Mice with a targeted deletion of the tetranectin gene exhibit a spinal deformity

Tetranectin is a plasminogen-binding, homotrimeric protein belonging to the C-type lectin family of proteins. Tetranectin has been suggested to play a role in tissue remodeling, due to its ability to stimulate plasminogen activation and its expression in developing tissues such as developing bone and muscle. To test the functional role of tetranectin directly, we have generated mice with a targeted disruption of the gene. We report that the tetranectin-deficient mice exhibit kyphosis, a type of spinal deformity characterized by an increased curvature of the thoracic spine. The kyphotic angles were measured on radiographs. In 6-month-old normal mice (n = 27), the thoracic angle was 73 degrees +/- 2 degrees, while in tetranectin-deficient 6-month-old mice (n = 35), it was 93 degrees +/- 2 degrees (P < 0.0001). In approximately one-third of the mutant mice, X-ray analysis revealed structural changes in the morphology of the vertebrae. Histological analysis of the spines of these mice revealed an apparently asymmetric development of the growth plate and of the intervertebral disks of the vertebrae. In the most advanced cases, the growth plates appeared disorganized and irregular, with the disk material protruding through the growth plate. Tetranectin-null mice had a normal peak bone mass density and were not more susceptible to ovariectomy-induced osteoporosis than were their littermates as determined by dual-emission X-ray absorptiometry scanning. These results demonstrate that tetranectin plays a role in tissue growth and remodeling. The tetranectin-deficient mouse is the first mouse model that resembles common human kyphotic disorders, which affect up to 8% of the population.     

Estradiol-induced attenuation of pulmonary hypertension is not associated with altered eNOS expression

Female rats develop less severe pulmonary hypertension (PH) in response to chronic hypoxia compared with males, thus implicating a potential role for ovarian hormones in mediating this gender difference. Considering that estrogen upregulates endothelial nitric oxide (NO) synthase (eNOS) in systemic vascular tissue, we hypothesized that estrogen inhibits hypoxic PH by increasing eNOS expression and activity. To test this hypothesis, we examined responses to the endothelium-derived NO-dependent dilator ionomycin and the NO donors S-nitroso-N-acetylpenicillamine and spermine NONOate in U-46619-constricted, isolated, saline-perfused lungs from the following groups: 1) normoxic rats with intact ovaries, 2) chronic hypoxic (CH) rats with intact ovaries, 3) CH ovariectomized rats given 17 beta-estradiol (E(2)beta), and 4) CH ovariectomized rats given vehicle. Additional experiments assessed pulmonary eNOS levels in each group by Western blotting. Our findings indicate that E(2)beta attenuated chronic hypoxia-induced right ventricular hypertrophy, pulmonary arterial remodeling, and polycythemia. Furthermore, although CH augmented vasodilatory responsiveness to ionomycin and increased pulmonary eNOS expression, these responses were not potentiated by E(2)beta. Finally, responses to S-nitroso-N-acetylpenicillamine and spermine NONOate were similarly attenuated in all CH groups compared with normoxic control groups. We conclude that the inhibitory influence of E(2)beta on chronic hypoxia-induced PH is not associated with increased eNOS expression or activity.     

Phosphorylation of eNOS initiates excessive NO production in early phases of portal hypertension

Akt, also known as protein kinase B, is a serine/threonine kinase. Akt becomes active when phosphorylated by the activation of receptor tyrosine kinases, G protein-coupled receptors, and mechanical forces such as shear stress. Studies in vitro have shown that Akt can directly phosphorylate endothelial nitric oxide (NO) synthase (eNOS) and activate the enzyme, leading to NO production. The aim of this study was to test the hypothesis that the phosphorylation of eNOS plays a role in the enhanced NO production observed in early portal hypertension. Male Sprague-Dawley rats were subjected to either sham or portal vein ligation (PVL), and mesenteric arterial beds were used for ex vivo perfusion studies. Mesenteric arterial beds from PVL rats had an approximately 60-70% decrease in response to methoxamine (an alpha(1)-agonist and vasoconstrictor) compared with the sham group (P < 0.01). When N(G)-monomethyl-L-arginine (a NOS inhibitor) was added to the perfusion, the difference in perfusion pressure between the two groups was abolished, suggesting that enhanced NO production in the PVL group blunted the response to the vasoconstrictor. The reduced responsiveness in PVL was not due to changes in eNOS expression but was due to an increase in enzyme-specific activity, suggesting posttranslational modification of eNOS. The phosphorylation of eNOS at Ser(1176) was significantly increased by twofold (P < 0.05) in the PVL group. Furthermore, PVL significantly increased Akt phosphorylation (an active form of Akt) by threefold (P < 0.05). When vessels were treated with wortmannin (10 nM) to block the phosphatidylinositol-3-OH-kinase/Akt pathway, NO-induced vasodilatation was significantly reduced. These results suggest that the phosphorylation of eNOS by Akt activates the enzyme and may be the first step leading to an initial increase in NO production in portal hypertension.     

Mice with targeted mutation of peroxiredoxin 6 develop normally but are susceptible to oxidative stress

Reactive oxygen species, especially hydrogen peroxide, are important in cellular signal transduction. However, excessive amounts of these species damage tissues and cells by oxidizing virtually all important biomolecules. Peroxiredoxin 6 (PRDX6) (also called antioxidant protein 2, or AOP2) is a novel peroxiredoxin family member whose function in vivo is unknown. Through immunohistochemistry, we have determined that the PRDX6 protein was widely expressed in every tissue examined, most abundantly in epithelial cells. It was found in cytosol, but not in membranes, organelles, and nuclei fractions. Prdx6 mRNA was also expressed in every tissue examined. The widespread expression of Prdx6 suggested that its functions were quite important. To determine these functions, we generated Prdx6-targeted mutant (Prdx6-/-) mice, confirmed the gene disruption by Southern blots, PCR, RT-PCR, Western blots, and immunohistochemistry, and compared the effects of paraquat, hydrogen peroxide, and t-butyl hydroperoxide on Prdx6-/- and wild-type (Prdx6+/+) macrophages, and of paraquat on Prdx6-/- and Prdx6+/+ mice. Prdx6-/- macrophages had higher hydrogen peroxide levels, and lower survival rates; Prdx6-/- mice had significantly lower survival rates, more severe tissue damage, and higher protein oxidation levels. Additionally, there were no differences in the mRNA expression levels of other peroxiredoxins, glutathione peroxidases, catalase, superoxide dismutases, thioredoxins, and glutaredoxins between normal Prdx6-/- and Prdx6+/+ mice and those injected with paraquat. Our study provides in vivo evidence that PRDX6 is a unique non-redundant antioxidant that functions independently of other peroxiredoxins and antioxidant proteins.     

Mice with a targeted mutation of patched2 are viable but develop alopecia and epidermal hyperplasia

Hedgehog (Hh) signaling plays pivotal roles in tissue patterning and development in Drosophila melanogaster and vertebrates. The Patched1 (Ptc1) gene, encoding the Hh receptor, is mutated in nevoid basal cell carcinoma syndrome, a human genetic disorder associated with developmental abnormalities and increased incidences of basal cell carcinoma (BCC) and medulloblastoma (MB). Ptc1 mutations also occur in sporadic forms of BCC and MB. Mutational studies with mice have verified that Ptc1 is a tumor suppressor. We previously identified a second mammalian Patched gene, Ptc2, and demonstrated its distinct expression pattern during embryogenesis, suggesting a unique role in development. Most notably, Ptc2 is expressed in an overlapping pattern with Shh in the epidermal compartment of developing hair follicles and is highly expressed in the developing limb bud, cerebellum, and testis. Here, we describe the generation and phenotypic analysis of Ptc2(tm1/tm1) mice. Our molecular analysis suggests that Ptc2(tm1) likely represents a hypomorphic allele. Despite the dynamic expression of Ptc2 during embryogenesis, Ptc2(tm1/tm1) mice are viable, fertile, and apparently normal. Interestingly, adult Ptc2(tm1/tm1) male animals develop skin lesions consisting of alopecia, ulceration, and epidermal hyperplasia. While functional compensation by Ptc1 might account for the lack of a strong mutant phenotype in Ptc2-deficient mice, our results suggest that normal Ptc2 function is required for adult skin homeostasis.     

Matrix metalloproteinase-9 inhibition or deletion attenuates portal hypertension in rodents

Liver cirrhosis and portal hypertension are accompanied by hyperdynamic circulation, angiogenesis and portosystemic collaterals. Matrix metalloproteinases (MMPs) participate in fibrogenesis and angiogenesis, however, whether they can be targeted in cirrhosis treatment is unclear. Therefore, we performed three series of experiments to investigate this issue. Liver cirrhosis was induced by common bile duct ligation (BDL) in Sprague-Dawley rats. Sham-operated rats served as controls. Rats were randomly allocated to receive vehicle, minocycline (a nonselective MMP inhibitor) or SB-3CT (MMP-2 and −9 inhibitor) for 28 days in the first and second series, respectively. MMP-9 knockout mice were used in the third series. The results showed that minocycline ameliorated portal hypertension, hemodynamic abnormalities, reduced collateral shunting, mesenteric vascular density, plasma VEGF level and alleviated liver fibrosis. SB-3CT attenuated portal hypertension, hemodynamic derangements, reduced shunting, mesenteric vascular density, mesenteric VEGF protein expression, and liver fibrosis. Knockout BDL mice had significantly alleviated portal hypertension, liver fibrosis, liver α-SMA and mesenteric eNOS protein expressions compared to wild-type BDL mice. Liver SMAD2 phosphorylation was down-regulated in all series with MMP inhibition or knock-out. In conclusion, MMP-9 inhibition or deletion ameliorated the severity of cirrhosis, portal hypertension, and associated derangements. MMP-9 may be targeted in the treatment of liver cirrhosis.     

In vitro labeling and tissue autoradiography of splenomegaly associated with portal hypertension

Sinus hyperplasia occurring in the congestive splenomegaly associated with portal hypertension is a interesting model of steady-state hyperplasia in an adult tissue. In vitro labeling of human splenic tissue with 3H-thymidine and the demonstration of S-phase cells by autoradiography was performed to investigate the proliferative activity of sinus-lining and cordal reticular cells in congestive splenomegaly compared with that in the normal spleen. The labeling index (%) of sinus-lining cells after 2 h incubation was 0.07 +/- 0.02 in the normal spleen and 0.26 +/- 0.03 in congestive splenomegaly (t = 4.553, P less than 0.005, n = 11). This result indicated that sinus-lining cells have a long life span and that their proliferative activity is increased in congestive splenomegaly. On the other hand, the labeling of cordal reticular cells and arterial and venous endothelial cells was sparse or absent in both congestive splenomegaly controls, and these cells do not appear to be involved in sinus hyperplasia.     

Role of NPY for vasoregulation in the splanchnic circulation during portal hypertension

Vascular dysfunction in the splanchnic circulation during portal hypertension is characterized by enhanced NO-mediated vasorelaxation and vascular hyporeactivity to norepinephrine that lead to arterial vasodilation. NPY most likely counteracts both of these key features. Firstly, NPY appears to inhibit Ach- and PNS-induced vasorelaxation in mesenteric arteries. This effect is more pronounced in portal hypertensive rats as compared to control, and most likely reflects the inhibition of increased e- and nNOS-derived NO-synthesis during portal hypertensive conditions. Secondly, NPY sensitizes the mesenteric vasculature to alpha(1)-adrenergic vasoconstriction. Most importantly, in portal hypertensive rats but not in sham rats NPY markedly augments vascular contractility and thereby corrects vascular hyporeactivity. Both actions of NPY increase vascular tone and may well act synergistically in the splanchnic circulation during portal hypertension. Moreover, the vasoconstrictive effects of NPY are most pronounced at particularly high levels of alpha(1)-adrenergic activity. Therefore, it appears that NPY becomes increasingly important for optimizing adrenergic vasoconstriction at particularly high adrenergic drive and also for playing a predominant role for vascular homeostasis. Cirrhotic patients present with elevated circulating plasma levels of NPY, which appears to be independent from the severity of liver dysfunction and to correlate with portal pressure. This finding indicates enhanced NPY release during portal hypertension that may represent a compensatory mechanism aimed at counterbalancing arterial vasodilation by restoring the efficacy of endogenous catecholamines and inhibiting vasodilative drive in the splanchnic circulation.     

Mice with a targeted deletion of the type 2 deiodinase are insulin resistant and susceptible to diet induced obesity

Background

The type 2 iodothyronine deiodinase (D2) converts the pro-hormone thyroxine into T3 within target tissues. D2 is essential for a full thermogenic response of brown adipose tissue (BAT), and mice with a disrupted Dio2 gene (D2KO) have an impaired response to cold. BAT is also activated by overfeeding.

Methodology/Principal Findings

After 6-weeks of HFD feeding D2KO mice gained 5.6% more body weight and had 28% more adipose tissue. Oxygen consumption (V0₂) was not different between genotypes, but D2KO mice had an increased respiratory exchange ratio (RER), suggesting preferential use of carbohydrates. Consistent with this, serum free fatty acids and β-hydroxybutyrate were lower in D2KO mice on a HFD, while hepatic triglycerides were increased and glycogen content decreased. Neither genotype showed glucose intolerance, but D2KO mice had significantly higher insulin levels during GTT independent of diet. Accordingly, during ITT testing D2KO mice had a significantly reduced glucose uptake, consistent with insulin resistance. Gene expression levels in liver, muscle, and brown and white adipose tissue showed no differences that could account for the increased weight gain in D2KO mice. However, D2KO mice have higher PEPCK mRNA in liver suggesting increased gluconeogenesis, which could also contribute to their apparent insulin resistance.

Conclusions/Significance

We conclude that the loss of the Dio2 gene has significant metabolic consequences. D2KO mice gain more weight on a HFD, suggesting a role for D2 in protection from diet-induced obesity. Further, D2KO mice appear to have a greater reliance on carbohydrates as a fuel source, and limited ability to mobilize and to burn fat. This results in increased fat storage in adipose tissue, hepatic steatosis, and depletion of liver glycogen in spite of increased gluconeogenesis. D2KO mice are also less responsive to insulin, independent of diet-induced obesity.     

Characterization of mice with targeted deletion of glycine receptor alpha 2

Glycine receptors are ligand-gated chloride channels that mediate inhibitory neurotransmission in the adult nervous system. During development, glycine receptor alpha 2 (GlyRalpha2) is expressed in the retina, in the spinal cord, and throughout the brain. Within the cortex, GlyRalpha2 is expressed in immature cells and these receptors have been shown to be active and excitatory. In the developing retina, inhibition of glycine receptor activity prevents proper rod photoreceptor development. These data suggest that GlyRalpha2, the developmentally expressed glycine receptor, may play an important role in neuronal development. We have generated mice with a targeted deletion of glycine receptor alpha 2 (Glra2). Although these mice lack expression of GlyRalpha2, no gross morphological or molecular alterations were observed in the nervous system. In addition, the cerebral cortex does not appear to require glycine receptor activity for proper development, as Glra2 knockout mice did not show any electrophysiological responses to glycine.     

肝硬化门脉高压症大鼠模型制作方法的探讨

目的：建立稳定可靠的肝硬化门脉高压大鼠动物模型。材料与方法：50只雄性sD大鼠,随机分为3组。正常对照组10只,行假手术,给予正常饮食。肝硬化A组20只,先行左肾上腺静脉结扎,然后给予初始浓度为0．03％的硫代乙酰胺（thioacetamide,TAA）溶液作为饮用水并根据大鼠体重变化调节给药浓度。肝硬化B组20只,行假手术,给予固定浓度为0．03％TAA溶液。给药时间为14周。结果：肝硬化A组大鼠死亡率为0,肝硬化形成率达到100％。肝硬化B组大鼠死亡率15％,肝硬化形成率75％。肝硬化A组大鼠的门静脉压力明显高于肝硬化B组和对照组。结论：采用左肾上腺静脉结扎并根据体重变化调节TAA给药浓度可建立稳定可靠的肝硬化门脉高压大鼠动物模型。     

Effects of somatostatin in patients with portal hypertension

Portal hypertension is a common complication of chronic liver disease. Conventional therapy consists of surgery and palliative measures for the hemodynamic problem. It has been recently reported that somatostatin may reduce portal pressure without altering the systemic circulation and so reducing hepatic blood flow. This peptide also causes a significant fall in azygos circulation in patients with esophageal varices. The mechanism of this effect is unclear although suppression of intestinal vasodilating hormones and of glucagon have been claimed to play a role. Comparative clinical studies have shown somatostatin to be superior to the standard vasopressin treatment. Recent findings suggest that the efficacy of somatostatin can be increased by administering this peptide in repeated intravenous bolus injections. New derivatives, specially long-acting peptides, may eventually prove beneficial in the chronic treatment of this complication.     

eNOS polymorphism associated with metabolic syndrome in children and adolescents

We investigated whether genetic polymorphisms in the endothelial nitric oxide (eNOS) gene (T⁷⁸⁶C in the promoter region, Glu298Asp in exon 7, and 4b/4a in intron 4) or eNOS haplotypes are associated with metabolic syndrome (MetS) in obese children and adolescents. We studied 242 subjects: 108 healthy (controls), 64 normotensive obese, and 70 obese children and adolescents with MetS. Genotypes were determined by Taqman^? allele discrimination assay and real-time polymerase chain reaction (PCR), and PCR followed by fragment separation by electrophoresis. We compared the distribution of eNOS genotypes, alleles, and haplotypes in the three groups of subjects. The CC genotype for the T⁷⁸⁶C polymorphism was more common in the MetS group than in the control group (OR?=?3.27; CI 1.81?C9.07; P?<?0.05). However, we found no significant differences in the distribution of eNOS haplotypes (P?>?0.00625; P for significance after correction for multiple comparisons). Our findings suggest that while eNOS haplotypes are not relevant, the CC genotype for the T⁷⁸⁶C polymorphism is associated with MetS in obese children and adolescents. Further studies examining interactions of eNOS haplotypes with environmental factors and other genetic markers are warranted.