immunofluorescence analyses demonstrate increased co-localization of Ki67 with both nestin and Sox2 in the SVZ of Cav-1 KO brains. Remarkably similar results were also obtained with Cav-2 and Cav-3 KO mouse brains as well, with increased proliferation of adult neural stem cells. Thus, the SVZ of caveolin KO mouse brains displays an increased proliferation of adult neural stem cells. Caveolin proteins might represent new crucial regulators of adult neural stem cell proliferation.

Abstract 5355: An Alpha-1A-Adrenergic Receptor Subtype Agonist Prevents Cardiomyopathy Without Increasing Blood Pressure

Background: Alpha-1-adrenergic receptor (AR) agonists classically increase blood pressure (BP). Among the 3 alpha-1-AR subtypes, A, B, and D, the alpha-1A is required for cardiac protection in a knockout (KO) mouse, and is sufficient for protection of cultured cardiac myocytes, via ERK activation. However, it is unknown if activation of the alpha-1A-subtype by a drug can protect the heart in vivo. Hypothesis: At a dose that does not increase BP, an agonist selective for the alpha-1A-AR subtype can prevent cardiomyopathy. Methods: We gave the alpha-1A agonist A61603 (A6) to 11 week-old wild type (WT) male C57Bl6J mice by osmotic minipump. We induced cardiomyopathy with a single dose of doxorubicin (DOX) (25 mg/kg IP), a cardiotoxic cancer drug. We measured BP by tail cuff, activated (phosphorylated, P)-ERK by immunoblot, heart mRNAs by RT-qPCR, fractional shortening (FS) by echocardiog-raphy (ECHO), myocyte necrosis by serum creatine kinase (CK), apoptosis by TUNEL stain, and fibrosis by sirius red stain. Results : In dose-finding experiments (0.01–100 ug/kg/d), A6 at 10 ng/kg/d over 7 days had no effect on daily tail cuff BP (average mmHg Vehicle 115 ± 4; A6 119 ± 4), but increased heart P-ERK (1.7-fold) and the mRNAs for beta-MyHC and ANF (5-fold). A6 at higher doses increased BP. Next, A6 at the non-hypertensive dose (10 ng/kg/d) or vehicle was infused over 7 days after a single DOX injection. The TABLE shows that DOX caused cardiomyopathy, with reduced survival and FS, and increased necrosis, apoptosis, and fibrosis (data are mean ± SE). A6 prevented all of these abnormalities. In alpha-1A-subtype KO mice, DOX caused increased apoptosis and mortality compared with WT mice, and A6 had no beneficial effect (not shown), indicating specificity of A6. Conclusions: A very low dose of an agonist selective for the alpha-1A-AR subtype can activate cardiac survival signaling (P-ERK), induce cardiac fetal genes, and prevent DOX-induced cardiomyopathy, all without increasing BP.

Glial Cell Line-Derived Neurotrophic Factor Increases β-Cell Mass and Improves Glucose Tolerance

Pancreatic beta-cell mass increases in response to increased demand for insulin, but the factors involved are largely unknown. Glial cell line-derived neurotrophic factor (GDNF) is a growth factor that plays a role in the development and survival of the enteric nervous system. We investigated the role of GDNF in regulating beta-cell survival. Studies were performed using the beta-TC-6 pancreatic beta-cell line, isolated mouse pancreatic beta cells, and in vivo in transgenic mice that overexpress GDNF in pancreatic glia. GDNF receptor family alpha1 and c-Ret receptor expression were assessed by reverse-transcription polymerase chain reaction and immunofluorescence microscopy. Apoptosis was evaluated by assessing caspase-3 cleavage. Phosphoinositol-3-kinase signaling pathway was analyzed by Akt phosphorylation. Glucose homeostasis was assessed by performing intraperitoneal glucose tolerance tests. Insulin sensitivity was assessed using intraperitoneal injection of insulin. We demonstrate the presence of receptors for GDNF, GFRalpha1, and c-Ret on beta cells. GDNF promoted beta-cell survival and proliferation and protected them from thapsigargin-induced apoptosis (P<.0001) in vitro. Exposure of beta-cells to GDNF also resulted in phosphorylation of Akt and GSK3beta. Transgenic mice that overexpress GDNF in glia exhibit increased beta-cell mass, proliferation, and insulin content. No differences in insulin sensitivity and c-peptide levels were noted. Compared with wild-type mice, GDNF-transgenic mice have significantly lower blood glucose levels and improved glucose tolerance (P<.01). GDNF-transgenic mice are resistant to streptozotocin-induced beta-cell loss (P<.001) and subsequent hyperglycemia. We demonstrate that over expression of GDNF in pancreatic glia improves glucose tolerance and that GDNF may be a therapeutic target for improving beta-cell mass.