A mutation in Ampd2 is associated with nephrotic syndrome and hypercholesterolemia in mice.

BACKGROUND: Previously, we identified three loci affecting HDL-cholesterol levels in a screen for ENU-induced mutations in mice and discovered two mutated genes. We sought to identify the third mutated gene and further characterize the mouse phenotype. METHODS: We engaged, DNA sequencing, gene expression profiling, western blotting, lipoprotein characterization, metabolomics assessment, histology and electron microscopy in mouse tissues. RESULTS: We identify the third gene as Ampd2, a liver isoform of AMP Deaminase (Ampd), a central component of energy and purine metabolism pathways. The causative mutation was a guanine-to-thymine transversion resulting in an A341S conversion in Ampd2. Ampd2 homozygous mutant mice exhibit a labile hypercholesterolemia phenotype, peaking around 9 weeks of age (251 mg/dL vs. wildtype control at 138 mg/dL), and was evidenced by marked increases in HDL, VLDL and LDL. In an attempt to determine the molecular connection between Ampd2 dysfunction and hypercholesterolemia, we analyzed hepatic gene expression and found the downregulation of Ldlr, Hmgcs and Insig1 and upregulation of Cyp7A1 genes. Metabolomic analysis confirmed an increase in hepatic AMP levels and a decrease in allantoin levels consistent with Ampd2 deficiency, and increases in campesterol and ß-sitosterol. Additionally, nephrotic syndrome was observed in the mutant mice, through proteinuria, kidney histology and effacement and blebbing of podocyte foot processes by electron microscopy. CONCLUSION: In summary we describe the discovery of a novel genetic mouse model of combined transient nephrotic syndrome and hypercholesterolemia, resembling the human disorder.

NSAID-based γ-secretase modulators do not bind to the amyloid-ß polypeptide.

γ-Secretase modulators (GSMs) have received much attention as potential therapeutic agents for Alzheimer's disease (AD). GSMs increase the ratio between short and long forms of the amyloid-ß (Aß) polypeptides produced by γ-secretase and thereby decrease the amount of the toxic amyloid species. However, the mechanism of action of these agents is still poorly understood. One recent paper [Richter et al. (2010) Proc. Natl. Acad. Sci. U. S. A.107, 14597-14602] presented data that were interpreted to support direct binding of the GSM sulindac sulfide to Aß(42), supporting the notion that GSM action is linked to direct binding of these compounds to the Aß domain of its immediate precursor, the 99-residue C-terminal domain of the amyloid precursor protein (C99, also known as the ß-CTF). Here, contrasting results are presented that indicate there is no interaction between monomeric sulindac sulfide and monomeric forms of Aß42. Instead, it was observed that sulindac sulfide is itself prone to form aggregates that can bind nonspecifically to Aß42 and trigger its aggregation. This observation, combined with data from previous work [Beel et al. (2009) Biochemistry48, 11837-11839], suggests both that the poor behavior of some NSAID-based GSMs in solution may obscure results of binding assays and that NSAID-based GSMs do not function by directly targeting C99. It was also observed that another GSM, flurbiprofen, fails to bind to monomeric Aß42 or to C99 reconstituted into bilayered lipid vesicles. These results disfavor the hypothesis that these NSAID-based GSMs exert their modulatory effect by directly targeting a site located in the Aß42 domain of free C99.

A volumetric method for quantifying atherosclerosis in mice by using microCT: comparison to en face.

Precise quantification of atherosclerotic plaque in preclinical models of atherosclerosis requires the volumetric assessment of the lesion(s) while maintaining in situ architecture. Here we use micro-computed tomography (microCT) to detect ex vivo aortic plaque established in three dyslipidemic mouse models of atherosclerosis. All three models lack the low-density lipoprotein receptor (Ldlr(-/-)), each differing in plaque severity, allowing the evaluation of different plaque volumes using microCT technology. From clearly identified lesions in the thoracic aorta from each model, we were able to determine plaque volume (0.04-3.1 mm(3)), intimal surface area (0.5-30 mm(2)), and maximum plaque (intimal-medial) thickness (0.1-0.7 mm). Further, quantification of aortic volume allowed calculation of vessel occlusion by the plaque. To validate microCT for future preclinical studies, we compared microCT data to intimal surface area (by using en face methodology). Both plaque surface area and plaque volume were in excellent correlation between microCT assessment and en face surface area (r(2) = 0.99, p<0.0001 and r(2) = 0.95, p<0.0001, respectively). MicroCT also identified internal characteristics of the lipid core and fibrous cap, which were confirmed pathologically as Stary type III-V lesions. These data validate the use of microCT technology to provide a more exact empirical measure of ex vivo plaque volume throughout the entire intact aorta in situ for the quantification of atherosclerosis in preclinical models.

Generation and characterization of two novel mouse models exhibiting the phenotypes of the metabolic syndrome: Apob48-/-Lepob/ob mice devoid of ApoE or Ldlr.

The metabolic syndrome is a group of disorders including obesity, insulin resistance, atherogenic dyslipidemia, hyperglycemia, and hypertension. To date, few animal models have been described to recapitulate the phenotypes of the syndrome. In this study, we generated and characterized two lines of triple-knockout mice that are deficient in either apolipoprotein E (Apoe(-/-)) or low-density lipoprotein receptor (Ldlr(-/-)) and express no leptin (Lep(ob/ob)) or apolipoprotein B-48 but exclusively apolipoprotein B-100 (Apob(100/100)). These two lines are referred to as Apoe triple-knockout-Apoe 3KO (Apoe(-/-)Apob(100/100)Lep(ob/ob)) and Ldlr triple-knockout-Ldlr 3KO (Ldlr(-/-)Apob(100/100)Lep(ob/ob)) mice. Both lines develop obesity, hyperinsulinemia, hyperlipidemia, hypertension, and atherosclerosis. However, only Apoe 3KO mice are hyperglycemic and glucose intolerant and are more obese than Ldlr 3KO mice. To evaluate the utility of these lines as pharmacological models, we treated both with leptin and found that leptin therapy ameliorated most metabolic derangements. Leptin was more effective in improving glucose tolerance in Ldlr 3KO than Apoe 3KO animals. The reduction of plasma cholesterol by leptin in Ldlr 3KO mice can be accounted for by its suppressive effect on food intake. However, in Apoe 3KO mice, leptin further reduced plasma cholesterol independently of its effect on food intake, and this improvement correlated with a smaller plaque lesion area. These effects suggest a direct role of leptin in modulating VLDL levels and, likewise, the lesion areas in VLDL-enriched animals. These two lines of mice represent new models with features of the metabolic syndrome and will be useful in testing therapies targeted for combating the human condition.

Design, synthesis, and evaluation of orally active benzimidazoles and benzoxazoles as vascular endothelial growth factor-2 receptor tyrosine kinase inhibitors.

Inhibition of the VEGF signaling pathway has become a valuable approach in the treatment of cancers. Guided by X-ray crystallography and molecular modeling, a series of 2-aminobenzimidazoles and 2-aminobenzoxazoles were identified as potent inhibitors of VEGFR-2 (KDR) in both enzymatic and HUVEC cellular proliferation assays. In this report we describe the synthesis and structure-activity relationship of a series of 2-aminobenzimidazoles and benzoxazoles, culminating in the identification of benzoxazole 22 as a potent and selective VEGFR-2 inhibitor displaying a good pharmacokinetic profile. Compound 22 demonstrated efficacy in both the murine matrigel model for vascular permeability (79% inhibition observed at 100 mg/kg) and the rat corneal angiogenesis model (ED(50) = 16.3 mg/kg).

Regulatory effects of novel neurotrophin-1/b cell-stimulating factor-3 (cardiotrophin-like cytokine) on B cell function.

We describe regulatory effects that a novel neurotrophin-1/B cell-stimulating factor-3 (NNT-1/BSF-3; also reported as cardiotrophin-like cytokine) has on B cell function. NNT-1/BSF-3 stimulates B cell proliferation and Ig production in vitro. NNT-1/BSF-3-transgenic mice, engineered to express NNT-1/BSF-3 in the liver under control of the apolipoprotein E promoter, show B cell hyperplasia with particular expansion of the mature follicular B cell subset in the spleen and the prominent presence of plasma cells. NNT-1/BSF-3-transgenic mice show high serum levels of IgM, IgE, IgG2b, IgG3, anti-dsDNA Abs, and serum amyloid A. NNT-1/BSF-3-transgenic mice also show non-amyloid mesangial deposits that contain IgM, IgG, and C3 and are characterized by a distinctive ultrastructure similar to that of immunotactoid glomerulopathy. NNT-1/BSF-3-transgenic mice produce high amounts of Ag-specific IgM, IgA, and IgE and low amounts of IgG2a and IgG3. Normal mice treated with NNT-1/BSF-3 also produce high amounts of Ag-specific IgE. NNT-1/BSF-3 regulates immunity by stimulating B cell function and Ab production, with preference for Th2 over Th1 Ig types.