HO Mice Research Articles

Enamel Structure Defects in Kdf1 Missense Mutation Knock-in Mice.

The Keratinocyte differentiation factor 1 (KDF1) is reported to take part in tooth formation in humans, but the dental phenotype of Kdf1 mutant mice has not been understood. Additionally, the role of the KDF1 gene in dental hard tissue development is rarely known. In this study, we constructed a Kdf1 missense mutation knock-in mouse model through CRISPR/Cas9 gene-editing technology. Enamel samples from wildtypes (WT) and Kdf1 homozygous mutants (HO) were examined using micro-computed tomography (micro-CT), scanning electron microscopy (SEM), an atomic force microscope (AFM) and Raman microspectroscopy. The results showed that a novel Kdf1 missense mutation (c. 908G>C, p.R303P) knock-in mice model was constructed successfully. The enamel of HO mice incisors appeared chalky and defective, exposing the rough interior of the inner enamel and dentin. Micro-CT showed that HO mice had lower volume and mineral density in their tooth enamel. In addition, declined thickness was found in the unerupted enamel layer of incisors in the HO mice. Using SEM and AFM, it was found that enamel prisms in HO mice enamel were abnormally and variously shaped with loose decussating crystal arrangement, meanwhile the enamel rods were partially fused and collapsed, accompanied by large gaps. Furthermore, misshapen nanofibrous apatites were disorderly combined with each other. Raman microspectroscopy revealed a compromised degree of order within the crystals in the enamel after the Kdf1 mutation. To conclude, we identified enamel structure defects in the Kdf1 missense mutation knock-in mice, which displayed fragmentary appearance, abnormally shaped prism structure, decreased mineral density, altered crystal ordering degree and chemical composition of the enamel layer. This may support the potential role of the KDF1 gene in the natural development of enamel.

In vivo Crispr-Cas9 targeting matrix metalloproteinase-9 improves bladder voiding in prediabetic female Tally Ho mice

In vivo Crispr-Cas9 targeting matrix metalloproteinase-9 improves bladder voiding in prediabetic female Tally Ho mice

Tff3 Deficiency Protects against Hepatic Fat Accumulation after Prolonged High-Fat Diet.

Trefoil factor 3 (Tff3) protein is a small secretory protein expressed on various mucosal surfaces and is involved in proper mucosal function and recovery via various mechanisms, including immune response. However, Tff3 is also found in the bloodstream and in various other tissues, including the liver. Its complete attenuation was observed as the most prominent event in the early phase of diabetes in the polygenic Tally Ho mouse model of diabesity. Since then, its role in metabolic processes has emerged. To elucidate the complex role of Tff3, we used a new Tff3-deficient mouse model without additional metabolically relevant mutations (Tff3-/-/C57BL/6NCrl) and exposed it to a high-fat diet (HFD) for a prolonged period (8 months). The effect was observed in male and female mice compared to wild-type (WT) counter groups (n = 10 animals per group). We monitored the animals’ general metabolic parameters, liver morphology, ultrastructure and molecular genes in relevant lipid and inflammatory pathways. Tff3-deficient male mice had reduced body weight and better glucose utilization after 17 weeks of HFD, but longer HFD exposure (32 weeks) resulted in no such change. We found a strong reduction in lipid accumulation in male Tff3-/-/C57BL/6NCrl mice and a less prominent reduction in female mice. This was associated with downregulated peroxisome proliferator-activated receptor gamma (Pparγ) and upregulated interleukin-6 (Il-6) gene expression, although protein level difference did not reach statistical significance due to higher individual variations. Tff3-/-/C57Bl6N mice of both sex had reduced liver steatosis, without major fatty acid content perturbations. Our research shows that Tff3 protein is clearly involved in complex metabolic pathways. Tff3 deficiency in C57Bl6N genetic background caused reduced lipid accumulation in the liver; further research is needed to elucidate its precise role in metabolism-related events.

PP2A-mTOR-p70S6K/4E-BP1 axis regulates M1 polarization of pulmonary macrophages and promotes ambient particulate matter induced mouse lung injury

To identify key signaling pathways involved in ambient particulate matter (PM)-induced pulmonary injury, we generated a mouse model with myeloid-specific deletion of Ppp2r1a gene (encoding protein phosphatase 2 A (PP2A) A subunit), and conducted experiments in a real-ambient PM exposure system. PP2A Aα-/- homozygote (Aα HO) mice and matched wild-type (WT) littermates were exposed to PM over 3-week and 6-week. The effects of PM exposure on pulmonary inflammation, oxidative stress, and apoptosis were significantly enhanced in Aα HO compared to WT mice. The number of pulmonary macrophages increased by 74.8~88.0% and enhanced M1 polarization appeared in Aα HO mice upon PM exposure. Secretion of M1 macrophage-related inflammatory cytokines was significantly increased in Aα HO vs. WT mice following PM exposure. Moreover, we demonstrated that PP2A-B56α holoenzyme regulated M1 polarization and that the mTOR signaling pathway mediated the persistent M1 polarization upon PM2.5 exposure. Importantly, PP2A-B56α holoenzyme was shown to complex with mTOR/p70S6K/4E-BP1, and suppression of B56α led to enhanced phosphorylation of mTOR, p70S6K, and 4E-BP1. These observations demonstrate that the PP2A-mTOR-p70S6K/4E-BP1 signaling is a critical pathway in mediating macrophage M1 polarization, which contributes to PM-induced pulmonary injury.

Muscle injury promotes heterotopic ossification by stimulating local bone morphogenetic protein-7 production

BackgroundHeterotopic ossification (HO) is a pathological condition of abnormal bone formation in soft tissue, which causes pain and restricted range of motion in patients. There are two broad categories of HO, hereditary and acquired. Although different types of HO do not use identical mechanistic pathways of pathogenesis, muscle injury appears to be a unifying feature for all types of HO. However, little is known about the mechanisms by which muscle injury facilitates HO formation. Objective and methodThis study aimed to explore the cellular and molecular mechanisms linking muscle injury to HO by using cardiotoxin to induce muscle injury in a bone morphogenetic protein-2 (BMP-2)–induced HO mouse model. ResultsWe found that muscle injury augmented HO formation and that this effect was correlated with BMP signalling activation and upregulation of BMP-7 expression at the early phase of HO progression. We further demonstrated that inhibition of BMP-7 activity in vitro suppressed the osteogenesis-promoting effect of conditioned medium derived from injured muscle tissue and in vivo reduced the volume of HO formation. We also showed that antiinflammatory drug treatment reduced the volume of HO with concomitant reduction in BMP-7 production. ConclusionIn summary, our study has identified BMP-7 as a key osteoinductive factor in injured muscle that facilitates HO formation. The translational potential of this articleOur results provide a candidate mechanistic rationale for the use of antiinflammatory drugs in the prevention of HO.

Marinobufagenin, a mammalian endogenous cardiotonic steroid, is synthesized from the intermediates in the classical bile acid pathway in Cyp27A1 knockout mice

ObjectivesThe bioactive steroidal Na/K‐ATPase ligand marinobufagenin (MBG) participates in renal Na+ transport, cell homeostasis, regulates blood pressure and arterial wall collagen production. MBG biosynthesis in mammals is controlled by the Cyp27a1 enzyme (cytochrome P450, family 27, subfamily a, polypeptide 1), which initiates the acidic bile acid (BA) pathway in rodent adrenal cortex. Nevertheless, the knockout of Cyp27a1 in mice did not reduce MBG production. In addition, the homozygous Cyp27a1(−/−) mice (HO) exhibited an upregulation of adrenal Akr1d1 enzyme (aldo‐keto reductase family 1, member D1) vs. wild type mice (WT). Akr1d1 participates in the final steps of both acidic and classical BA pathways. We hypothesized (i) that MBG may be synthesized from the intermediates in the classical BA pathway under Akr1d1 control, and (ii) that inhibition of Akr1d1 in HO will decrease MBG production.MethodsAll‐trans retinoic acid (ATRA) suppresses Akr1d1 and the enzymes controlled classical BA pathway. Four‐to‐six months old male HO and wild type mice (WT) were fed ATRA diet (150 mg/kg of diet) or control diet (CTRL) for 10 days (n=8/group). Adrenal Cyp27a1 and Akr1d1, and hepatic Cyp27a1, Akr1d1, Cyp7a1, and Cyp8b1 mRNA (by qPCR), and MBG excretion were assessed before (baseline, BL) and after ATRA diet. Data were analyzed by t‐test or 2‐way ANOVA and are presented as mean±SE.ResultsAdrenal and hepatic Cyp27a1 mRNAs were down‐regulated by 80% in HO vs. WT (p<0.01). Adrenal Akr1d1 mRNA was 3.5‐fold overexpressed in HO vs. WT (p<0.01) (Figure 1A). Hepatic mRNAs were overexpressed in HO vs. WT (Figure 1B; Table). ATRA diet decreased expression of hepatic Akr1d1 mRNA in both strains vs. CTRL (HO: by 180%, p<0.01; WT: by 170%, p<0.01). Hepatic Cyp7a1 and Cyp8b1 mRNAs were also significantly down‐regulated by ATRA in both WT and HO but remained significantly higher in HO than in WT (Table). Baseline urine MBG level was 2‐fold higher in HO vs. WT (p<0.05); inhibition of Akr1d1 by ATRA was associated with a decrease in urine MBG in HO (5.3±0.5 vs. 2.9±0.2 pmol/24hr; BL vs. ATRA; p<0.05), but not in WT (2.1±0.4 vs. 2.6±0.9 pmol/24hr; BL vs. ATRA, n/s).ConclusionsThe up‐regulation of hepatic Akr1d1, Cyp7a1 and Cyp8b1 enzymes in Cyp27a1(−/−) mice may compensate for a lack of Cyp27a1 and promote production of a bioactive steroid MBG from the intermediates in the classical BA pathway. Inhibition of Akr1d1 resulted in a decrease of MBG production in Cyp27a1(−/−) mice only. MBG production was not changed by ATRA in WT with normal expression of Cyp27a1, which indicates that Cyp27a1 is a predominant enzyme in MBG biosynthesis.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Benzene-induced mouse hematotoxicity is regulated by a protein phosphatase 2A complex that stimulates transcription of cytochrome P4502E1

Chronic benzene exposure is associated with hematotoxicity and the development of aplastic anemia and leukemia. However, the signaling pathways underlying benzene-induced hematotoxicity remain to be defined. Here, we investigated the role of protein phosphatase 2A (PP2A) in the regulation of benzene-induced hematotoxicity in a murine model. Male mice with a hepatocyte-specific homozygous deletion of the Ppp2r1a gene (encoding PP2A Aα subunit) (HO) and matched wildtype (WT) mice were exposed to benzene via inhalation at doses of 1, 10, and 100 ppm for 28 days. Peripheral white blood cell counts and activation of bone marrow progenitors were attenuated in the HO mice, indicating that Ppp2r1a deletion protects against benzene-induced hematotoxicity. Moreover, elevation of urinary S-phenyl mercapturic acid, a benzene metabolite, was much greater in WT mice than in HO mice. Real-time exhalation analysis revealed more exhaled benzene but fewer benzene metabolites in HO mice than in WT mice, possibly because of the down-regulation of Cyp2e1, encoding cytochrome P4502E1, in hepatocytes of the HO mice. Loss-of-function screening disclosed that PP2A complexes containing the B56α subunit participate in regulating Cyp2e1 expression. Notably, PP2A-B56α suppression in HepG2 cells resulted in persistent β-catenin phosphorylation at Ser33-Ser37-Thr41 in response to CYP2E1 agonists. In parallel, nuclear translocation of β-catenin was inhibited, concomitant with a remarkable decrease of Cyp2e1 expression. These findings support the notion that a regulatory cascade comprising PP2A-B56α, β-catenin, and Cyp2e1 is involved in benzene-induced hematotoxicity, providing critical insight into the role of PP2A in responses to the environmental chemicals.

Peripheral denervation participates in heterotopic ossification in a spinal cord injury model

We previously reported the development of a new acquired neurogenic HO (NHO) mouse model, combining spinal cord transection (SCI) and chemical muscle injury. Pathological mechanisms responsible for ectopic osteogenesis after central neurological damage are still to be elucidated. In this study, we first hypothesized that peripheral nervous system (PNS) might convey pathological signals from injured spinal cord to muscles. Secondly, we sought to determine whether SCI could lead to intramuscular modifications of BMP2 signalling pathways. Twenty-one C57Bl6 mice were included in this protocol. Bilateral cardiotoxin (CTX) injection in hamstring muscles was associated with a two-stage surgical procedure, combining thoracic SCI with unilateral peripheral denervation. Volumes of HO (bone volume, BV) were measured 28 days after surgery using micro-computed tomography imaging techniques and histological analyses were made to confirm intramuscular osteogenesis. Volume comparisons were conducted between right and left hind limb of each animal, using a Wilcoxon signed rank test. Quantitative polymerase chain reaction (qPCR) was performed to explore intra muscular expression of BMP2, Alk3 and Id1. Nineteen mice survive the complete SCI and peripheral denervation procedure. When CTX injections were done right after surgery ( n = 7), bilateral HO were detected in all animals after 28 days. Micro-CT measurements showed significantly increased BV in denervated paws (1.47 mm 3 ± 0.5) compared to contralateral sides (0.56 mm 3 ± 0.4), P = 0.03. When peripheral denervation and CTX injections were performed after sham SCI surgery ( n = 6), bilateral HO were present in three mice at day 28. qPCR analyses showed no changes in intra muscular BMP2 expression after SCI as compared to control mice. Peripheral denervation can be reliably added to spinal cord transection in NHO mouse model. This new experimental design confirms that neuro-inflammatory mechanisms induced by central or peripheral nervous system injury plays a key role in triggering ectopic osteogenesis.

Enzyme replacement therapy prevents loss of bone and fat mass in murine homocystinuria.

Skeletal and connective tissue defects are the most striking symptoms in patients suffering from classical homocystinuria (HCU). Here, we determined body composition and bone mass in three mouse models of HCU and assessed whether a long-term administration of enzyme replacement therapy (ERT) corrected the phenotype. The mouse models of HCU were analyzed using dual-energy X-ray absorptiometry and the data were complemented by plasma biochemical profiles. Both the mouse model lacking CBS (KO) and the one expressing human CBS mutant transgene on a mouse CBS null background (I278T) showed marked bone loss and decreased weight mostly due to a lower fat content compared with negative controls. In contrast, the HO mouse expressing the human CBS WT transgene on a mouse CBS null background showed no such phenotype despite similar plasma biochemical profile to the KO and I278T mice. More importantly, administration of ERT rescued bone mass and changes in body composition in the KO mice treated since birth and reversed bone loss and improved fat content in the I278T mice injected after the development of clinical symptoms. Our study suggests that ERT for HCU may represent an effective way of preventing the skeletal problems in patients without a restricted dietary regime.

Targeted Inhibition of PI3K and MEK in Combination With Radiation Therapy in UT15: A Murine Model of Head and Neck Squamous Cell Carcinoma

To study the effectiveness of dual targeted PI3K and MEK inhibition alone and in combination with radiotherapy in HNSCC models as a novel clinical treatment strategy. MTT assays were used to establish growth inhibition and schedule of RT with Buparlisib (BKM120) a pan PI3K-inhibitor. Clonogenic survival assays were used to assess the impact of BKM120 alone, Binimetnib (MEK162), a MEK1/2-inhibitor, alone, and both drugs in combination. Subcutaneous xenografts were established in female NIH III HO mice using the UT15 cell line. Tumors grew to 200-400 mm3 before treatment: +BKM120 (10mg/kg oral gavage), +MEK162 (5mg/kg oral gavage), or combined +BKM120 (10mg/kg), +MEK162 (5mg/kg oral gavage) ± RT. In radiation treated cohorts, a sub-curative 45 Gy dose (3 Gy/day 5 days a week) was delivered 4 hours before drug treatment. The primary endpoint was tumor regrowth at 90 days post treatment. For UT15 cell line the IC50 concentration for BKM120 was 0.5 μM and for MEK162 was above 10 μM. Delivery of BKM120 (0.5 μM) 4 hours post radiotherapy produced the greatest reduction in cell viability compared to radiation alone 100% to 70%. Clonogenic survival assay confirmed UT15 to be more sensitive to MEK162 than BKM120 with survivals of 40% at 0.1 μM versus 90% at 0.2 μM, respectively. In the UT15 flank tumor model, RT alone, (BKM120 & MEK162), RT+ BKM120, RT+ MEK162, and RT+ (BKM120 & MEK162) had slower growth rates compared to controls (p=0.004). There was no difference in growth rates between the experimental arms. The time needed to reach twice normalized volume from the start of treatment is listed in Table 1. Western analysis confirmed the targeted agents were acting as expected based on their mechanism of action. When assessing tumor regrowth past the 90 days post-treatment it did appear that RT+ BKM120 cohort never surpasses the 3x normalized treatment volume raising the possibility of permanent change in tumor growth and viability. However, the standard error of the means (SEM) becomes increasing variable and strong conclusions cannot be drawn at this time. In previous experiments, there was a significant difference in growth rates between RT alone and RT+ MEK162 ± BKM120, primarily driven by the combination of RT and MEK162. In our UT15 model, radiation in combination with PI3K and/or MEK inhibition did not significant delay tumor growth rates. There was, however, a suggestion of permanent tumor change, in UT15, after 90 days in particular with PI3K blockade. This is contrary to our UT14 model where MEK inhibition produced the greatest delay in growth patterns. Further experiments with quantitative PCR and other agents are ongoing to determine if this difference is related to varying expression levels or differences in drug efficacy.Abstract 3377; Table 1Treatment cohortsMean Time to 2x volume (days)SEMControl12.11.05RT alone85.75.60BKM12060.33.44MEK16261.15.25BKM120 & MEK16275.911.7RT + BKM120988.78RT + MEK1621017.97RT + (BKM120 & MEK162)87.16.09 Open table in a new tab

Peripheral denervation participates in heterotopic ossification in a spinal cord injury model.

We previously reported the development of a new acquired neurogenic HO (NHO) mouse model, combining spinal cord transection (SCI) and chemical muscle injury. Pathological mechanisms responsible for ectopic osteogenesis after central neurological damage are still to be elucidated. In this study, we first hypothesized that peripheral nervous system (PNS) might convey pathological signals from injured spinal cord to muscles in NHO mouse model. Secondly, we sought to determine whether SCI could lead to intramuscular modifications of BMP2 signaling pathways. Twenty one C57Bl6 mice were included in this protocol. Bilateral cardiotoxin (CTX) injection in hamstring muscles was associated with a two-stage surgical procedure, combining thoracic SCI with unilateral peripheral denervation. Volumes of HO (Bone Volume, BV) were measured 28 days after surgery using micro-computed tomography imaging techniques and histological analyses were made to confirm intramuscular osteogenesis. Volume comparisons were conducted between right and left hind limb of each animal, using a Wilcoxon signed rank test. Quantitative polymerase chain reaction (qPCR) was performed to explore intra muscular expression of BMP2, Alk3 and Id1. Nineteen mice survive the complete SCI and peripheral denervation procedure. When CTX injections were done right after surgery (n = 7), bilateral HO were detected in all animals after 28 days. Micro-CT measurements showed significantly increased BV in denervated paws (1.47 mm3 +/- 0.5) compared to contralateral sides (0.56 mm3 +/-0.4), p = 0.03. When peripheral denervation and CTX injections were performed after sham SCI surgery (n = 6), bilateral HO were present in three mice at day 28. Quantitative PCR analyses showed no changes in intra muscular BMP2 expression after SCI as compared to control mice (shamSCI). Peripheral denervation can be reliably added to spinal cord transection in NHO mouse model. This new experimental design confirms that neuro inflammatory mechanisms induced by central or peripheral nervous system injury plays a key role in triggering ectopic osteogenesis.

Cystathionine beta-synthase deficiency alters hepatic phospholipid and choline metabolism: Post-translational repression of phosphatidylethanolamine N-methyltransferase is a consequence rather than a cause of liver injury in homocystinuria.

Classical homocystinuria (HCU) due to inactivating mutation of cystathionine β-synthase (CBS) is a poorly understood life-threatening inborn error of sulfur metabolism. A previously described cbs-/- mouse model exhibits a semi-lethal phenotype due to neonatal liver failure. The transgenic HO mouse model of HCU exhibits only mild liver injury and recapitulates multiple aspects of the disease as it occurs in humans. Disruption of the methionine cycle in HCU has the potential to impact multiple aspect of phospholipid (PL) metabolism by disruption of both the Kennedy pathway and phosphatidylethanolamine N-methyltransferase (PEMT) mediated synthesis of phosphatidylcholine (PC). Comparative metabolomic analysis of HO mouse liver revealed decreased levels of choline, and choline phosphate indicating disruption of the Kennedy pathway. Alterations in the relative levels of multiple species of PL included significant increases in PL degradation products consistent with enhanced membrane PL turnover. A significant decrease in PC containing 20:4n6 which primarily formed by the methylation of phosphatidylethanolamine to PC was consistent with decreased flux through PEMT. Hepatic expression of PEMT in both the cbs-/- and HO models is post-translationally repressed with decreased levels of PEMT protein and activity that inversely-correlates with the scale of liver injury. Failure to induce further repression of PEMT in HO mice by increased homocysteine, methionine and S-adenosylhomocysteine or depletion of glutathione combined with examination of multiple homocysteine-independent models of liver injury indicated that repression of PEMT in HCU is a consequence rather than a cause of liver injury. Collectively, our data show significant alteration of a broad range of hepatic PL and choline metabolism in HCU with the potential to contribute to multiple aspects of pathogenesis in this disease.

Mast cells and exosomes in hyperoxia-induced neonatal lung disease.

Chronic lung disease of prematurity (CLD) is a frequent sequela of premature birth and oxygen toxicity is a major associated risk factor. Impaired alveolarization, scarring, and inflammation are hallmarks of CLD. Mast cell hyperplasia is a feature of CLD but the role of mast cells in its pathogenesis is unknown. We hypothesized that mast cell hyperplasia is a consequence of neonatal hyperoxia and contributes to CLD. Additionally, mast cell products may have diagnostic and prognostic value in preterm infants predisposed to CLD. To model CLD, neonatal wild-type and mast cell-deficient mice were placed in an O2 chamber delivering hyperoxic gas mixture [inspired O2 fraction (FiO2 ) of 0.8] (HO) for 2 wk and then returned to room air (RA) for an additional 3 wk. Age-matched controls were kept in RA (FiO2 of 0.21). Lungs from HO mice had increased numbers of mast cells, alveolar simplification and enlargement, and increased lung compliance. Mast cell deficiency proved protective by preserving air space integrity and lung compliance. The mast cell mediators β-hexosaminidase (β-hex), histamine, and elastase increased in the bronchoalveolar lavage fluid of HO wild-type mice. Tracheal aspirate fluids (TAs) from oxygenated and mechanically ventilated preterm infants were analyzed for mast cell products. In TAs from infants with confirmed cases of CLD, β-hex was elevated over time and correlated with FiO2 Mast cell exosomes were also present in the TAs. Collectively, these data show that mast cells play a significant role in hyperoxia-induced lung injury and their products could serve as potential biomarkers in evolving CLD.

A role for Sarcolemmal Membrane‐Associated Protein (SLMAP) in mediating autophagy in endothelial cells through an AMPK‐dependent mechanism

ObjectiveSarcolemmal membrane associated protein (SLMAP) belongs to the superfamily of tail‐anchored membrane proteins and is a component of endoplasmic reticulum (ER)/sarcoplasmic reticulum as well as mitochondria, cell surface membranes and the nuclear envelope. It has been reported to be involved in various physiological functions mainly in, for example, the regulation of ion channels, membrane fusion and vesicle transport. Previously, we have shown that the up‐regulation of SLMAP protein in diabetic db/db and Tally Ho mice was linked to endothelial dysfunction; however, until now no mechanistic evidence has been provided as to how an up‐regulation of SLMAP disrupts endothelial function in type 2 diabetes mellitus. The primary objective for the current study was to elucidate the cellular pathway(s) whereby an overexpression of SLMAP in human umbilical vein endothelial cells (HUVECs) results in endothelial dysfunction.Materials and MethodsHUVECs were cultured in M199 medium (Sigma Aldrich, USA) supplemented with 15% FBS, heparin (0.1% of 100mg/ml), ECGS (0.01% of 100 mg/ml), 100 μg/mL penicillin, and 100 U/mL streptomycin, at 37°C in a 5% CO2—95% air‐humidified incubator. The 41kDa isoform of SLMAP was overexpressed in HUVECs using SLMAP Lentiviral particles designed for humans (Accession number: AF304450.1; Genecopoeia), and SLMAP was partially knocked using small hairpin RNA (shRNA) lentiviral particles for humans (Santa Cruz Biotechnology, Catalogue number: sc‐78464‐V). The procedures were performed according to the manufacturer's instructions using polybrene at a concentration of 5μg/ml. After overexpression, the HUVECs were treated with, or without, the AMP‐activated protein kinase activator (AMPK), AICAR, 500μM, for 24 h. Protein markers of apoptosis, oxidative stress and autophagy were evaluated by Western Blotting, FACS, DHE staining and immunofluorescence techniques respectively.ResultsThe ER chaperone protein, GRP78; the unfolded protein response (UPR) signaling proteins, such as IRE‐1α, PERK, XBP1s, cleaved caspase‐3, p‐JNK, Bcl‐XL; and markers of autophagy, such as LC3B‐II, Beclin‐1 were found to be significantly elevated (p<0.05), while protein levels of p‐AMPKα (Thr172) and SERCA2 were significantly decreased (p<0.05) in HUVECs overexpressed with SLMAP. Interestingly, treatment with the AMPK activator, AICAR, significantly (p<0.05) reversed all of the changes in HUVECs overexpressed with SLMAP. Moreover, the partial knock down of SLMAP significantly reduced IRE‐1α, PERK, XBP1s, p‐JNK, Bcl‐XL, LC3B‐II, Beclin‐1 proteins level, and increased p‐AMPKα (Thr172) and SERCA2 proteins level in SLMAP shRNA‐transfected HUVECs.ConclusionCollectively, this study has demonstrated for the first time that changes in the expression of SLMAP protein (41kDa) play an important role in ER stress and autophagy in endothelial cells via an AMPK‐dependent mechanism.Support or Funding InformationThis abstract is part of the project funded by NPRP: 5‐149‐3‐040

Altered expression of apoA-I, apoA-IV and PON-1 activity in CBS deficient homocystinuria in the presence and absence of treatment: Possible implications for cardiovascular outcomes

Classical homocystinuria (HCU) is caused by mutations in cystathionine beta-synthase (CBS) which, if untreated, typically results in cognitive impairment, thromboembolic complications and connective tissue disturbances. Paraoxonase-1 (PON1) and apolipoprotein apoA-I are both synthesized in the liver and contribute to much of the cardioprotective effects of high density lipoprotein. Additionally, apoA-I exerts significant neuro-protective effects that act to preserve cognition. Previous work in a Cbs null mouse model that incurs significant liver injury, reported that HCU dramatically decreases PON1 expression. Conflicting reports exist in the literature concerning the relative influence of homocysteine and cysteine upon apoA-I expression. We investigated expression of PON1 and apoA-I in the presence and absence of homocysteine lowering therapy, in both the HO mouse model of HCU and human subjects with this disorder. We observed no significant change in plasma PON1 paraoxonase activity in either mice or humans with HCU indicating that this enzyme is unlikely to contribute to the cardiovascular sequelae of HCU. Plasma levels of apoA-I were unchanged in mice with mildly elevated homocysteine due to CBS deficiency but were significantly diminished in both mice and humans with HCU. Subsequent experiments revealed that HCU acts to dramatically decrease apoA-I levels in the brain. Cysteine supplementation in HO mice had no discernible effect on plasma levels of apoA-I while treatment to lower homocysteine normalized plasma levels of this lipoprotein in both HO mice and humans with HCU. Our results indicate that plasma apoA-I levels in HCU are inversely related to homocysteine and are consistent with a plausible role for decreased expression of apoA-I as a contributory factor for both cardiovascular disease and cognitive impairment in HCU.

Long-term betaine therapy in a murine model of cystathionine beta-synthase deficient homocystinuria: Decreased efficacy over time reveals a significant threshold effect between elevated homocysteine and thrombotic risk

Classical homocystinuria (HCU) is caused by deficiency of cystathionine β-synthase and is characterized by connective tissue disturbances, mental retardation and cardiovascular disease. Treatment for pyridoxine non-responsive HCU typically involves lowering homocysteine levels with a methionine-restricted diet and dietary supplementation with betaine. Compliance with the methionine-restricted diet is difficult and often poor. Investigating optimization of the efficacy of long-term betaine treatment in isolation from a methionine-restricted diet is precluded by ethical considerations regarding patient risk. The HO mouse model of HCU developed in our laboratory, exhibits constitutive expression of multiple pro-inflammatory cytokines and a hypercoagulative phenotype both of which respond to short-term betaine treatment. Investigation of the effects of long-term betaine treatment in the absence of methionine-restriction in HO HCU mice revealed that the ability of betaine treatment to lower homocysteine diminished significantly over time. Plasma metabolite analysis indicated that this effect was due at least in part, to reduced betaine-homocysteine S-methyltransferase (BHMT) mediated remethylation of homocysteine. Western blotting analysis revealed that BHMT protein levels are significantly repressed in untreated HCU mice but are significantly induced in the presence of betaine treatment. The observed increase in plasma homocysteine during prolonged betaine treatment was accompanied by a significant increase in the plasma levels of TNF-alpha and IL-1beta and reversion to a hypercoagulative phenotype. Our findings are consistent with a relatively sharp threshold effect between severely elevated plasma homocysteine and thrombotic risk in HCU and indicate that the HO mouse model can serve as a useful tool for both testing novel treatment strategies and examining the optimal timing and dosing of betaine treatment with a view toward optimizing clinical outcome.

67 Spontaneous cardiac hypertrophy and adverse LV remodelling in a novel human relevant mouse model of diabetes; a mechanistic insight

<h3></h3> Heart failure (HF) is one of the commonest cardiovascular complications of Diabetes Mellitus (DM) with the prevalence of DM reported at around 30% in many pivotal heart failure studies. DM is an independent predictor of mortality in patients with HF, however molecular mechanisms that contribute to HF development in the diabetic population are poorly understood. Using a novel human relevant mouse model of DM (GENA348), identified through the MRC mouse mutagenesis programme with a point mutation in the pancreatic glucokinase (GLK) gene we investigate the molecular mechanisms that contribute to the HF phenotype in DM. GLK is the glucose sensor which regulates insulin secretion and GLK activity is reduced by 90% by the GENA348 point mutation resulting in severe hyperglycaemia. Similar mutations underlie Maturity Onset Diabetes of the Young Type 2 (MODY 2) in humans. Mean random blood glucose was found to be increased in the GENA348 mutant (HO) mice compared to wild type (WT) littermates (WT 6.9±0.3 mmol/l vs HO 20.6±0.8 mmol/l, p&lt;0.001). Serial echocardiography was performed, at 3, 6 and 12 months. No significant changes in echocardiographic parameters were observed at 3 months, although by 6 months development of significant cardiac hypertrophy in HO mice was observed. At 12 months of age left ventricular dilatation was evident, characterised by an 8% increase in diastolic diameter (WT 4.08±0.10 vs HO 4.41±0.12, p&lt;0.05). Systolic function was preserved although significant diastolic dysfunction was evident at 6 and 12 months with a 31% reduction in the E:A ratio. Histological staining illustrated significant cellular hypertrophy with real time PCR data demonstrating a relative 150% increase in the hypertrophic marker BNP. Hypertrophic pathways were examined through western blot analysis revealing an age dependant increase in Akt phosphorylation (3 months- no increase, 6 months-140%, 12 months-460%). Serum levels of advanced glycation end products (AGE) were also elevated by 86% (WT 21±3.5 ng/ml vs HO 39±8.3 ng/ml, p&lt;0.05) as was the protein expression level of the receptor for AGEs (RAGE). In vitro cellular experiments also revealed AGEs directly activate Akt through phosphorylation and increase levels of the receptor RAGE. AGE induced phosphorylation of Akt is inhibited in the presence of wortmannin, suggesting a PI3K dependent signalling mechanism. This was further confirmed in vivo where a bolus injection of wortmannin in 6-month old mutant mice returned Akt phosphorylation levels to those seen in WT mice. In conclusion, using the first human relevant mouse model of diabetes, GENA348 we demonstrate the development of a progressive cardiac phenotype including cardiac hypertrophy, LV dilatation and diastolic dysfunction similar to the clinical manifestations of diabetic cardiomyopathy. We propose that the RAGE/PI3K/Akt pathway contributes to the molecular mechanisms associated with the cardiac phenotype.

A novel transgenic mouse model of CBS-deficient homocystinuria does not incur hepatic steatosis or fibrosis and exhibits a hypercoagulative phenotype that is ameliorated by betaine treatment

Cystathionine beta-synthase (CBS) catalyzes the condensation of homocysteine (Hcy) and serine to cystathionine, which is then hydrolyzed to cysteine by cystathionine gamma-lyase. Inactivation of CBS results in CBS-deficient homocystinuria more commonly referred to as classical homocystinuria, which, if untreated, results in mental retardation, thromboembolic complications, and a range of connective tissue disorders. The molecular mechanisms that underlie the pathology of this disease are poorly understood. We report here the generation of a new mouse model of classical homocystinuria in which the mouse cbs gene is inactivated and that exhibits low-level expression of the human CBS transgene under the control of the human CBS promoter. This mouse model, designated “human only” (HO), exhibits severe elevations in both plasma and tissue levels of Hcy, methionine, S-adenosylmethionine, and S-adenosylhomocysteine and a concomitant decrease in plasma and hepatic levels of cysteine. HO mice exhibit mild hepatopathy but, in contrast to previous models of classical homocystinuria, do not incur hepatic steatosis, fibrosis, or neonatal death with approximately 90% of HO mice living for at least 6months. Tail bleeding determinations indicate that HO mice are in a hypercoagulative state that is significantly ameliorated by betaine treatment in a manner that recapitulates the disease as it occurs in humans. Our findings indicate that this mouse model will be a valuable tool in the study of pathogenesis in classical homocystinuria and the rational design of novel treatments.

008 Spontaneous cardiac hypertrophy and identification of novel hypertrophic pathways in a genetic mouse model that emulates human diabetes

Animal models are key in the exploration of the pathophysiological mechanisms and complications of diabetes mellitus (DM). Most current models have considerable limitations in that they do not faithfully replicate human forms of DM. Recently, a novel human relevant mouse model of diabetes (GENA 348) was identified through the Medical Research Council, Harwell, UK mouse mutagenesis programme in which a point mutation in the glucokinase gene results in severely impaired glucokinase function and significant hyperglycaemia. Similar mutations in the glucokinase gene are known to underlie Maturity Onset Diabetes of the Young Type 2 (MODY 2) in humans. We studied the GENA 348 mouse to determine whether it expresses a cardiac phenotype to provide insight into the pathophysiological mechanisms underlying the development of diabetic cardiomyopathy. Fifteen wild type (WT) controls and eight homozygote mutant (HO) mice had serial echocardiography performed at 3 and 6 months. At 3 months no evidence of cardiac hypertrophy or contractile dysfunction was demonstrated in HO compared to WT mice. By 6 months of age, echo demonstrated development of cardiac hypertrophy in GENA 348 HO mice characterised by a 16% increase in left ventricular mass/body weight (wt 4.28±0.15 vs HO 4.95±0.26, p&lt;0.05), a 25% increase in dPW/dD (WT 23.3±1.0 vs HO 29.1±1.1, p&lt;0.01) and an 18% increase in left ventricular relative wall thickness (WT 0.45±0.01 vs HO 0.53±0.02, p&lt;0.05). No differences in systolic function were observed although significant diastolic dysfunction was also evident with a 31% reduction in the E:A ratio (WT 2.65±0.2 vs HO 1.84±0.19, p&lt;0.05) and a 34% increase in the IVRT (WT 15.2±1.1 ms vs HO 20.5±0.37 ms, p&lt;0.01). Histological staining illustrated cellular hypertrophy with a 60% increase in cardiomyocyte size (WT 238±14 μm² vs HO 383±26 μm², p&lt;0.001). Hypertrophic pathways were examined through western blot analysis, a 140% increase in Akt phosphorylation, a key mediator of cardiac hypertrophy and a 65% increase in GSK3β phosphorylation one of its key regulators was observed. We also demonstrate that advanced glycation end products (AGE) were elevated by 86% in the serum of mutant GENA 348 mice. Collectively, these data indicate GENA 348 mutant mice develop a cardiac phenotype including hypertrophy and diastolic dysfunction similar to the clinical manifestations of diabetic cardiomyopathy. Furthermore, a novel hypertrophic pathway has been identified in which AGE stimulation through GSK3β leads to activation of the intracellular Akt pathway. GENA 348 may thus provide a valuable, human-relevant tool for studying the molecular determinants of DM related cardiovascular complications.

Searching QTL by gene expression: analysis of diabesity

BackgroundRecent developments in sequence databases provide the opportunity to relate the expression pattern of genes to their genomic position, thus creating a transcriptome map. Quantitative trait loci (QTL) are phenotypically-defined chromosomal regions that contribute to allelically variant biological traits, and by overlaying QTL on the transcriptome, the search for candidate genes becomes extremely focused.ResultsWe used our novel data mining tool, ExQuest, to select genes within known diabesity QTL showing enriched expression in primary diabesity affected tissues. We then quantified transcripts in adipose, pancreas, and liver tissue from Tally Ho mice, a multigenic model for Type II diabetes (T2D), and from diabesity-resistant C57BL/6J controls. Analysis of the resulting quantitative PCR data using the Global Pattern Recognition analytical algorithm identified a number of genes whose expression is altered, and thus are novel candidates for diabesity QTL and/or pathways associated with diabesity.ConclusionTranscription-based data mining of genes in QTL-limited intervals followed by efficient quantitative PCR methods is an effective strategy for identifying genes that may contribute to complex pathophysiological processes.