Gene expression changes in the secondary palate and mandible of Prdm16 −/− mice

Loss of Prdm16 expression in the mouse leads to a complete cleft of the secondary palate. We have now determined changes in gene expression in the secondary palates of Prdm16 ^?/? fetuses in an attempt to reveal the mechanism(s) leading to the failure of palate closure in these mice. Defined pathway-based polymerase chain reaction arrays were used to analyze the expression of genes associated with the extracellular matrix and the transforming growth factor-β and bone morphogenetic protein signaling networks, perturbations of which can lead to palatal clefting. Loss of Prdm16 expression in the secondary palate leads to alterations in numerous genes within these groups, many of which have been linked to chondrogenesis and osteogenesis. The expression of several genes linked to bone development was significantly changed in the developing secondary palate. Analysis of gene expression in the mandibles of Prdm16 ^?/? fetuses revealed similar alterations in the same gene set. These data suggest that one function of Prdm16 is the regulation of genes that play a role in the differentiation of mesenchymal cells into chondro-/osteocytes.     

Reduced VLDL clearance in Apoe−/−Npc1−/− mice is associated with increased Pcsk9 and Idol expression and decreased hepatic LDL-receptor levels

Niemann-Pick type C1 (NPC1) promotes the transport of LDL receptor (LDL-R)-derived cholesterol from late endosomes/lysosomes to other cellular compartments. NPC1-deficient cells showed impaired regulation of liver_X receptor (LXR) and sterol regulatory element-binding protein (SREBP) target genes. We observed that Apoe^−/−Npc1^−/− mice displayed a marked increase in total plasma cholesterol mainly due to increased VLDL, reflecting decreased clearance. Although nuclear SREBP-2 and Ldlr mRNA levels were increased in Apoe^−/−Npc1^−/− liver, LDL-R protein levels were decreased in association with marked induction of proprotein convertase subtilisin/kexin type 9 (Pcsk9) and inducible degrader of the LDL-R (Idol), both known to promote proteolytic degradation of LDL-R. While Pcsk9 is known to be an SREBP-2 target, marked upregulation of IDOL in Apoe^−/−Npc1^−/− liver was unexpected. However, several other LXR target genes also increased in Apoe^−/−Npc1^−/− liver, suggesting increased synthesis of endogenous LXR ligands secondary to activation of sterol biosynthesis. In conclusion, we demonstrate that NPC1 deficiency has a major impact on VLDL metabolism in Apoe^−/− mice through modulation of hepatic LDL-R protein levels. In contrast to modest induction of hepatic IDOL with synthetic LXR ligands, a striking upregulation of IDOL in Apoe^−/−Npc1^−/− mice could indicate a role of endogenous LXR ligands in regulation of hepatic IDOL.     

Ontogenic changes in lung cholesterol metabolism,lipid content,and histology in mice with Niemann–Pick type C disease

Niemann–Pick Type C (NPC) disease is caused by a deficiency of either NPC1 or NPC2. Loss of function of either protein results in the progressive accumulation of unesterified cholesterol in every tissue leading to cell death and organ damage. Most literature on NPC disease focuses on neurological and liver manifestations. Pulmonary dysfunction is less well described. The present studies investigated how Npc1 deficiency impacts the absolute weight, lipid composition and histology of the lungs of Npc1^−/− mice (Npc1^nih) at different stages of the disease, and also quantitated changes in the rates of cholesterol and fatty acid synthesis in the lung over this same time span (8 to 70 days of age). Similar measurements were made in Npc2^−/− mice at 70 days. All mice were of the BALB/c strain and were fed a basal rodent chow diet. Well before weaning, the lung weight, cholesterol and phospholipid (PL) content, and cholesterol synthesis rate were all elevated in the Npc1^−/− mice and remained so at 70 days of age. In contrast, lung triacylglycerol content was reduced while there was no change in lung fatty acid synthesis. Despite the elevated PL content, the composition of PL in the lungs of the Npc1^−/− mice was unchanged. H&E staining revealed an age-related increase in the presence of lipid-laden macrophages in the alveoli of the lungs of the Npc1^−/− mice starting as early as 28 days. Similar metabolic and histologic changes were evident in the lungs of the Npc2^−/− mice. Together these findings demonstrate an intrinsic lung pathology in NPC disease that is of early onset and worsens over time.     

Rutaecarpine suppresses atherosclerosis in ApoE−/− mice through upregulating ABCA1 and SR-BI within RCT

ABCA1 and scavenger receptor class B type I (SR-BI)/CD36 and lysosomal integral membrane protein II analogous 1 (CLA-1) are the key transporter and receptor in reverse cholesterol transport (RCT). Increasing the expression level of ABCA1 and SR-BI/CLA-1 is antiatherogenic. The aim of the study was to find novel antiatherosclerotic agents upregulating expression of ABCA1 and SR-BI/CLA-1 from natural compounds. Using the ABCA1p-LUC and CLA-1p-LUC HepG2 cell lines, we found that rutaecarpine (RUT) triggered promoters of ABCA1 and CLA-1 genes. RUT increased ABCA1 and SR-BI/CLA-1 expression in vitro related to liver X receptor alpha and liver X receptor beta. RUT induced cholesterol efflux in RAW264.7 cells. ApoE-deficient (ApoE^−/−) mice treated with RUT for 8 weeks showed ∼68.43, 70.23, and 85.56% less en face lesions for RUT (L), RUT (M), and RUT (H) groups, respectively, compared with the model group. Mouse macrophage-specific antibody and filipin staining indicated that RUT attenuated macrophages and cholesterol accumulations in atherosclerotic lesions, respectively. Additionally, ABCA1 and SR-BI expression was highly induced by RUT in livers of ApoE^−/− mice. Meanwhile, RUT treatment significantly increased the fecal ³H-cholesterol excretion, which demonstrated that RUT could promote RCT in vivo. RUT was identified to be a candidate that protected ApoE^−/− mice from developing atherosclerosis through preferentially promoting activities of ABCA1 and SR-BI within RCT.     

Impact of the loss of caveolin-1 on lung mass and cholesterol metabolism in mice with and without the lysosomal cholesterol transporter,Niemann–Pick type C1

Caveolin-1 (Cav-1) is a major structural protein in caveolae in the plasma membranes of many cell types, particularly endothelial cells and adipocytes. Loss of Cav-1 function has been implicated in multiple diseases affecting the cardiopulmonary and central nervous systems, as well as in specific aspects of sterol and lipid metabolism in the liver and intestine. Lungs contain an exceptionally high level of Cav-1. Parameters of cholesterol metabolism in the lung were measured, initially in Cav-1-deficient mice (Cav-1^−/−), and subsequently in Cav-1^−/− mice that also lacked the lysosomal cholesterol transporter Niemann–Pick C1 (Npc1) (Cav-1^−/−:Npc1^−/−). In 50-day-old Cav-1^−/− mice fed a low- or high-cholesterol chow diet, the total cholesterol concentration (mg/g) in the lungs was marginally lower than in the Cav-1^+/+ controls, but due to an expansion in their lung mass exceeding 30%, whole-lung cholesterol content (mg/organ) was moderately elevated. Lung mass (g) in the Cav-1^−/−:Npc1^−/− mice (0.356 ± 0.022) markedly exceeded that in their Cav-1^+/+:Npc1^+/+ controls (0.137 ± 0.009), as well as in their Cav-1^−/−:Npc1^+/+ (0.191 ± 0.013) and Cav-1^+/+:Npc1^−/− (0.213 ± 0.022) littermates. The corresponding lung total cholesterol contents (mg/organ) in mice of these genotypes were 6.74 ± 0.17, 0.71 ± 0.05, 0.96 ± 0.05 and 3.12 ± 0.43, respectively, with the extra cholesterol in the Cav-1^−/−:Npc1^−/− and Cav-1^+/+:Npc1^−/− mice being nearly all unesterified (UC). The exacerbation of the Npc1 lung phenotype and increase in the UC level in the Cav-1^−/−:Npc1^−/− mice imply a regulatory role of Cav-1 in pulmonary cholesterol metabolism when lysosomal sterol transport is disrupted.     

Characterization of intestinal inflammation and identification of related gene expression changes in mdr1a−/− mice

Multidrug resistance targeted mutation (mdr1a (-/-) ) mice spontaneously develop intestinal inflammation. The aim of this study was to further characterize the intestinal inflammation in mdr1a (-/-) mice. Intestinal samples were collected to measure inflammation and gene expression changes over time. The first signs of inflammation occurred around 16 weeks of age and most mdr1a (-/-) mice developed inflammation between 16 and 27 weeks of age. The total histological injury score was the highest in the colon. The inflammatory lesions were transmural and discontinuous, revealing similarities to human inflammatory bowel diseases (IBD). Genes involved in inflammatory response pathways were up-regulated whereas genes involved in biotransformation and transport were down-regulated in colonic epithelial cell scrapings of inflamed mdra1 (-/-) mice at 25 weeks of age compared to non-inflamed FVB mice. These results show overlap to human IBD and strengthen the use of this in vivo model to study human IBD. The anti-inflammatory regenerating islet-derived genes were expressed at a lower level during inflammation initiation in non-inflamed colonic epithelial cell scrapings of mdr1a (-/-) mice at 12 weeks of age. This result suggests that an insufficiently suppressed immune response could be crucial to the initiation and development of intestinal inflammation in mdr1a (-/-) mice.     

Hypercholesterolemia in young adult APOE−/− mice alters epidermal lipid composition and impairs barrier function

Long-term exposure to hypercholesterolemia induces the development of skin xanthoma's characterized by the accumulation of lipid-laden foam cells in humans and in mice. Early skin changes in response to hypercholesterolemia are however unknown. In this study, we investigated the skin lipid composition and associated barrier function in young adult low-density lipoprotein receptor knockout (LDLR^−/−) and apolipoprotein E knockout (APOE^−/−) mice, two commonly used hypercholesterolemic mouse models characterized by the accumulation of apolipoprotein B containing lipoproteins. No differences were observed on cholesterol content in the epidermis in LDLR^−/− mice nor in the more extremely hypercholesterolemic APOE^−/− mice. Interestingly, the free fatty acid profile in the APOE^−/− epidermis shifted towards shorter and unsaturated chains. Genes involved in the synthesis of cholesterol and fatty acids were downregulated in APOE^−/− skin suggesting a compensation for the higher influx of plasma lipids, most probably as cholesteryl esters. Importantly, in vivo transepidermal water loss and permeability studies with murine lipid model membranes revealed that the lipid composition of the APOE^−/− skin resulted in a reduced skin barrier function. In conclusion, severe hypercholesterolemia associated with increased apolipoprotein B containing lipoproteins affects the epidermal lipid composition and its protective barrier.     

Properties of the permeability transition in VDAC1−/− mitochondria

Opening of the permeability transition pore (PTP), a high-conductance mitochondrial channel, causes mitochondrial dysfunction with Ca²⁺ deregulation, ATP depletion, release of pyridine nucleotides and of mitochondrial apoptogenic proteins. Despite major efforts, the molecular nature of the PTP remains elusive. A compound library screening led to the identification of a novel high affinity PTP inhibitor (Ro 68-3400), which labeled a ∼32 kDa protein that was identified as isoform 1 of the voltage-dependent anion channel (VDAC1) [A.M. Cesura, E. Pinard, R. Schubenel, V. Goetschy, A. Friedlein, H. Langen, P. Polcic, M.A. Forte, P. Bernardi, J.A. Kemp, The voltage-dependent anion channel is the target for a new class of inhibitors of the mitochondrial permeability transition pore. J. Biol. Chem. 278 (2003) 49812–49818]. In order to assess the role of VDAC1 in PTP formation and activity, we have studied the properties of mitochondria from VDAC1^−/− mice. The basic properties of the PTP in VDAC1^−/− mitochondria were indistinguishable from those of strain-matched mitochondria from wild-type CD1 mice, including inhibition by Ro 68-3400, which labeled identical proteins of 32 kDa in both wild-type and VDAC1^−/− mitochondria. The labeled protein could be separated from all VDAC isoforms. While these results do not allow to exclude that VDAC is part of the PTP, they suggest that VDAC is not the target for PTP inhibition by Ro 68-3400.     

Melatonin through blockade of Hif-1α signaling mediates the anti-fibrosis under hypoxia in canine Sertoli cells

The hypoxic microenvironment of cryptorchidism is an important factor in the impairment and fibrosis of Sertoli cells which result in blood-testis barrier (BTB) destruction and spermatogenesis loss. Recent studies have shown that melatonin, a well-known pineal hormone exerts beneficial effects against pathological fibrosis in a various of organs. However, it is still unknown whether melatonin can regulate hypoxia-induced fibrosis of Sertoli cells. In this study we evaluate melatonin levels, and its synthesizing enzymes, AANAT and HIOMT expression patterns in canine cryptorchidism and contralateral normal testis. Results show abdominal testes presented low melatonin levels and AANAT and HIOMT expression compared with testes located in the scrotum. Moreover, we established a hypoxia-induced fibrosis model in canine Sertoli cells induced by cobalt chloride (CoCl₂) and found that melatonin inhibited the EMT markers expression and ECM production as well as Hif-1α expression of Sertoli cells in a dose-dependent manner. Furthermore, use of Lificiguat (synonyms YC-1, Hif-1α inhibitor) to interfere with the Hif-1α pathway showed a similar effect with melatonin suppression of the fibrosis in Sertoli cells. The results indicate that melatonin supplementation can alleviate the fibrosis process of Sertoli cells caused by hypoxia, which is associated with regulating the inhibition of Hif-1α signaling.     

Impaired instrumental learning in Spred1−/− mice,a model for a rare RASopathy

RASopathies are neuro-cardio-facio-cutaneous disorders stemming from mutations in genes regulating the RAS-MAPK pathway. Legius syndrome is a rare RASopathy disorder caused by mutations in the SPRED1 gene. SPRED1 protein negatively regulates activation of Ras by inhibiting RAS/RAF and by its interaction with neurofibromin, a Ras GTPase-activating protein (RAS-GAP). Cognitive impairments have been reported in Legius syndrome as well as in other RASopathy disorders. Modelling these cognitive deficits in a Spred1 mouse model for Legius syndrome has demonstrated spatial learning and memory deficits, but other cognitive domains remained unexplored. Here, we attempted to utilize a cognitive touchscreen battery to investigate if Spred1^−/− mice exhibit deficits in other cognitive domains. We show that Spred1^−/− mice had heterogeneous performance in instrumental operant learning, with a large subgroup (n = 9/20) failing to reach the standard criterion on touchscreen operant pretraining, precluding further cognitive testing. To examine whether targeting the RAS-MAPK signalling pathway could rescue these cognitive impairments, Spred1^−/− mice were acutely treated with the clinically relevant mitogen-activated protein kinase (MEK) inhibitor PD325901. However, MEK inhibition did not improve their instrumental learning. We conclude that Spred1^−/− mice can model severe cognitive impairments that cannot be reversed in adulthood.     

The role of hepatic Surf4 in lipoprotein metabolism and the development of atherosclerosis in apoE−/− mice

Elevated plasma levels of low-density lipoprotein-C (LDL-C) increase the risk of atherosclerotic cardiovascular disease. Circulating LDL is derived from very low-density lipoprotein (VLDL) metabolism and cleared by LDL receptor (LDLR). We have previously demonstrated that cargo receptor Surfeit 4 (Surf4) mediates VLDL secretion. Inhibition of hepatic Surf4 impairs VLDL secretion, significantly reduces plasma LDL-C levels, and markedly mitigates the development of atherosclerosis in LDLR knockout (Ldlr^?/?) mice. Here, we investigated the role of Surf4 in lipoprotein metabolism and the development of atherosclerosis in another commonly used mouse model of atherosclerosis, apolipoprotein E knockout (apoE^?/?) mice. Adeno-associated viral shRNA was used to silence Surf4 expression mainly in the liver of apoE^?/? mice. In apoE^?/? mice fed a regular chow diet, knockdown of Surf4 expression significantly reduced triglyceride secretion and plasma levels of non-HDL cholesterol and triglycerides without causing hepatic lipid accumulation or liver damage. When Surf4 was knocked down in apoE^?/? mice fed the Western-type diet, we observed a significant reduction in plasma levels of non-HDL cholesterol, but not triglycerides. Knockdown of Surf4 did not increase hepatic cholesterol and triglyceride levels or cause liver damage, but significantly diminished atherosclerosis lesions. Therefore, our findings indicate the potential of hepatic Surf4 inhibition as a novel therapeutic strategy to reduce the risk of atherosclerotic cardiovascular disease.     

Loss of the nutrient receptor Tas1R3 reduces atherosclerotic plaque accumulation and hepatic steatosis in ApoE−/− mice

Journal of Physiology and Biochemistry - The taste receptor type I (Tas1R) family consists of three G protein-coupled receptors (T1R1, T1R2, and T1R3) that form heterodimers recognizing sweet...     

On the regulatory role of side-chain hydroxylated oxysterols in the brain. Lessons from CYP27A1 transgenic and Cyp27a1−/− mice

The two oxysterols, 27-hydroxycholesterol (27OH) and 24S-hydroxycholesterol (24OH), are both inhibitors of cholesterol synthesis and activators of the liver X receptor (LXR) in vitro. Their role as physiological regulators under in vivo conditions is controversial, however. In the present work, we utilized a previously described mouse model with overexpressed human sterol 27-hydroxylase (CYP27A1). The levels of 27OH were increased about 12-fold in the brain. The brain levels of HMG-CoA reductase mRNA and HMG-CoA synthase mRNA levels were increased. In accordance with increased cholesterol synthesis, most of the cholesterol precursors were also increased. The level of 24OH, the dominating oxysterol in the brain, was decreased by about 25%, most probably due to increased metabolism by CYP27A1. The LXR target genes were unaffected or slightly changed in a direction opposite to that expected for LXR activation. In the brain of Cyp27^−/− mice, cholesterol synthesis was slightly increased, with increased levels of cholesterol precursors but normal mRNA levels of HMG-CoA reductase and HMG-CoA synthase. The mRNA levels corresponding to LXR target genes were not affected. The results are consistent with the possibility that both 24OH and 27OH are physiological suppressors of cholesterol synthesis in the brain. The results do not support the contention that 27OH is a general activator of LXR target genes in this organ.     

Counteract of bone marrow of blotchy mice against the increases of plasma copper levels induced by high-fat diets in LDLR−/− mice

BackgroundBone marrow of blotchy mouse (blotchy marrow) reflects the function of transmembrane domain and relevant intramembrane sites of ATP7A in myeloid cells. By chronic infusion of angiotensin II, we previously found that blotchy marrow plays a minor role in regulating plasma copper. Moreover, the recipients of blotchy marrow presented a moderate reduction of plasma lipids and inflammatory mediator production. Little is known about whether these changes are a specific response to angiotensin II or reveal a more general role of ATP7A.Objective and designWe investigated if blotchy marrow reduces plasma lipids and inflammatory mediators induced by high-fat diets. To test this hypothesis, blotchy and control marrows were reconstituted to the recipient mice (irradiated male LDLR−/− mice), followed by high-fat-diet feeding for 4 months. At the end points, plasma metals (copper, zinc and iron), lipid profiling (cholesterol, triglyceride, phospholipids and lipoprotein) and six inflammatory mediators (lymphotacin, MCP3, MCP5, TIMP1, VEGF-A and IP-10) were measured. Parallel experiments were performed using male LDLR−/− mice fed either high-fat diets or chow diets for 4 months.ResultsIn addition to hyperlipidemia and low-grade inflammation, high-fat diets selectively increased plasma copper concentration compared to chow diets in LDLR−/− mice. After high-fat-diet feeding, the recipients with blotchy marrow showed a decrease in plasma copper (p < 0.01) and an increase in plasma iron (p < 0.05). The recipients with blotchy marrow also presented decreases in cholesterol (p < 0.01) and phospholipids (p < 0.05) in plasma. Surprisingly, plasma levels of MCP3 (p < 0.05), MCP5 (p < 0.05), TIMP1 (p < 0.01), VEGF-A (p < 0.01) and IP-10 (p < 0.01) were significantly increased in the recipients with blotchy marrow compared to controls; the increased levels of MCP3, MCP5 and TIMP1 were more than 50%.ConclusionOur studies showed that blotchy marrow counteracts the increased copper levels induced by high-fat diets, indicating that circulating myeloid cells can regulate blood copper levels via ATP7A. Moreover, transplantation of blotchy marrow followed by high-fat diets leads to a decrease in lipid profile and an increase in inflammatory mediator production. Overall, blotchy marrow mediates divergent responses to angiotensin II and high-fat diets in vivo.