Regulation of the cardiac sodium channel Nav1.5 by utrophin in dystrophin-deficient mice

Duchenne muscular dystrophy (DMD) is a severe striated muscle disease due to the absence of dystrophin. Dystrophin deficiency results in dysfunctional sodium channels and conduction abnormalities in hearts of mdx mice. Disease progression in the mdx mouse only modestly reflects that of DMD patients, possibly due to utrophin up-regulation. Here, we investigated mice deficient in both dystrophin and utrophin [double knockout (DKO)] to assess the role of utrophin in the regulation of the cardiac sodium channel (Na(v)1.5) in mdx mice.

Xanthohumol ameliorates atherosclerotic plaque formation, hypercholesterolemia, and hepatic steatosis in ApoE-deficient mice

SCOPE: Xanthohumol (XN), a prenylated antioxidative and anti-inflammatory chalcone from hops, exhibits positive effects on lipid and glucose metabolism. Based on its favorable biological properties, we investigated whether XN attenuates atherosclerosis in western-type diet-fed apolipoprotein-E-deficient (ApoE(-/-) ) mice. METHODS AND RESULTS: XN supplementation markedly reduced plasma cholesterol concentrations, decreased atherosclerotic lesion area, and attenuated plasma concentrations of the proinflammatory cytokine monocyte chemoattractant protein 1. Decreased hepatic triglyceride and cholesterol content, activation of AMP-activated protein kinase, phosphorylation and inactivation of acetyl-CoA carboxylase, and reduced expression levels of mature sterol regulatory element-binding protein (SREBP)-2 and SREBP-1c mRNA indicate reduced lipogenesis in the liver of XN-fed ApoE(-/-) mice. Concomitant induction of hepatic mRNA expression of carnitine palmitoyltransferase-1a in ApoE(-/-) mice-administered XN suggests increased fatty acid beta-oxidation. Fecal cholesterol concentrations were also markedly increased in XN-fed ApoE(-/-) mice compared with mice fed western-type diet alone. CONCLUSION: The atheroprotective effects of XN might be attributed to combined beneficial effects on plasma cholesterol and monocyte chemoattractant protein 1 concentrations and hepatic lipid metabolism via activation of AMP-activated protein kinase.

Sensitivity to endothelin-1 is decreased in isolated livers of endothelial constitutive nitric oxide synthase knockout mice

ABSTRACT: BACKGROUND: Hepatic sinusoidal resistance is regulated by vasoactive factors including endothelin-1 (ET-1) and nitric oxide (NO). In the absence of NO, vasoconstrictor response to endothelin is expected to predominate. Therefore, we hypothesized sensitivity to endothelin to be increased in mice lacking the endothelial cell NO synthase gene. Response of vascular resistance to endothelin was assessed in the in situ perfused liver of endothelial constitutive nitric oxide synthase (ecNOS) knockout and wild type mice. Livers were also harvested for RNA and protein isolation for quantitative PCR and Western blotting, respectively. The expression of endothelin receptors, isoenzymes of NO synthase, heme-oxygenase and adrenomedullin was quantified. RESULTS: Endothelin increased hepatic vascular resistance in a dose-dependent manner in both strains; however, this increase was significantly less in ecNOS knockout mice at physiologic concentrations. Expression of heme-oxygenases and adrenomedullin was similar in both groups, whereas inducible nitric oxide synthase (iNOS) protein was not detectable in either strain. mRNA levels of pre-pro-endothelin-1 and ETB receptor were comparable in both strains, while mRNA for ETA receptor was decreased in ecNOS knockouts. CONCLUSION: Livers of ecNOS knockout mice have a decreased sensitivity to endothelin at physiologic concentrations; this is associated with a decreased expression of ETA receptors, but not with other factors, such as iNOS, ETB receptors, adrenomedullin or heme-oxygenase. Further studies targeting adaptive changes in ETA receptor distribution and/or intracellular signaling downstream of the receptor are indicated.

Truncated recombinant human SP-D attenuates emphysema and type II cell changes in SP-D deficient mice

BACKGROUND: Surfactant protein D (SP-D) deficient mice develop emphysema-like pathology associated with focal accumulations of foamy alveolar macrophages, an excess of surfactant phospholipids in the alveolar space and both hypertrophy and hyperplasia of alveolar type II cells. These findings are associated with a chronic inflammatory state. Treatment of SP-D deficient mice with a truncated recombinant fragment of human SP-D (rfhSP-D) has been shown to decrease the lipidosis and alveolar macrophage accumulation as well as production of proinflammatory chemokines. The aim of this study was to investigate if rfhSP-D treatment reduces the structural abnormalities in parenchymal architecture and type II cells characteristic of SP-D deficiency. METHODS: SP-D knock-out mice, aged 3 weeks, 6 weeks and 9 weeks were treated with rfhSP-D for 9, 6 and 3 weeks, respectively. All mice were sacrificed at age 12 weeks and compared to both PBS treated SP-D deficient and wild-type groups. Lung structure was quantified by design-based stereology at the light and electron microscopic level. Emphasis was put on quantification of emphysema, type II cell changes and intracellular surfactant. Data were analysed with two sided non-parametric Mann-Whitney U-test. MAIN RESULTS: After 3 weeks of treatment, alveolar number was higher and mean alveolar size was smaller compared to saline-treated SP-D knock-out controls. There was no significant difference concerning these indices of pulmonary emphysema within rfhSP-D treated groups. Type II cell number and size were smaller as a consequence of treatment. The total volume of lamellar bodies per type II cell and per lung was smaller after 6 weeks of treatment. CONCLUSION: Treatment of SP-D deficient mice with rfhSP-D leads to a reduction in the degree of emphysema and a correction of type II cell hyperplasia and hypertrophy. This supports the concept that rfhSP-D might become a therapeutic option in diseases that are characterized by decreased SP-D levels in the lung.

Abnormal collagen fibrils in cartilage of matrilin-1/matrilin-3-deficient mice

Matrilins are oligomeric extracellular matrix adaptor proteins mediating interactions between collagen fibrils and other matrix constituents. All four matrilins are expressed in cartilage and mutations in the human gene encoding matrilin-3 (MATN3) are associated with different forms of chondrodysplasia. Surprisingly, however, Matn3-null as well as Matn1- and Matn2-null mice do not show an overt skeletal phenotype, suggesting a dominant negative pathomechanism for the human disorders and redundancy/compensation among the family members in the knock-out situation. Here, we show that mice lacking both matrilin-1 and matrilin-3 develop an apparently normal skeleton, but exhibit biochemical and ultrastructural abnormalities of the knee joint cartilage. At the protein level, an altered SDS-PAGE band pattern and a clear up-regulation of the homotrimeric form of matrilin-4 were evident in newborn Matn1/Matn3 and Matn1 knock-out mice, but not in Matn3-null mice. The ultrastructure of the cartilage matrix after conventional chemical fixation was grossly normal; however, electron microscopy of high pressure frozen and freeze-substituted samples, revealed two consistent observations: 1) moderately increased collagen fibril diameters throughout the epiphysis and the growth plate in both single and double mutants; and 2) increased collagen volume density in Matn1(-/-)/Matn3(-/-) and Matn3(-/-) mice. Taken together, our results demonstrate that matrilin-1 and matrilin-3 modulate collagen fibrillogenesis in cartilage and provide evidence that biochemical compensation might exist between matrilins.

Progression of atherosclerosis in ApoE-deficient mice that express distinct molecular forms of TNF-alpha

TNFalpha (TNF) critically regulates inflammation-driven atherosclerosis. Because the transmembrane (tmTNF) and soluble (sTNF) forms of TNF possess distinct immuno-modulatory properties, we hypothesized that they might differentially regulate atherosclerosis progression. Three groups of male ApoE(-/-) mice were studied: one expressing wild-type TNF (WT-TNF); one expressing exclusively a mutated non-cleavable form of TNF (KI-TNF); and one deficient in TNF (KO-TNF). Mice aged 5 weeks were fed the high-fat diet for 5 (T5) and 15 weeks (T15) or a standard chow diet for 15 weeks. At T5, in mice fed the high-fat diet, no significant differences in lesion area were observed among the three groups, either in valves or in aortas. At T15, lesion areas in valves were significantly lower in KO-TNF mice compared with those in WT-TNF mice, whereas in KI-TNF mice, they were intermediate between KO- and WT-TNF mice but not significantly different from these two groups. In aortas, lesions in KI-TNF were comparable to those of KO-TNF, both being significantly lower than those in WT-TNF. Theses differences were not linked to circulating lipids, or to macrophage, actin, and collagen contents of lesions. At T15, in mice fed the chow diet, lesion areas in valves and the aortic arch were not significantly different between the three groups. Levels of IL-6, IFNgamma, IL-10, and Foxp3 mRNAs in spleens and production of IL-6, IL-10, MCP-1, RANTES, and TNFR-2 by peritoneal macrophages at T15 of the high-fat diet showed a decrease in pro-inflammatory status, more marked in KO-TNF than in KI-TNF mice. Apoptosis was reduced only in KO-TNF mice. In conclusion, these data show that TNF effects on atherosclerosis development are detectable at stages succeeding fatty streaks and that wild-type TNF is superior to tmTNF alone in promoting atherosclerosis. TNF-dependent progression of atherosclerosis is probably linked to the differential production of pro-inflammatory mediators whether tmTNF is preponderant or essentially cleaved. Copyright (c) 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley ; Sons, Ltd.

Impairment of gastric acid secretion and increase of embryonic lethality in Foxq1-deficient mice

The mouse Foxq1 gene, also known as Hfh1, encodes a winged helix/forkhead transcription factor. In adult mice, Foxq1 is highly expressed in kidney and stomach. Here, we report that Foxq1 is expressed during prenatal and postnatal stomach development and the transcripts are restricted to acid secreting parietal cells. Mice homozygous for a deletion of the Foxq1 locus on a 129/Sv x C57BL/6J hybrid genetic background display variable phenotypes consistent with requirement of the gene during embryogenesis. Approximately 50% of Foxq1-/- embryos die in utero. Surviving homozygous mutants are normal and fertile, and have a silky shiny coat. Although the parietal cell development is not affected in the absence of Foxq1, there is a lack of gastric acid secretion in response to various secretagogue stimuli. Ultrastructural analysis suggests that the gastric acid secretion defect in Foxq1-deficient mice might be due to impairment in the fusion of cytoplasmic tubulovesicles to the apical membrane of secretory canaliculi.

The etiology of cleft palate formation in BMP7-deficient mice

Palatogenesis is a complex process implying growth, elevation and fusion of the two lateral palatal shelves during embryogenesis. This process is tightly controlled by genetic and mechanistic cues that also coordinate the growth of other orofacial structures. Failure at any of these steps can result in cleft palate, which is a frequent craniofacial malformation in humans. To understand the etiology of cleft palate linked to the BMP signaling pathway, we studied palatogenesis in Bmp7-deficient mouse embryos. Bmp7 expression was found in several orofacial structures including the edges of the palatal shelves prior and during their fusion. Bmp7 deletion resulted in a general alteration of oral cavity morphology, unpaired palatal shelf elevation, delayed shelf approximation, and subsequent lack of fusion. Cell proliferation and expression of specific genes involved in palatogenesis were not altered in Bmp7-deficient embryos. Conditional ablation of Bmp7 with Keratin14-Cre or Wnt1-Cre revealed that neither epithelial nor neural crest-specific loss of Bmp7 alone could recapitulate the cleft palate phenotype. Palatal shelves from mutant embryos were able to fuse when cultured in vitro as isolated shelves in proximity, but not when cultured as whole upper jaw explants. Thus, deformations in the oral cavity of Bmp7-deficient embryos such as the shorter and wider mandible were not solely responsible for cleft palate formation. These findings indicate a requirement for Bmp7 for the coordination of both developmental and mechanistic aspects of palatogenesis.

Glomerular and renal vascular structural changes in alpha8 integrin-deficient mice

Integrins are matrix receptors that regulate cell-matrix interactions during development and in adult tissue. In the adult kidney, the alpha8 chain is specifically expressed in glomerular mesangial cells and vascular smooth muscle cells. alpha8-deficient (alpha8-/-) mice demonstrate reductions in renal mass, which can range from complete renal agenesis to the development of kidneys that are only slightly smaller than wild-type kidneys. No histologic abnormalities of these kidneys have been described. However, considering the prominent expression of alpha8 in glomeruli and renal vessels, it seemed unlikely that the kidneys of alpha8-/- mice would be completely normal. Therefore, the renal phenotype of adult alpha8-/- mice was investigated, for assessment of more subtle morphologic alterations in kidney tissue. alpha8-/- mice displayed a significant reduction in nephron number and an increase in glomerular volume, compared with wild-type control animals. Albuminuria was not different in wild-type and alpha8-/- mice. Quantitative morphologic analyses revealed that the glomeruli of alpha8-/- mice were hypercellular, with an increased number of mesangial cells, compared with wild-type mice. Mesangial matrix deposition (as demonstrated for collagen IV and the alpha8 ligand fibronectin) was expanded in alpha8-/- mice, compared with wild-type mice. Collagens I and III, which are not normally present in glomeruli, were detected in the glomeruli of alpha8-/- mice. Staining for other glomerular integrins demonstrated an increased abundance of the collagen receptor alpha2 integrin in alpha8-/- mice. The glomerular capillary length density was significantly greater in alpha8-/- mice than in wild-type mice. Cortical arterial vessel walls were not altered in alpha8-/- mice, but the capillaries of the peritubular network were widened. Despite the strong mesangial and vascular expression of alpha8, glomerular and renal vascular alterations in alpha8-/- mice were relatively mild. Only aged alpha8-/- mice demonstrated increased glomerular capillary widening, compared with control animals. The results suggest that the lack of alpha8 can be largely compensated for, at least in younger alpha8-/- mice. It is not yet clear whether the occurrence of collagens that are not normally present in glomeruli and the increased abundance of the collagen receptor alpha2 contribute to maintaining the glomerular structure in alpha8-/- mice. The compensatory mechanisms involved will be the subject of future research.

Fetal lungs of tenascin-C-deficient mice grow well, but branch poorly in organ culture

Tenascin-C (TNC) is a multidomain extracellular matrix protein that contributes to organogenesis and tumorgenesis. To elucidate its developmental function in the context of TNC deficiency, lung lobes of TNC null mice were obtained at Embryonic Days E11.5 and E12.5 and cultured for 3 d. In lung explants of homozygote TNC-deficient embryos (E12.5) the number of future airway branches was reduced by 36% as compared with wild-type. In heterozygote explants only half of the reduction (18%) was observed. No significant alteration, neither of the explant growth nor of the pattern of airway branching, was noticed in TNC-null explants. However, the terminal endbuds of the transgenic explants were enlarged. The results are supported by a morphologic investigation at Postnatal Day P2, where the airspaces of TNC-deficient lungs appeared larger than in wild-type lungs. Taken together, our results represent the first developmental phenotype of TNC-null mice. We conclude that TNC takes part in the control of fetal lung branching, and that not only the presence of TNC but also its amount is important. Because TNC is predominantly expressed at the growing tip of the future airways, we hypothesize that TNC promotes the penetration into the surrounding mesenchyme and the branching of the growing airways.

Toll-like receptor 2-deficient mice are highly susceptible to Streptococcus pneumoniae meningitis because of reduced bacterial clearing and enhanced inflammation

Toll-like receptor-2 (TLR2) mediates host responses to gram-positive bacterial wall components. TLR2 function was investigated in a murine Streptococcus pneumoniae meningitis model in wild-type (wt) and TLR2-deficient (TLR2(-/-)) mice. TLR2(-/-) mice showed earlier time of death than wt mice (P<.02). Plasma interleukin-6 levels and bacterial numbers in blood and peripheral organs were similar for both strains. With ceftriaxone therapy, none of the wt but 27% of the TLR2(-/-) mice died (P<.04). Beyond 3 hours after infection, TLR2(-/-) mice had higher bacterial loads in brain than did wt mice, as assessed with luciferase-tagged S. pneumoniae by means of a Xenogen-CCD (charge-coupled device) camera. After 24 h, tumor necrosis factor activity was higher in cerebrospinal fluid of TLR2(-/-) than wt mice (P<.05) and was related to increased blood-brain barrier permeability (Evans blue staining, P<.02). In conclusion, the lack of TLR2 was associated with earlier death from meningitis, which was not due to sepsis but to reduced brain bacterial clearing, followed by increased intrathecal inflammation.

Regular exercise decreases liver tumors development in hepatocyte-specific PTEN-deficient mice independently of steatosis.

BACKGROUND & AIMS Unhealthy lifestyles predispose to non-alcoholic steatohepatitis (NASH), which may further result in the development of hepatocellular carcinoma (HCC). Although NASH patients benefit from physical activity, it is unknown whether regular exercise reduces the risk of developing HCC. Therefore, we studied the effect of regular exercise on the development of HCC in male hepatocyte-specific PTEN-deficient mice (AlbCrePten(flox/flox)), which develop steatohepatitis and HCC spontaneously. METHODS Mice were fed a standardized 10% fat diet and were randomly divided into exercise or sedentary groups. The exercise group ran on a motorized treadmill for 60 minutes/day, 5 days/week during 32 weeks. RESULTS After 32 weeks of regular exercise, 71% of exercised mice developed nodules larger than 15 mm(3) vs 100% of mice in the sedentary group. The mean number of tumors per liver was reduced by exercise, as well as the total tumoral volume per liver. Exercise did not affect steatosis and had no effect on the Non-alcoholic fatty liver disease (NAFLD) Activity Score (NAS). Exercise decreased tumor cell proliferation. Mechanistically, exercise stimulated the phosphorylation of AMPK and its substrate raptor, which decreased the kinase activity of mTOR. CONCLUSIONS These data show a benefit of regular exercise on the development of HCC in an experimental model of NASH and offer a rationale for encouraging predisposed patients to increase their physical activity for the prevention of HCC.

Activating enhancer binding protein 2 epsilon (AP-2ε)-deficient mice exhibit increased matrix metalloproteinase 13 expression and progressive osteoarthritis development.

INTRODUCTION The transcription factor activating enhancer binding protein 2 epsilon (AP-2ε) was recently shown to be expressed during chondrogenesis as well as in articular chondrocytes of humans and mice. Furthermore, expression of AP-2ε was found to be upregulated in affected cartilage of patients with osteoarthritis (OA). Despite these findings, adult mice deficient for AP-2ε (Tfap2e(-/-)) do not exhibit an obviously abnormal cartilaginous phenotype. We therefore analyzed embryogenesis of Tfap2e(-/-) mice to elucidate potential transient abnormalities that provide information on the influence of AP-2ε on skeletal development. In a second part, we aimed to define potential influences of AP-2ε on articular cartilage function and gene expression, as well as on OA progression, in adult mice. METHODS Murine embryonic development was accessed via in situ hybridization, measurement of skeletal parameters and micromass differentiation of mesenchymal cells. To reveal discrepancies in articular cartilage of adult wild-type (WT) and Tfap2e(-/-) mice, light and electron microscopy, in vitro culture of cartilage explants, and quantification of gene expression via real-time PCR were performed. OA was induced via surgical destabilization of the medial meniscus in both genotypes, and disease progression was monitored on histological and molecular levels. RESULTS Only minor differences between WT and embryos deficient for AP-2ε were observed, suggesting that redundancy mechanisms effectively compensate for the loss of AP-2ε during skeletal development. Surprisingly, though, we found matrix metalloproteinase 13 (Mmp13), a major mediator of cartilage destruction, to be significantly upregulated in articular cartilage of adult Tfap2e(-/-) mice. This finding was further confirmed by increased Mmp13 activity and extracellular matrix degradation in Tfap2e(-/-) cartilage explants. OA progression was significantly enhanced in the Tfap2e(-/-) mice, which provided evidence for in vivo relevance. This finding is most likely attributable to the increased basal Mmp13 expression level in Tfap2e(-/-) articular chondrocytes that results in a significantly higher total Mmp13 expression rate during OA as compared with the WT. CONCLUSIONS We reveal a novel role of AP-2ε in the regulation of gene expression in articular chondrocytes, as well as in OA development, through modulation of Mmp13 expression and activity.