Nitroglycerin Tolerance in Caveolin-1 Deficient Mice

Nitrate tolerance developed after persistent nitroglycerin (GTN) exposure limits its clinical utility. Previously, we have shown that the vasodilatory action of GTN is dependent on endothelial nitric oxide synthase (eNOS/NOS3) activity. Caveolin-1 (Cav-1) is known to interact with NOS3 on the cytoplasmic side of cholesterol-enriched plasma membrane microdomains (caveolae) and to inhibit NOS3 activity. Loss of Cav-1 expression results in NOS3 hyperactivation and uncoupling, converting NOS3 into a source of superoxide radicals, peroxynitrite, and oxidative stress. Therefore, we hypothesized that nitrate tolerance induced by persistent GTN treatment results from NOS3 dysfunction and vascular toxicity. Exposure to GTN for 48–72 h resulted in nitrosation and depletion (>50%) of Cav-1, NOS3 uncoupling as measured by an increase in peroxynitrite production (>100%), and endothelial toxicity in cultured cells. In the Cav-1 deficient mice, NOS3 dysfunction was accompanied by GTN tolerance (>50% dilation inhibition at low GTN concentrations). In conclusion, GTN tolerance results from Cav-1 modification and depletion by GTN that causes persistent NOS3 activation and uncoupling, preventing it from participating in GTN-medicated vasodilation.     

Impaired Bone Formation in Pdia3 Deficient Mice

1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] is crucial for normal skeletal development and bone homeostasis. Protein disulfide isomerase family A, member 3 (PDIA3) mediates 1α,25(OH)₂D₃ initiated-rapid membrane signaling in several cell types. To understand its role in regulating skeletal development, we generated Pdia3-deficient mice and examined the physiologic consequence of Pdia3-disruption in embryos and Pdia3 ^+/− heterozygotes at different ages. No mice homozygous for the Pdia3-deletion were found at birth nor were there embryos after E12.5, indicating that targeted disruption of the Pdia3 gene resulted in early embryonic lethality. Pdia3-deficiency also resulted in skeletal manifestations as revealed by µCT analysis of the tibias. In comparison to wild type mice, Pdia3 heterozygous mice displayed expanded growth plates associated with decreased tether formation. Histomorphometry also showed that the hypertrophic zone in Pdia3 ^+/− mice was more cellular than seen in wild type growth plates. Metaphyseal trabecular bone in Pdia3 ^+/− mice exhibited an age-dependent phenotype with lower BV/TV and trabecular numbers, which was most pronounced at 15 weeks of age. Bone marrow cells from Pdia3 ^+/− mice exhibited impaired osteoblastic differentiation, based on reduced expression of osteoblast markers and mineral deposition compared to cells from wild type animals. Collectively, our findings provide in vivo evidence that PDIA3 is essential for normal skeletal development. The fact that the Pdia3 ^+/− heterozygous mice share a similar growth plate and bone phenotype to nVdr knockout mice, suggests that PDIA3-mediated rapid membrane signaling might be an alternative mechanism responsible for 1α,25(OH)₂D₃’s actions in regulating skeletal development.     

Subtle Neuromuscular Defects in Utrophin-deficient Mice

Utrophin is a large cytoskeletal protein that is homologous to dystrophin, the protein mutated in Duchenne and Becker muscular dystrophy. In skeletal muscle, dystrophin is broadly distributed along the sarcolemma whereas utrophin is concentrated at the neuromuscular junction. This differential localization, along with studies on cultured cells, led to the suggestion that utrophin is required for synaptic differentiation. In addition, utrophin is present in numerous nonmuscle cells, suggesting that it may have a more generalized role in the maintenance of cellular integrity. To test these hypotheses we generated and characterized utrophin-deficient mutant mice. These mutant mice were normal in appearance and behavior and showed no obvious defects in muscle or nonmuscle tissue. Detailed analysis, however, revealed that the density of acetylcholine receptors and the number of junctional folds were reduced at the neuromuscular junctions in utrophin-deficient skeletal muscle. Despite these subtle derangements, the overall structure of the mutant synapse was qualitatively normal, and the specialized characteristics of the dystrophin-associated protein complex were preserved at the mutant neuromuscular junction. These results point to a predominant role for other molecules in the differentiation and maintenance of the postsynaptic membrane.     

Defective Secretion of Islet Hormones in Chromogranin-B Deficient Mice

Granins are major constituents of dense-core secretory granules in neuroendocrine cells, but their function is still a matter of debate. Work in cell lines has suggested that the most abundant and ubiquitously expressed granins, chromogranin A and B (CgA and CgB), are involved in granulogenesis and protein sorting. Here we report the generation and characterization of mice lacking chromogranin B (CgB-ko), which were viable and fertile. Unlike neuroendocrine tissues, pancreatic islets of these animals lacked compensatory changes in other granins and were therefore analyzed in detail. Stimulated secretion of insulin, glucagon and somatostatin was reduced in CgB-ko islets, in parallel with somewhat impaired glucose clearance and reduced insulin release, but normal insulin sensitivity in vivo. CgB-ko islets lacked specifically the rapid initial phase of stimulated secretion, had elevated basal insulin release, and stored and released twice as much proinsulin as wildtype (wt) islets. Stimulated release of glucagon and somatostatin was reduced as well. Surprisingly, biogenesis, morphology and function of insulin granules were normal, and no differences were found with regard to β-cell stimulus-secretion coupling. We conclude that CgB is not required for normal insulin granule biogenesis or maintenance in vivo, but is essential for adequate secretion of islet hormones. Consequentially CgB-ko animals display some, but not all, hallmarks of human type-2 diabetes. However, the molecular mechanisms underlying this defect remain to be determined.     

Gender Affects Skin Wound Healing in Plasminogen Deficient Mice

The fibrinolytic activity of plasmin plays a fundamental role in resolution of blood clots and clearance of extravascular deposited fibrin in damaged tissues. These vital functions of plasmin are exploited by malignant cells to accelerate tumor growth and facilitate metastases. Mice lacking functional plasmin thus display decreased tumor growth in a variety of cancer models. Interestingly, this role of plasmin has, in regard to skin cancer, been shown to be restricted to male mice. It remains to be clarified whether gender also affects other phenotypic characteristics of plasmin deficiency or if this gender effect is restricted to skin cancer. To investigate this, we tested the effect of gender on plasmin dependent immune cell migration, accumulation of hepatic fibrin depositions, skin composition, and skin wound healing. Gender did not affect immune cell migration or hepatic fibrin accumulation in neither wildtype nor plasmin deficient mice, and the existing differences in skin composition between males and females were unaffected by plasmin deficiency. In contrast, gender had a marked effect on the ability of plasmin deficient mice to heal skin wounds, which was seen as an accelerated wound closure in female versus male plasmin deficient mice. Further studies showed that this gender effect could not be reversed by ovariectomy, suggesting that female sex-hormones did not mediate the accelerated skin wound healing in plasmin deficient female mice. Histological examination of healed wounds revealed larger amounts of fibrotic scars in the provisional matrix of plasmin deficient male mice compared to female mice. These fibrotic scars correlated to an obstruction of cell infiltration of the granulation tissue, which is a prerequisite for wound healing. In conclusion, the presented data show that the gender dependent effect of plasmin deficiency is tissue specific and may be secondary to already established differences between genders, such as skin thickness and composition.     

Drastically Abnormal Gluco- and Galactosylceramide Composition Does Not Affect Ganglioside Metabolism in the Brain of Mice Deficient in Galactosylceramide Synthase

Mice that are genetically deficient in UDP-galactose: ceramide galactosyltransferase are unable to synthesize galactosylceramide. Consequently, sulfatide, which can be synthesized only by sulfation of galactosylceramide, is also totally absent in affected mouse brain. -Hydroxy fatty acid-containing glucosylceramide partially replaces the missing galactosylceramide. A substantial proportion of sphingomyelin, which normally contains only non-hydroxy fatty acids, also contains -hydroxy fatty acids. These findings indicate that -hydroxy fatty acid-containing ceramide normally present only in galactosylceramide and sulfatide is diverted to other compounds because they cannot be synthesized into galactosylceramide due to the lack of the galactosyltransferase. We have examined brain gangliosides in order to determine if -hydroxy fatty acid-containing glucosylceramide present in an abnormally high concentration is also incorporated into gangliosides. The brain ganglioside composition, however, is entirely normal in both the total amount and molecular distribution in these mice. One feasible explanation is that UDP-galactose: glucosylceramide galactosyltransferase does not recognize -hydroxy fatty acid-containing glucosylceramide as acceptor. This analytical finding is consistent with the relative sparing of gray matter in the affected mice and provides an insight into sphingolipid metabolism in the mouse brain.     

Development of Fetal Testicular Cells in Androgen Receptor Deficient Mice

During mammalian development, androgens produced by the fetal testis are the most important hormones controlling the masculinization of the reproductive tract and the genitalia. New findings show that the male germ line is the most sensitive to anti-androgenic endocrine disruptors during the embryonic period. In a recent study, we reported that endogenous androgens physiologically control germ cell growth in the male mouse fetus during early fetal life. In the present study, we extended this result by showing the presence of a functional androgen receptor in the gonocytes in the latter part of the fetal life. We also studied the effect of androgens on the development of the somatic testicular cells using the Tfm mice which carry a naturally inactivating mutation of the androgen receptor. Fetal Leydig cell are largely independent of endogenous androgens during fetal development whereas fetal Sertoli cell number is decreased following a default of peritubular myoid cells differenciation. They also point to the gonocyte as a special target for androgens during the embryonic period and indicate a novel mechanism of androgen action on gonocytes. Elucidation of this new pathway in the fetal testis will clarify not only fetal testis physiology but also the effects of environmental anti-androgens that act during fetal life and open new perspectives for future investigations into the sensitivity of fetal germ cell to androgens.     

IL-25 Inhibits Atherosclerosis Development in Apolipoprotein E Deficient Mice

Objective

IL-25 has been implicated in the initiation of type 2 immunity and in the protection against autoimmune inflammatory diseases. Recent studies have identified the novel innate lymphoid type 2 cells (ILC2s) as an IL-25 target cell population. The purpose of this study was to evaluate if IL-25 has any influence on atherosclerosis development in mice.

Methods and Results

Administration of 1 μg IL-25 per day for one week to atherosclerosis-prone apolipoprotein (apo)E deficient mice, had limited effect on the frequency of T cell populations, but resulted in a large expansion of ILC2s in the spleen. The expansion was accompanied by increased levels of anti-phosphorylcholine (PC) natural IgM antibodies in plasma and elevated levels of IL-5 in plasma and spleen. Transfer of ILC2s to apoE deficient mice elevated the natural antibody-producing B1a cell population in the spleen. Treatment of apoE/Rag-1 deficient mice with IL-25 was also associated with extensive expansion of splenic ILC2s and increased plasma IL-5, suggesting ILC2s to be the source of IL-5. Administration of IL-25 in IL-5 deficient mice resulted in an expanded ILC2 population, but did not stimulate generation of anti-PC IgM, indicating that IL-5 is not required for ILC2 expansion but for the downstream production of natural antibodies. Additionally, administration of 1 μg IL-25 per day for 4 weeks in apoE deficient mice reduced atherosclerosis in the aorta both during initiation and progression of the disease.

Conclusions

The present findings demonstrate that IL-25 has a protective role in atherosclerosis mediated by innate responses, including ILC2 expansion, increased IL-5 secretion, B1a expansion and natural anti-PC IgM generation, rather than adaptive Th2 responses.     

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2^-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. Wesley S. Bond and Lalita Wadhwa are co-first authors.     

ALKBH4 Depletion in Mice Leads to Spermatogenic Defects

ALKBH4, an AlkB homologue in the 2-oxoglutarate and Fe²⁺ dependent hydroxylase family, has previously been shown to regulate the level of monomethylated lysine-84 in actin and thereby indirectly influences the ability of non-muscular myosin II to bind actin filaments. ALKBH4 modulates fundamental processes including cytokinesis and cell motility, and its depletion is lethal during early preimplantation embryo stage. The aim of this study was to investigate the effect of ALKBH4 deficiency in a physiological context, using inducible Alkbh4 knockout mice. Here, we report that ALKBH4 is essential for the development of spermatocytes during the prophase of meiosis, and that ALKBH4 depletion leads to insufficient establishment of the synaptonemal complex. We also show that ALKBH4 is localized in nucleolar structures of Sertoli cells, spermatogonia and primary spermatocytes.     

Defects in Ultrasonic Vocalization of Cadherin-6 Knockout Mice

Background

Although some molecules have been identified as responsible for human language disorders, there is still little information about what molecular mechanisms establish the faculty of human language. Since mice, like songbirds, produce complex ultrasonic vocalizations for intraspecific communication in several social contexts, they can be good mammalian models for studying the molecular basis of human language. Having found that cadherins are involved in the vocal development of the Bengalese finch, a songbird, we expected cadherins to also be involved in mouse vocalizations.

Methodology/Principal Findings

To examine whether similar molecular mechanisms underlie the vocalizations of songbirds and mammals, we categorized behavioral deficits including vocalization in cadherin-6 knockout mice. Comparing the ultrasonic vocalizations of cadherin-6 knockout mice with those of wild-type controls, we found that the peak frequency and variations of syllables were differed between the mutant and wild–type mice in both pup-isolation and adult-courtship contexts. Vocalizations during male-male aggression behavior, in contrast, did not differ between mutant and wild–type mice. Open-field tests revealed differences in locomotors activity in both heterozygote and homozygote animals and no difference in anxiety behavior.

Conclusions/Significance

Our results suggest that cadherin-6 plays essential roles in locomotor activity and ultrasonic vocalization. These findings also support the idea that different species share some of the molecular mechanisms underlying vocal behavior.     

Alkaline Phosphatase Protects Lipopolysaccharide-Induced Early Pregnancy Defects in Mice

Excessive cytokine inflammatory response due to chronic or superphysiological level of microbial infection during pregnancy leads to pregnancy complications such as early pregnancy defects/loss and preterm birth. Bacterial toxin lipopolysaccharide (LPS), long recognized as a potent proinflammatory mediator, has been identified as a risk factor for pregnancy complications. Alkaline phosphatase (AP) isozymes have been shown to detoxify LPS by dephosphorylation. In this study, we examined the role of alkaline phosphatase (AP) in mitigating LPS-induced early pregnancy complications in mice. We found that 1) the uterus prior to implantation and implantation sites following embryo implantation produce LPS recognition and dephosphorylation molecules TLR4 and tissue non-specific AP (TNAP) isozyme, respectively; 2) uterine TNAP isozyme dephosphorylates LPS at its sites of production; 3) while LPS administration following embryo implantation elicits proinflammatory cytokine mRNA levels at the embryo implantation sites (EISs) and causes early pregnancy loss, dephosphorylated LPS neither triggers proinflammatory cytokine mRNA levels at the EISs nor induces pregnancy complications; 4) AP isozyme supplementation to accelerate LPS detoxification attenuates LPS-induced pregnancy complications following embryo implantation. These findings suggest that a LPS dephosphorylation strategy using AP isozyme may have a unique therapeutic potential to mitigate LPS- or Gram-negative bacteria-induced pregnancy complications in at-risk women.     

Hyperplasia of Interstitial Cells of Cajal in Sprouty Homolog 4 Deficient Mice

Gastrointestinal stromal tumors, which are thought to derive from interstitial cells of Cajal or their precursors, often harbor an oncogenic mutation of the KIT receptor tyrosine kinase. Sprouty homolog 4, a known negative regulator of ERK pathway, has been identified in the interstitial cells of Cajal in the Kit^K641E murine model of gastrointestinal stromal tumors. Sprouty homolog 4 was upregulated both at the mRNA and protein level in these cells, suggesting that Sprouty homolog 4 is downstream of oncogenic KIT activation and potentially engaged in the negative feedback loop of ERK activation in this model. Here, we used Kit^K641E heterozygous and Sprouty homolog 4 knock out animals to quantify interstitial cells of Cajal in situ, using quantitative immunofluorescence for the receptor tyrosine kinase Kit and for phosphodiesterase 3a (PDE3A). In the antrum of Sprouty homolog 4 knock out mice, hyperplasia of interstitial cells of Cajal was reminiscent of the Kit^K641E heterozygous mice antrum. Additionally, the density of interstitial cells of Cajal was higher in the colon of adult Sprouty homolog 4 knock out mice than in WT littermates, although hyperplasia seemed more severe in Kit^K641E heterozygous mice. Functional transit studies also show similarities between Sprouty homolog 4 knock out and Kit^K641E heterozygous mice, as the total transit time in 9 month old animals was significantly increased in both genotypes compared to WT littermates. We concluded that the lack of Sprouty homolog 4 expression leads to hyperplasia of the interstitial cells of Cajal and is functionally associated with a delayed transit time.     

Embryonic Heart and Skin Defects in Mice Lacking Plakoglobin

Plakoglobin is the only component common to both the desmosomal plaque and the cadherin–catenin cell adhesion complex in the adherens junction. It is highly homologous to vertebrate β-catenin and toDrosophilaarmadillo protein and may—like these proteins—be also involved in signaling pathways. To analyze the role of plakoglobin during mouse development we inactivated theplakoglobingene by homologous recombination in embryonic stem cells and generated transgenic mice.Plakoglobinnull-mutant embryos died from Embryonic Day 10.5 onward, due to severe heart defects. Some mutant embryos developed further, especially on a C57BL/6 genetic background, and died around birth, presumably due to cardiac dysfunction, and with skin blistering and subcorneal acantholysis. Ultrastructural analysis revealed that here desmosomes were greatly reduced in number and structurally altered. Thus, using reversed genetics we demonstrate that plakoglobin is an essential structural component for desmosome function. The skin phenotype in plakoglobin-deficient mice is reminiscent of the human blistering disease, epidermolytic hyperkeratosis.     

Signs of Cardiac Autonomic Imbalance and Proarrhythmic Remodeling in FTO Deficient Mice

In humans, variants of the fat mass and obesity associated (FTO) gene have recently been associated with obesity. However, the physiological function of FTO is not well defined. Previous investigations in mice have linked FTO deficiency to growth retardation, loss of white adipose tissue, increased energy metabolism and enhanced systemic sympathetic activation. In this study we investigated for the first time the effects of global knockout of the mouse FTO gene on cardiac function and its autonomic neural regulation. ECG recordings were acquired via radiotelemetry in homozygous knockout (n = 12) and wild-type (n = 8) mice during resting and stress conditions, and analyzed by means of time- and frequency-domain indexes of heart rate variability. In the same animals, cardiac electrophysiological properties (assessed by epicardial mapping) and structural characteristics were investigated. Our data indicate that FTO knockout mice were characterized by (i) higher heart rate values during resting and stress conditions, (ii) heart rate variability changes (increased LF to HF ratio), (iii) larger vulnerability to stress-induced tachyarrhythmias, (iv) altered ventricular repolarization, and (v) cardiac hypertrophy compared to wild-type counterparts. We conclude that FTO deficiency in mice leads to an imbalance of the autonomic neural modulation of cardiac function in the sympathetic direction and to a potentially proarrhythmic remodeling of electrical and structural properties of the heart.