Amelogenin- and enamelysin (Mmp-20)-deficient mice display altered birefringence in the secretory-stage enamel organic extracellular matrix

Dental enamel is the most mineralized tissue of vertebrate organisms. Enamel biosynthesis is initiated by the secretion, processing, and self-assembly of a complex mixture of proteins. The formation of an ordered enamel organic extracellular matrix (ECM) seems be a crucial step for the proper formation of mineral phase. Polarizing microscopy demonstrates that the ordered supramolecular structure of the secretory-stage enamel organic ECM is strongly birefringent. In the present work we analyzed the birefringence of secretory-stage enamel organic ECM in amelogenin (Amelx)- and enamelysin (Mmp20)-deficient mice. Female Amelx+/- animals showed significant reduction in optical retardation values when compared with the Amelx+/+ subgroup (p=0.0029). The secretory-stage enamel organic ECM of the Amelx-/- subgroup did not exhibit birefringence. The secretory-stage enamel organic ECM of Mmp20-/- mice showed a significant decrease in optical retardation as compared with Mmp20+/+ and Mmp20+/- mice (p=0.0000). Mmp20+/- and Mmp20+/+ mice exhibited similar birefringence (p=1.0000). The results presented here support growing evidence for the idea that the birefringence of secretory-stage enamel organic ECM is influenced by the ordered supramolecular organization of its components.     

Birefringence of the Secretory-Stage Enamel Organic Extracellular Matrix from Rats Submitted to Successive Injections of Bisphosphonates

The aim of the present study was to assess birefringence of the secretory-stage enamel organic extracellular matrix (ECM) and mechanical properties of mature enamel from rats treated with bisphosphonates. Longitudinal sections were obtained from upper incisors of rats that had been submitted to injections of bisodic etidronate (8 mg/Kg/day), sodium alendronate (30 μg/Kg/day), or sodium chloride as control (8 mg/Kg/day) for 42 days. Sections were immersed in 80% glycerin for 30 min and optical retardation of birefringence brightness in the secretory-stage enamel organic ECM was determined in nanometers. Etidronate-treated rats exhibited extensive morphological changes in the secretory-stage enamel organic ECM inclusive nonbirefringent conspicuous incremental lines, but presented optical retardation values similar to those showed by control rats (p > 0.05). Birefringence of secretory enamel organic ECM from etidronate rats presented an irregular aspect. Alendronate-treated rats did not show morphological alterations in the secretory-stage enamel organic ECM, however, they presented significant reduction in optical retardation of birefringence brightness when compared with control and etidronate rats (p < 0.01). Alendronate and etidronate groups exhibited reductions of ≈6–10% in mature enamel cross-sectional microhardness when compared with control group (p < 0.01). Scanning electron microscopy analysis showed extensive alterations in mature enamel only from etidronate group with absence of enamel rods. The present work shows that bisphosphonates can affect the birefringence of the secretory-stage enamel organic ECM. The results presented here suggest that alterations in the supramolecular organization of the secretory-stage enamel organic ECM are a plausible mechanism by which environmental factors may cause enamel defects.     

The induction of apoptosis in ovaries and uteri ofbcl-2-deficient mice

To elucidate the essential role ofbcl-2 in female reproductive organs,bcl-2-deficient mice (bcl-2 ^?/? mice), heterozygotes (bcl-2 ^+/?), and wild-type (bcl-2 ^+/+) mice were investigated. The ovaries of 4-week-oldbcl-2 ^?/? mice contained only preantral follicles, although follicles in various developmental stages were present in the ovaries of both 7-and 12-week-oldbcl-2 ^?/? mice. The number of apoptotic granulosa cells inbcl-2 ^?/? mice was higher than in both thebcl-2 ^+/? and thebcl-2 ^+/+ mice. The uteri of thebcl-2-deficient mice were characterized by growth retardation, including a lack of bifurcation of the organ and poorly developed lamina propria and myometrium. The most interesting finding was the presence of apoptotic cells in both the glandular epithelium and the myometrium. Therefore,bcl-2 is an essential gene for the survival of ovarian granulosa cells and of both endometrial glandular cells and myometrial smooth muscle cells in the uteri.     

Under‐expression of α8 integrin aggravates experimental atherosclerosis

Integrins play an important role in vascular biology. The α8 integrin chain attenuates smooth muscle cell migration but its functional role in the development of atherosclerosis is unclear. Therefore, we studied the contribution of α8 integrin to atherosclerosis and vascular remodelling. We hypothesized that α8 integrin expression is reduced in atherosclerotic lesions, and that its under‐expression leads to a more severe course of atherosclerosis. α8 Integrin was detected by immunohistochemistry and qPCR and α8 integrin‐deficient mice were used to induce two models of atherosclerotic lesions. First, ligation of the carotid artery led to medial thickening and neointima formation, which was quantified in carotid cross‐sections. Second, after crossing into ApoE‐deficient mice, the formation of advanced vascular lesions with atherosclerotic plaques was quantified in aortic en face preparations stained with Sudan IV. Parameters of renal physiology and histopathology were assessed: α8 integrin was detected in the media of human and murine vascular tissue and was down‐regulated in arteries with advanced atherosclerotic lesions. In α8 integrin‐deficient mice (α8^?/?) as well as α8^+/? and α8^+/+ littermates, carotid artery ligation increased media:lumen ratios in all genotypes, with higher values in ligated α8^?/? and α8^+/? compared to ligated α8^+/+ animals. Carotid artery ligation increased smooth muscle cell number in the media of α8^+/+ mice and, more prominently, of α8^?/? or α8^+/? mice. On an ApoE^?/? background, α8^+/? and α8^?/? mice developed more atherosclerotic plaques than α8^+/+ mice. α8 Integrin expression was reduced in α8^+/? animals. Renal damage with increased serum creatinine and glomerulosclerosis was detected in α8^?/? mice only. Thus, under‐expression of α8 integrin aggravates vascular lesions, while a complete loss of α8 integrin results in reduced renal mass and additional renal disease in the presence of generalized atherosclerosis. Our data support the hypothesis that integrin α8β1 has a protective role in arterial remodelling and atherosclerosis. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

SGK1-dependent salt appetite in pregnant mice

Aim: Pregnancy is typically paralleled by substantial increase in maternal extracellular fluid volume, requiring net accumulation of water and NaCl. The positive water and salt balance is accomplished at least in part by increased uptake of salt secondary to enhanced salt appetite. Little is known about the underlying cellular mechanisms. Stimulation of salt appetite by mineralocorticoids, however, is known to be dependent on the serum‐ and glucocorticoid‐inducible kinase SGK1. Methods: To test for a role of SGK1 in the stimulation of salt appetite during pregnancy, fluid intake was recorded in pregnant SGK1 knockout mice (sgk1^?/?) and their wild type littermates (sgk1^+/+). The mice were offered two bottles, one with plain water and the other with isotonic saline. Results: In early pregnancy, i.e. up to 10 days prior to parturition, the sgk1^+/+ mice displayed a significant preference for saline, whereas the sgk1^?/? mice preferred water. Accordingly, the water intake was significantly smaller and saline intake was significantly larger in sgk1^+/+ mice than in sgk1^?/? mice and the preference for water was significantly stronger in sgk1^?/? mice than in sgk1^+/+ mice. Plasma aldosterone levels were higher in sgk1^?/? mice than in sgk1^+/+ mice, a difference contrasting the enhanced salt appetite of sgk1^+/+ mice. Conclusions: SGK1 participates in the stimulation of salt appetite during pregnancy.     

Fluoride effect on the secretory-stage enamel organic extracellular matrix of mice

The formation of an ordered enamel organic extracellular matrix (EOECM) seems to be a crucial step for the proper formation of the enamel mineral phase. The ordered supramolecular structure of the EOECM in the secretory stage can be analyzed using polarizing microscopy, as it is strongly birefringent. Excessive fluoride (F) ingestion during tooth development can cause enamel fluorosis, leading to increased porosity in mature enamel. We analyzed the effects of F on the birefringence of the EOECM in the A/J, CBA, and DBA/2 strains of mice given 0, 11.25, and 45 ppm of fluoride in drinking water. In the CBA and DBA/2 strains, the 11.25 and 45 ppmF groups presented a significant decrease in optical retardation (OR) when compared with the respective 0 (CBA 11.25 ppmF p = 0.0056 and 45 ppmF p < 0.0001; DBA/2 11.25 and 45 ppmF p < 0.05). ORs in A/J 0 ppmF were significantly higher than in 45 (p < 0.0001). The enamel of the A/J strain was more severely affected by fluoride than it was in the other strains of mice and exhibited the lowest levels of fluoride in plasma, whereas its normal secretory enamel presented a significantly higher protein absorbance than it did in CBA and DBA mice (p = 0.0099 and p = 0.0025, respectively). The results showed that experimental fluorosis can alter the supramolecular organization of EOECM in the secretory stage of amelogenesis and that the susceptibility to dental fluorosis seems to be influenced by the inherent characteristics of the developing enamel.     

CRMP1 and CRMP2 have synergistic but distinct roles in dendritic development

Collapsin response mediator protein 2, CRMP2, has been identified as an intracellular signaling mediator for Semaphorin 3A (Sema3A). CRMP2 plays a key role in axon guidance, dendritic morphogenesis, and cell polarization. It has been also implicated in a variety of neurological and psychiatric disorders. However, the in vivo functions of CRMP2 remain unknown. We generated CRMP2 gene‐deficient (crmp2^?/?) mice. The crmp2^?/? mice showed irregular development of dendritic spines in cortical neurons. The density of dendritic spines was reduced in the cortical layer V pyramidal neurons of crmp2^?/? mice as well as in those of sema3A^?/? and crmp1^?/? mice. However, no abnormality was found in dendritic patterning in crmp2^?/? compared to wild‐type (WT) neurons. The level of CRMP1 was increased in crmp2^?/?, but the level of CRMP2 was not altered in crmp1^?/? compared to WT cortical brain lysates. Dendritic spine density and branching were reduced in double‐heterozygous sema3A^+/?;crmp2^+/? and sema3A^+/?;crmp1^+/? mice. The phenotypic defects had no genetic interaction between crmp1 and crmp2. These findings suggest that both CRMP1 and CRMP2 mediate Sema3A signaling to regulate dendritic spine maturation and patterning, but through overlapping and distinct signaling pathways.     

Decreased levels of keratin 8 sensitize mice to streptozotocin‐induced diabetes

Aim

Diabetes is a result of an interplay between genetic, environmental and lifestyle factors. Keratin intermediate filaments are stress proteins in epithelial cells, and keratin mutations predispose to several human diseases. However, the involvement of keratins in diabetes is not well known. K8 and its partner K18 are the main β‐cell keratins, and knockout of K8 (K8^?/?) in mice causes mislocalization of glucose transporter 2, mitochondrial defects, reduced insulin content and altered systemic glucose/insulin control. We hypothesize that K8/K18 offer protection during β‐cell stress and that decreased K8 levels contribute to diabetes susceptibility.

Methods

K8‐heterozygous knockout (K8^+/?) and wild‐type (K8^+/+) mice were used to evaluate the influence of keratin levels on endocrine pancreatic function and diabetes development under basal conditions and after T1D streptozotocin (STZ)‐induced β‐cell stress and T2D high‐fat diet (HFD).

Results

Murine K8^+/? endocrine islets express ~50% less K8/K18 compared with K8^+/+. The decreased keratin levels have little impact on basal systemic glucose/insulin regulation, β‐cell health or insulin levels. Diabetes incidence and blood glucose levels are significantly higher in K8^+/? mice after low‐dose/chronic STZ treatment, and STZ causes more β‐cell damage and polyuria in K8^+/? compared with K8^+/+. K8 appears upregulated 5 weeks after STZ treatment in K8^+/+ islets but not in K8^+/?. K8^+/? mice showed no major susceptibility risk to HFD compared to K8^+/+.

Conclusion

Partial K8 deficiency reduces β‐cell stress tolerance and aggravates diabetes development in response to STZ, while there is no major susceptibility to HFD.

     

Altered dermal fibroblast behavior in a collagen V haploinsufficient murine model of classic Ehlers–Danlos syndrome

Mutations in collagen V are associated with classic Ehlers–Danlos syndrome (EDS). A significant percentage of these mutations result in haploinsufficiency for collagen V. The purpose of this work was to determine if changes in collagen V expression are associated with altered dermal fibroblast behavior contributing to the poor wound healing response. A haploinsufficient Col5a1^+/? mouse model of EDS was utilized. In vivo wound healing studies demonstrated that mutant mice healed significantly slower than Col5a1^+/+ mice. The basis for this difference was examined in vitro using dermal fibroblast strains isolated from Col5a1^+/? and Col5a1^+/+ mice. Fibroblast proliferation was determined for each strain by counting cells at different time points after seeding as well as using the proliferation marker Ki-67. Fibroblast attachment to collagens I and III and fibronectin also was analyzed. In addition, in vitro scratch wounds were used to analyze fibroblast wound closure. Significantly decreased fibroblast proliferation was observed in Col5a1^+/? compared to Col5a1^+/+ fibroblasts. Our data indicate that the decreased fibroblast number was not due to apoptosis. Wildtype Col5a1^+/+ fibroblasts attached significantly better to components of the wound matrix (collagens I and III and fibronectin) than Col5a1^+/? fibroblasts. A significant difference in in vitro scratch wound closure rates also was observed. Col5a1^+/+ fibroblasts closed wounds in 22 h, while Col5a1^+/? fibroblasts demonstrated ~80% closure. There were significant differences in closure at all time points analyzed. Our data suggest that decreased fibroblast proliferation, extracellular matrix attachment, and migration contribute to the decreased wound healing response in classic EDS.     

PKCη deficiency improves lipid metabolism and atherosclerosis in apolipoprotein E‐deficient mice

Genomewide association studies have shown that a nonsynonymous single nucleotide polymorphism in PRKCH is associated with cerebral infarction and atherosclerosis‐related complications. We examined the role of PKCη in lipid metabolism and atherosclerosis using apolipoprotein E‐deficient (Apoe^?/?) mice. PKCη expression was augmented in the aortas of mice with atherosclerosis and exclusively detected in MOMA2‐positive macrophages within atherosclerotic lesions. Prkch^+/+Apoe^?/? and Prkch^?/?Apoe^?/? mice were fed a high‐fat diet (HFD), and the dyslipidemia observed in Prkch^+/+Apoe^?/? mice was improved in Prkch^?/?Apoe^?/? mice, with a particular reduction in serum LDL cholesterol and phospholipids. Liver steatosis, which developed in Prkch^+/+Apoe^?/? mice, was improved in Prkch^?/?Apoe^?/? mice, but glucose tolerance, adipose tissue and body weight, and blood pressure were unchanged. Consistent with improvements in LDL cholesterol, atherosclerotic lesions were decreased in HFD‐fed Prkch^?/?Apoe^?/? mice. Immunoreactivity against 3‐nitrotyrosine in atherosclerotic lesions was dramatically decreased in Prkch^?/?Apoe^?/? mice, accompanied by decreased necrosis and apoptosis in the lesions. ARG2 mRNA and protein levels were significantly increased in Prkch^?/?Apoe^?/? macrophages. These data show that PKCη deficiency improves dyslipidemia and reduces susceptibility to atherosclerosis in Apoe^?/? mice, showing that PKCη plays a role in atherosclerosis development.     

Type I interferons contribute to experimental cerebral malaria development in response to sporozoite or blood‐stage Plasmodium berghei ANKA

Cerebral malaria is a severe complication of Plasmodium falciparum infection. Although T‐cell activation and type II IFN‐γ are required for Plasmodium berghei ANKA (PbA)‐induced murine experimental cerebral malaria (ECM), the role of type I IFN‐α/β in ECM development remains unclear. Here, we address the role of the IFN‐α/β pathway in ECM devel‐opment in response to hepatic or blood‐stage PbA infection, using mice deficient for types I or II IFN receptors. While IFN‐γR1^?/? mice were fully resistant, IFNAR1^?/? mice showed delayed and partial protection to ECM after PbA infection. ECM resistance in IFN‐γR1^?/? mice correlated with unaltered cerebral microcirculation and absence of ischemia, while WT and IFNAR1^?/? mice developed distinct microvascular pathologies. ECM resistance appeared to be independent of parasitemia. Instead, key mediators of ECM were attenuated in the absence of IFNAR1, including PbA‐induced brain sequestration of CXCR3⁺‐activated CD8⁺ T cells. This was associated with reduced expression of Granzyme B, IFN‐γ, IL‐12Rβ2, and T‐cell‐attracting chemokines CXCL9 and CXCL10 in IFNAR1^?/? mice, more so in the absence of IFN‐γR1. Therefore, the type I IFN‐α/β receptor pathway contributes to brain T‐cell responses and microvascular pathology, although it is not as essential as IFN‐γ for the development of cerebral malaria upon hepatic or blood‐stage PbA infection.     

CCR6 regulates EAE pathogenesis by controlling regulatory CD4+ T‐cell recruitment to target tissues

The T‐cell subsets, characterized by their cytokine production profiles and immune regulatory functions, depend on correct in vivo location to interact with accessory or target cells for effective immune responses. Differentiation of naive CD4⁺ T cells into effectors is accompanied by sequentially regulated expression of the chemokine receptors responsible for cell recruitment to specific tissues. We studied CCR6 function in EAE, a CD4⁺ T‐cell‐mediated CNS disease characterized by mononuclear infiltration and demyelination. CCR6^?/? mice showed an altered time course of EAE development, with delayed onset, a higher clinical score, and more persistent symptoms than in controls. An imbalanced cytokine profile and reduced Foxp3⁺ cell frequency characterized CNS tissues from CCR6^?/? compared with CCR6^+/+ mice during the disease effector phase. Transfer of CCR6^+/+ Treg to CCR6^?/? mice the day before EAE induction reduced the clinical score associated with an increased in infiltrating Foxp3⁺ cells and recovery of the cytokine balance in CCR6^?/? mouse CNS. Competitive assays between CCR6^+/+ and CCR6^?/? Treg adoptively transferred to CCR6^?/? mice showed impaired ability of CCR6^?/? Treg to infiltrate CNS tissues in EAE‐affected mice. Our data indicate a CCR6 requirement by CD4⁺ Treg to downregulate the CNS inflammatory process and neurological signs associated with EAE.     

A novel phenotypic marker for ATM‐deficient 129S6/SvEvTac‐ATMtm1Awb/J mice

Ataxia‐telangiectasia (A‐T) is a human autosomal recessive disorder characterized by neuronal degeneration as well as many other physiological and somatic defects. ATM (A‐T, mutated), the gene mutated in A‐T, encodes a 370 kDa protein kinase. ATM knockout mouse models (ATM^?/?) show growth retardation, infertility, neurological dysfunction, defects in T‐lymphocytes, and extreme sensitivity to ionizing radiation. We have recently established multiple ATM^+/? breeding pairs and discovered that all ATM^?/? offspring exhibit a nonpigmented section of tail, usually at or near the tip. To our knowledge, this is the first time that a phenotype of nonpigmented tail has been reported in ATM^?/? knockout mice. We believe that the sections of nonpigmented tail of 129S6/SvEvTac‐ATM^tm1Awb/J mice provide a novel phenotypic marker for research using this ATM knockout mouse model. Results from histochemistry and immunoblotting analysis further demonstrate that while melanocyte precursors or melanoblasts are present in the nonpigmented tail tissue of ATM^?/? mice, they fail to differentiate fully into mature melanocytes. The potential connection between this phenotype and other clinical symptoms caused by ATM deficiency, such as progressive neuronal degeneration, is discussed in this article. Anat Rec, 2007. © 2007 Wiley‐Liss, Inc.     

IL-10 contributes to the suppressive function of tumour-associated myeloid cells and enhances myeloid cell accumulation in tumours

Studies have revealed that tumour‐associated myeloid cells (TAMC) are one of the major sources of IL‐10 in tumour‐bearing mice. However, the significance of TAMC‐derived IL‐10 in tumour immunity is poorly understood. Here, we show that IL‐10 blockade or IL‐10 deficiency reduces the capacity of TAMC in suppressing the proliferation of P1A‐specific CD8 T cells. In the spleen, IL‐10‐deficient and wild‐type (WT) mice bearing large tumour burdens have similar TAMC populations. The tumours from IL‐10‐deficient mice, however, have reduced numbers of TAMC compared with tumours from their WT counterparts. IL‐10^?/?RAG‐2^?/? mice also had reduced numbers of TAMC compared with tumours from IL‐10^+/+RAG‐2^?/? mice; therefore, the reduction in TAMC in IL‐10‐deficient tumours was not because of adaptive immune response in tumours. Adoptively transferred tumour antigen–specific CD8 T cells expanded more efficiently within tumours in IL‐10^?/?RAG‐2^?/? mice than in tumours from IL‐10^+/+RAG‐2^?/? mice. Cytotoxic T lymphocyte adoptive transfer therapy prevented tumour evasion in IL‐10^?/?RAG‐2^?/? mice more efficiently than in IL‐10^+/+RAG‐2^?/? mice. Thus, IL‐10 enhances the accumulation of myeloid cells in tumours, and TAMC‐derived IL‐10 suppresses the activation and expansion of tumour antigen–specific T cells.     

Poly (ADP‐ribose) polymerase‐1 deficiency does not affect ethylnitrosourea mutagenicity in liver and testis of lacZ transgenic mice

Poly (ADP‐ribose) polymerase‐1 (Parp1) has been implicated in DNA base excision repair, single‐ and double‐strand break repair pathways, as well as in cell death by apoptosis or necrosis. We used Parp1^?/? lacZ plasmid‐based transgenic mice to investigate whether Parp1 deficiency influences the in vivo mutagenic and clastogenic response to the alkylating agent N‐ethyl‐N‐Nitrosourea (ENU) in somatic and germ‐cell tissues. The comparison of the lacZ mutant frequencies (MFs) between Parp1^+/+ and Parp1^?/? mice showed that the ablation of Parp1 does not affect the spontaneous or ENU‐induced MFs in liver and testis. In addition, the spectrum of the ENU‐induced mutations was not dependent on the Parp1 status, given that similar spectra, consisting mostly of point mutations and a small fraction of deletions/insertions, wereobserved in organs of both Parp1^?/? and Parp1^+/+ mice. Sequencing of point mutations revealed a consistent significant increase in A:T → T:A base substitutions, typically induced by ENU. Overall, we observed that neither the frequency nor the spectrum of ENU‐induced mutations demonstrated a specificity that could be attributed to the Parp1 impairment in mice organs. The analysis of micronucleus frequency in peripheral blood reticulocytes showed that ENU was clastogenic in both Parp1^?/? and Parp1^+/+ mice and had a strong cytotoxic effect in Parp1^?/? mice only. The present data suggest that, at a whole‐organism level, Parp1‐independent repair mechanisms may be operative in the removal of ENU‐induced DNA lesions or that highly damaged cells may be preferentially committed to death when Parp1 is inactivated. Environ. Mol. Mutagen. 2010. © 2010 Wiley‐Liss, Inc.     

GRP78 and CHOP modulate macrophage apoptosis and the development of bleomycin‐induced pulmonary fibrosis

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have been associated with fibrotic lung disease, although exactly how they modulate this process remains unclear. Here we investigated the role of GRP78, the main UPR regulator, in an experimental model of lung injury and fibrosis. Grp78^+/?, Chop^?/? and wild type C57BL6/J mice were exposed to bleomycin by oropharyngeal intubation and lungs were examined at days 7 and 21. We demonstrate here that Grp78^+/? mice were strongly protected from bleomycin‐induced fibrosis, as shown by immunohistochemical analysis, collagen content and lung function measurements. In the inflammatory phase of this model, a reduced number of lung macrophages associated with an increased number of TUNEL‐positive cells were observed in Grp78^+/? mice. Dual immunohistochemical and in situ hybridization experiments showed that the macrophage population from the protected Grp78^+/? mice was also strongly positive for cleaved caspase‐3 and Chop mRNA, respectively. In contrast, the administration of bleomycin to Chop^?/? mice resulted in increased quasi‐static elastance and extracellular matrix deposition associated with an increased number of parenchymal arginase‐1‐positive macrophages that were negative for cleaved caspase‐3. The data presented indicate that the UPR is activated in fibrotic lung tissue and strongly localized to macrophages. GRP78‐ and CHOP‐mediated macrophage apoptosis was found to protect against bleomycin‐induced fibrosis. Overall, we demonstrate here that the fibrotic response to bleomycin is dependent on GRP78‐mediated events and provides evidence that macrophage polarization and apoptosis may play a role in this process. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Impaired Ca2+-handling in HIF-1α+/− mice as a consequence of pressure overload

The hypoxia-inducible factor (HIF)-1 is critically involved in the cellular adaptation to a decrease in oxygen availability. The influence of HIF-1α for the development of cardiac hypertrophy and cardiac function that occurs in response to sustained pressure overload has been mainly attributed to a challenged cardiac angiogenesis and cardiac hypertrophy up to now. Hif-1α ^+/+ and Hif-1α ^+/? mice were studied regarding left ventricular hypertrophy and cardiac function after being subjected to transverse aortic constriction (TAC). After TAC, both Hif-1α ^+/+ and Hif-1α ^+/? mice developed left ventricular hypertrophy with increased posterior wall thickness, septum thickness and increased left ventricular weight to a similar extent. No significant difference in cardiac vessel density was observed between Hif-1α ^+/+ and Hif-1α ^+/? mice. However, only the Hif-1α ^+/? mice developed severe heart failure as revealed by a significantly reduced fractional shortening mostly due to increased end-systolic left ventricular diameter. On the single cell level this correlated with reduced myocyte shortenings, decreased intracellular Ca²⁺-transients and SR-Ca²⁺ content in myocytes of Hif-1a ^+/? mice. Thus, HIF-1α can be critically involved in the preservation of cardiac function after chronic pressure overload without affecting cardiac hypertrophy. This effect is mediated via HIF-dependent modulation of cardiac calcium handling and contractility.     

Antigenotoxic effects of p53 on spontaneous and ultraviolet light B–induced deletions in the epidermis of gpt delta transgenic mice

Tumor development in the skin may be a multistep process where multiple genetic alterations occur successively. The p53 gene is involved in genome stability and thus is referred to as “the guardian of the genome.” To better understand the antigenotoxic effects of p53 in ultraviolet light B (UVB)‐induced mutagenesis, mutations were measured in the epidermis of UVB‐irradiated p53^+/+ and p53^?/? gpt delta mice. In the mouse model, point mutations and deletions are separately identified by the gpt and Spi^? assays, respectively. The mice were exposed to UVB at single doses of 0.5, 1.0, or 2.0 kJ/m². The mutant frequencies (MFs) were determined 4 weeks after the irradiation. All doses of UVB irradiation enhanced gpt MFs by about 10 times than that of unirradiated mice. There were no significant differences in gpt MFs and the mutation spectra between p53^+/+ and p53^?/? mice. The predominant mutations induced by UVB irradiation were G:C to A:T transitions at dipyrimidines. In contrast, in unirradiated p53^?/? mice, the frequencies of Spi^? large deletions of more than 1 kb and complex‐type deletions with rearrangements were significantly higher than those of the Spi^? large deletions in p53^+/+ counterparts. The specific Spi^? mutation frequency of more than 1 kb deletions and complex types increased in a dose‐dependent manner in the p53^+/+ mice. However, no increase of such large deletions was observed in irradiated p53^?/? mice. These results suggest that the antigenotoxic effects of p53 may be specific to deletions and complex‐type mutations induced by double‐strand breaks in DNA. Environ. Mol. Mutagen., 2011. © 2010 Wiley‐Liss, Inc.