Age-matched Littermate Controls Research Articles

QTc Interval is Prolonged in Hypercholesterolemic Mice

Hypercholesterolemia has been associated with ventricular arrhythmias and sudden cardiac death (SCD). Elevated cholesterol levels alter membrane properties and can affect cardiac ion channel function leading to rhythm disturbances. QT prolongation which reflects ventricular repolarization is a known risk factor for ventricular arrhythmia. Thus, the objective of this study was to investigate the effects of hypercholesterolemia on QTc interval using mice overexpressing human ApoB-100 (hAPOB+/+) which exhibit high levels of cholesterol. Surface ECG were recorded from anaesthetized male and female hAPOB+/+ mice and their age-matched littermate controls at 2, 4, 6, 9 and 12 mo. The 4mo male hAPOB+/+ mice displayed longer QTc intervals (ms) than the controls (4mo, CTL: 63.4±1.0, n=38; hAPOB+/+: 70.4±1.5, n=36, p<0.05). This difference was further accentuated at 9 and 12 mo (9mo, CTL: 63.8±0.6, n=12; hAPOB+/+: 76.5±2.4, n=12; 12mo, CTL: 66.1±1.6, n=10; hAPOB+/+: 79.8±2.1, n=12, p<0.05 vs 4mo). The increased QTc interval was independent of body weight (g) (12mo, CTL: 66±2, n=10; hAPOB+/+: 80±2, n=12, p=NS). In addition, female hAPOB+/+ mice also had a significantly longer QTc interval compared to their respective controls but unlike the males this difference became significant only at 6mo (6mo, CTL: 63.1±1.2 n=30; hAPOB+/+: 70.9±1.6, n=24, p<0.05) and did not increase further with age. Western Blot and qPCR analyses of ventricular K+ channels (Kv1.5, Kv2.1, Kv4.2, Kv4.3 and Kir2.1) showed no differences in the protein and mRNA expression of control and hAPOB+/+ mice. Altogether, these findings indicate that hypercholesterolemia lengthens ventricular repolarization in both males and females with a more severe phenotype in males. K+ channel expression were unchanged in hAPOB+/+ mice suggesting that the cholesterol-induced QT prolongation would be due to changes in membrane properties affecting K+ channel functions.

Abstract 17495: Thrombospondin-1/CD36 Pathway Critically Contributes to SHH Signaling Defect and EPC Dysfunction on Wound Healing in Type 1 Diabetes

Introduction: Refractory wounds in diabetic patients is a significant clinical problem. We reported previously that sonic hedgehog (SHH), a morphogenic protein central to wound repair, is deficient in diabetes, but the underlying mechanisms are poorly understood. We hypothesized that thrombospondin-1 (TSP-1) through its receptor CD36 contributes to SHH signaling defect in cutaneous tissue and endothelial progenitor cells (EPCs), resulting in impaired wound healing in type 1 diabetic mice. Methods and Results: Cutaneous expression of SHH and its signaling molecules including Patched (Ptc), Smoothened (Smo), and Gli were significantly decreased in streptozotocin (STZ)-induced type 1 diabetic mice (male, C57BL/6J, 12 weeks) vs. their age-matched control littermates (Western blot analysis, n=5, p&lt;0.05 vs. control), which were reversed in STZ-treated TSP-1 -/- mice (homozygote, C57BL/6J background, n=5, p&lt;0.05 vs. STZ-treated WT mice). Excisional wound (6-mm punch biopsy) closure was significantly delayed in STZ mice (n=6, p&lt;0.05 vs. control) with reduced wound blood flow (Laser Doppler), which was not observed in STZ-treated TSP-1 -/- mice (n=5, p&lt;0.05 vs. STZ-treated WT mice). The protective effect of TSP-1 depletion on wound repair was abolished following topical treatment with the selective SHH inhibitor cyclopamine (5 μ M, 30 min/day, n=5, p&lt;0.05 vs. STZ-treated TSP-1 -/- mice). Isolated EPCs from bone marrow of STZ mice showed significantly impaired angiogenesis (Matrigel tube formation, n=4, p&lt;0.05 vs. control), which was rescued by exogenous SHH (1 mg/L, 24 hours, n=4, p&lt;0.05 vs. STZ-control). Increased EPC angiogenesis in STZ-treated TSP-1 -/- mice (n=5, p&lt;0.05 vs. STZ-WT) was inhibited by the selective SHH inhibitor cyclopamine (5 μ M, 24 hours, n=5, p&lt;0.05). SHH, Ptc, Smo and Gli levels were significantly increased in EPCs from TSP-1 -/- mice and CD36 -/- mice, but not CD47 -/- mice (n=6, p&lt;0.05 vs. WT). Normal EPCs treated with recombinant human TSP-1 protein (2.2 nM, 24 hour) exhibited impaired angiogenesis, an effect that was not observed in EPCs from CD36 -/- mice (n=5, p&lt;0.05). Conclusion: TSP-1/CD36 pathway critically contributes to SHH signaling defect, resulting in EPC dysfunction and impaired wound healing in type 1 diabetic mice.

HIV-1 Tat-induced cerebrovascular toxicity is enhanced in mice with amyloid deposits

HIV-1-infected brains are characterized by elevated depositions of amyloid beta (Aβ); however, the interactions between Aβ and HIV-1 are poorly understood. In the present study, we administered specific HIV-1 protein Tat into the cerebral vasculature of 50–52-week-old double transgenic (B6C3-Tg) mice that express a chimeric mouse/human amyloid precursor protein (Mo/HuAPP695swe) and a mutant human presenilin 1 (PS1-dE9) and are characterized by increased Aβ depositions in the brain. Exposure to Tat increased permeability across cerebral capillaries, enhanced disruption of zonula occludens (ZO)-1 tight junction protein, and elevated brain expression of matrix metalloproteinase-9 (MMP-9) in B6C3-Tg mice as compared with age-matched littermate controls. These changes were associated with increased leukocyte attachment and their transcapillary migration. The majority of Tat-induced effects were attenuated by treatment with a specific Rho inhibitor, hydroxyfasudil. The results of animal experiments were reproduced in cultured brain endothelial cells exposed to Aβ and/or Tat. The present data indicate that increased brain levels of Aβ can enhance vascular toxicity and proinflammatory responses induced by HIV-1 protein Tat.

Osteoblast/osteocyte-specific inactivation of Stat3 decreases load-driven bone formation and accumulates reactive oxygen species

Signal transducers and activators of transcription 3 (Stat3) is a transcription factor expressed in many cell types including osteoblasts, osteocytes, and osteoclasts. STAT3 mutations cause a rare human immunodeficiency disease that presents reduced bone mineral density and recurrent pathological fractures. To investigate the role of Stat3 in load-driven bone metabolism, two strains of osteoblast/osteocyte-selective Stat3 knockout (KO) mice were generated. Compared to age-matched littermate controls, this selective inactivation of Stat3 significantly lowered bone mineral density (7-12%, p<0.05) as well as ultimate force (21-34%, p<0.01). In ulna loading (2.50-2.75N with 120 cycles/day at 2Hz for 3 consecutive days), Stat3 KO mice were less responsive than littermate controls as indicated by reduction in relative mineralizing surface (rMS/BS, 47-59%, p<0.05) and relative bone formation rate (rBFR/BS, 64-75%, p<0.001). Furthermore, inactivation of Stat3 suppressed load-driven mitochondrial activity, which led to an elevated level of reactive oxygen species (ROS) in cultured primary osteoblasts. Taken together, the results support the notion that the loss-of-function mutation of Stat3 in osteoblasts and osteocytes diminishes load-driven bone formation and impairs the regulation of oxidative stress in mitochondria.

Abstract 3166: Lung cancer bio-signatures in plasma based on the analysis of mouse models

Abstract Genetically engineered mouse models of cancer have been shown to recapitulate many of the molecular and biological features of human cancer. To identify plasma proteins that show altered abundance during lung cancer development, we applied in-depth quantitative proteomic analysis to plasma from three mouse models of lung adenocarcinoma driven by mutant EGFR, Kras or induced by urethane exposure; a mouse model of small cell lung cancer (SCLC); models of pancreatic, ovarian, colon, prostate and breast cancer; and two mouse models of inflammatory disease as confounders. Plasma proteins from mice with disease and age-matched littermate controls were analyzed by mass spectrometry (MS). Whole cell lysates and conditioned media from 12 lung adenocarcinoma cell lines were also analyzed by MS to identify enriched proteins in conditioned media in common with proteins with increased levels in plasmas from lung tumor bearing mice. Proteins corresponding to 3744 non-redundant IPIs (International Protein Index) that represented protein products from 2260 unique genes were quantified in one or more experiments. Unsupervised hierarchical clustering of plasma proteins revealed clustering of the four lung cancer models and the two breast cancer models, suggesting that protein signatures in the blood are organ-type specific. Fifty-eight percent of the elevated proteins in plasmas from mice with lung adenocarcinoma were identified in conditioned media of one or more human lung cell lines. A biosignature based on plasma proteins regulated by the Titf1/Nkx2-1 transcription factor was identified in plasmas from mouse models of lung adenocarcinoma. In addition, protein network analysis showed that many proteins whose abundance is altered in the plasma of the lung cancer mouse model engineered to express mutant human EGFR are associated with EGFR. A plasma protein signature that is enriched in proteins associated with neuroendocrine development was also identified in a SCLC model. Exploratory studies in human lung cancer and control plasmas of candidate markers identified in mouse models yielded altered levels in lung cancer plasmas concordant with findings in mouse models. Our studies of mouse models have identified plasma based signature for lung cancer that reflect developmental and signaling pathways applicable to human lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3166. doi:10.1158/1538-7445.AM2011-3166

Levels of membrane fluidity in the spinal cord and the brain in an animal model of amyotrophic lateral sclerosis

A mutant form of the copper/zinc superoxide dismutase (SOD1) protein is found in some patients with amyotrophic lateral sclerosis (ALS). Alteration of the activity of this antioxidant enzyme leads to an oxidative stress imbalance, which damages the structure of lipids and proteins in the CNS. Using fluorescence spectroscopy, we monitored membrane fluidity in the spinal cord and the brain in a widely used animal model of ALS, the SOD(G93A) mouse, which develops symptoms similar to ALS with an accelerated course. Our results show that the membrane fluidity of the spinal cord in this animal model significantly decreased in symptomatic animals compared with age-matched littermate controls. To the best of our knowledge, this is the first report showing that membrane fluidity is affected in the spinal cord of a SOD(G93A) animal model of ALS. Changes in membrane fluidity likely contribute substantially to alterations in cell membrane functions in the nervous tissue from SOD(G93A) mice.

Intrinsically Disordered Islet Amyloid Polypeptide Is a Pathogenic Link Between Type-2 diabetes and Heart Disease

Islet amyloid polypetide (IAPP) is an intrinsically disordered protein co-expressed and secreted with insulin by pancreatic β-cells. IAPP is produced at increased rates in patients with pre-diabetes, leading to proteotoxicity and extracellular amyloid deposition, a hallmark of type-2 diabetes. We found high IAPP levels in blood and failing hearts from pre-diabetic and diabetic humans, showing that IAPP accumulates in the heart and may contribute to heart dysfunction. Here, we tested the hypothesis that accumulation of IAPP in the heart alters Ca handling in cardiac myocytes, accelerating the occurrence of heart failure. We measured Ca transients in rats transgenic for human IAPP (HIP rats) and in wild-type RIP rats that bear only the non-diabetogenic IAPP isoform. Ca transient amplitude was significantly larger in cardiac myocytes from pre-diabetic HIP rats vs. age-matched control rats. In contrast, pre-diabetic RIP rats showed unaltered Ca transients. We also found increased Ca transients in myocytes incubated with exogenous human IAPP but not with rodent IAPP. Increased cellular Ca load is involved in hypertrophic signaling and pathological remodeling of the heart. We measured the level of brain natriuretic peptide (BNP), a molecular marker of hypertrophy, in heart protein homogenates from HIP rats using western blots. BNP expression was already elevated (by 70±21%) in hearts from pre-diabetic rats vs. age-matched control littermates and further increased with diabetes development. These data show that cardiac Ca dysregulation and hypertrophy correlates with toxic deposition of IAPP in the heart. In conclusion, our data suggest that accumulation of IAPP oligomers contribute significantly to the pathogenesis of diabetic heart failure. IAPP represents an effective target for diagnostic purposes and therapeutic strategies.

Impaired nigrostriatal function precedes behavioral deficits in a genetic mitochondrial model of Parkinson's disease

Parkinson's disease (PD) involves progressive loss of nigrostriatal dopamine (DA) neurons over an extended period of time. Mitochondrial damage may lead to PD, and neurotoxins affecting mitochondria are widely used to produce degeneration of the nigrostriatal circuitry. Deletion of the mitochondrial transcription factor A gene (Tfam) in C57BL6 mouse DA neurons leads to a slowly progressing parkinsonian phenotype in which motor impairment is first observed at ~12 wk of age. L-DOPA treatment improves motor dysfunction in these "MitoPark" mice, but this declines when DA neuron loss is more complete. To investigate early neurobiological events potentially contributing to PD, we compared the neurochemical and electrophysiological properties of the nigrostriatal circuit in behaviorally asymptomatic 6- to 8-wk-old MitoPark mice and age-matched control littermates. Release, but not uptake of DA, was impaired in MitoPark mouse striatal brain slices, and nigral DA neurons lacked characteristic pacemaker activity compared with control mice. Also, hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel function was reduced in MitoPark DA neurons, although HCN messenger RNA was unchanged. This study demonstrates altered nigrostriatal function that precedes behavioral parkinsonian symptoms in this genetic PD model. A full understanding of these presymptomatic cellular properties may lead to more effective early treatments of PD.

Targeted Proteomics of Isolated Glomeruli from the Kidneys of Diabetic Rats: Sorbin and SH3 Domain Containing 2 Is a Novel Protein Associated with Diabetic Nephropathy

To evaluate proteins associated with the development of diabetic nephropathy, a major cause of the end-stage renal disease, we analyzed protein expression in isolated glomeruli from spontaneous type 2 diabetic (OLETF) rats and their age-matched control littermates (LETO) in the early and proteinuric stages of diabetic nephropathy using QSTAR Elite LC-MS/MS. Among the 191 and 218 proteins that were altered significantly in the OLETF rats, twenty-four were actin cytoskeleton-associated proteins implicated in the formation of stress fibers, and the impairment of actin polymerization, intermediate filaments and microtubules. Importantly, sorbin and SH3 domain containing 2 (SORBS2), which is involved in the formation of stress fibers, was significantly upregulated in both stages of diabetic nephropathy (1.49- and 1.97-fold, resp.). Immunohistochemical and quantitative-PCR analyses revealed upregulation of SORBS2 in podocytes of glomeruli of OLETF rats. Our findings suggested that SORBS2 may be associated with the development of diabetic nephropathy possibility by reorganization of actin filaments.

Monitoring systemic oxidative stress in an animal model of amyotrophic lateral sclerosis

A mutant form of the ubiquitous copper/zinc superoxide dismutase (SOD1) protein has been found in some patients with amyotrophic lateral sclerosis (ALS). We monitored oxidative stress in an animal model of ALS, the SOD(G93A) mouse, which develops a disease similar to ALS with an accelerated course. The aim of this work was to show that ALS damages several organs and tissues, from an oxidative stress point of view. We measured lipid and protein oxidative damage in different tissue homogenates of SOD(G93A) mice. The biomarkers that we analyzed were malondialdehyde + 4-hydroxyalkenal (MDA + 4-HDA) and carbonyls, respectively. The spinal cord and brain of SOD(G93A) mice showed increased lipid peroxidation after 100 or 130 days compared to age-matched littermate controls. The CNS was most affected, but lipid peroxidation was also detected in the skeletal muscle and liver on day 130. No changes were observed in protein carbonylation in the homogenates. Our results are consistent with a multisystem etiology of ALS and suggest that oxidative stress may play a primary role in ALS pathogenesis. Thus, oxidative stress represents a potential biomarker that might be useful in developing new therapeutic strategies for ALS.

Characterisation of prostate cancer lesions in heterozygous Men1 mutant mice

BackgroundMutations of the MEN1 gene predispose to multiple endocrine neoplasia type 1 (MEN1) syndrome. Our group and others have shown that Men1 disruption in mice recapitulates MEN1 pathology. Intriguingly, rare lesions in hormone-dependent tissues, such as prostate and mammary glands, were also observed in the Men1 mutant mice.MethodsTo study the occurrence of prostate lesions, we followed a male mouse cohort of 47 Men1+/- mice and 23 age-matched control littermates, starting at 18 months of age, and analysed the prostate glands from the cohort.ResultsSix Men1+/- mice (12.8%) developed prostate cancer, including two adenocarcinomas and four in situ carcinomas, while none of the control mice developed cancerous lesions. The expression of menin encoded by the Men1 gene was found to be drastically reduced in all carcinomas, and partial LOH of the wild-type Men1 allele was detected in three of the five analysed lesions. Using immunostaining for the androgen receptor and p63, a basal epithelial cell marker, we demonstrated that the menin-negative prostate cancer cells did not display p63 expression and that the androgen receptor was expressed but more heterogeneous in these lesions. Furthermore, our data showed that the expression of the cyclin-dependent kinase inhibitor CDKN1B (p27), a Men1 target gene known to be inactivated during prostate cell tumorigenesis, was notably decreased in the prostate cancers that developed in the mutant mice.ConclusionOur work suggests the possible involvement of Men1 inactivation in the tumorigenesis of the prostate gland.

Deletion of the α-Arrestin Protein Txnip in Mice Promotes Adiposity and Adipogenesis While Preserving Insulin Sensitivity

OBJECTIVEThioredoxin interacting protein (Txnip), a regulator of cellular oxidative stress, is induced by hyperglycemia and inhibits glucose uptake into fat and muscle, suggesting a role for Txnip in type 2 diabetes pathogenesis. Here, we tested the hypothesis that Txnip-null (knockout) mice are protected from insulin resistance induced by a high-fat diet.RESEARCH DESIGN AND METHODSTxnip gene-deleted (knockout) mice and age-matched wild-type littermate control mice were maintained on a standard chow diet or subjected to 4 weeks of high-fat feeding. Mice were assessed for body composition, fat development, energy balance, and insulin responsiveness. Adipogenesis was measured from ex vivo fat preparations, and in mouse embryonic fibroblasts (MEFs) and 3T3-L1 preadipocytes after forced manipulation of Txnip expression.RESULTSTxnip knockout mice gained significantly more adipose mass than controls due to a primary increase in both calorie consumption and adipogenesis. Despite increased fat mass, Txnip knockout mice were markedly more insulin sensitive than controls, and augmented glucose transport was identified in both adipose and skeletal muscle. RNA interference gene-silenced preadipocytes and Txnip−/− MEFs were markedly adipogenic, whereas Txnip overexpression impaired adipocyte differentiation. As increased adipogenesis and insulin sensitivity suggested aspects of augmented peroxisome proliferator–activated receptor-γ (PPARγ) response, we investigated Txnip's regulation of PPARγ function; manipulation of Txnip expression directly regulated PPARγ expression and activity.CONCLUSIONSTxnip deletion promotes adiposity in the face of high-fat caloric excess; however, loss of this α-arrestin protein simultaneously enhances insulin responsiveness in fat and skeletal muscle, revealing Txnip as a novel mediator of insulin resistance and a regulator of adipogenesis.

Age-dependent endothelial nitric oxide synthase uncoupling in pulmonary arteries of endoglin heterozygous mice

Endoglin is a TGF-beta superfamily receptor critical for endothelial cell function. Mutations in this gene are associated with hereditary hemorrhagic telangiectasia type I (HHT1), and clinical signs of disease are generally more evident later in life. We previously showed that systemic vessels of adult Eng heterozygous (Eng(+/-)) mice exhibit increased vasorelaxation due to uncoupling of endothelial nitric oxide synthase (eNOS). We postulated that these changes may develop with age and evaluated pulmonary arteries from newborn and adult Eng(+/-) mice for eNOS-dependent, acetylcholine (ACh-induced) vasorelaxation, compared with that of age-matched littermate controls. While ACh-induced vasorelaxation was similar in all newborn mice, it was significantly increased in the adult Eng(+/-) vs. control vessels. The vasodilatory responses were inhibited by l-NAME suggesting eNOS dependence. eNOS uncoupling was observed in lung tissues of adult, but not newborn, heterozygous mice and was associated with increased production of reactive O(2) species (ROS) in adult Eng(+/-) vs. control lungs. Interestingly, ROS generation was higher in adult than newborn mice and so were the levels of NADPH oxidase 4 and SOD 1, 2, 3 isoforms. However, enzyme protein levels and NADPH activity were normal in adult Eng(+/-) lungs indicating that the developmental maturation of ROS generation and scavenging cannot account for the increased vasodilatation observed in adult Eng(+/-) mice. Our data suggest that eNOS-dependent H(2)O(2) generation in Eng(+/-) lungs accounts for the heightened pulmonary vasorelaxation. To the extent that these mice mimic human HHT1, age-associated pulmonary vascular eNOS uncoupling may explain the late childhood and adult onset of clinical lung manifestations.

A functional role for death receptor-3 in arthritis

TNF-like protein 1A (TL1A) ligation with death receptor-3 (DR3), a death domain containing member of the tumour necrosis factor receptor superfamily, activates multiple target cell responses. Published genetic and serological data implicate DR3 in rheumatoid arthritis, a chronic inflammatory joint disease. DR3’s function in rheumatoid arthritis has not been established. We question whether; DR3/TL1A activation triggers adverse joint pathology during inflammatory arthritis. We used two murine experimental arthritis models, antigen-induced arthritis (mAIA) and collagen-induced arthritis (mCIA), to study the histological progression of DR3 dependent joint pathology. Osteoclast differentiation assays were performed on both human and mouse macrophages. Osteoclast numbers were quantified in vivo and in vitro by tartrate resistant acid phosphatase staining. In the mAIA model all disease activity indices were significantly reduced in DR3-deficient mice (DR3ko) over age-matched DR3-sufficient wild-type littermate controls. Most striking was the fact that DR3ko were completely protected from the development of the focal bone erosions characteristically seen in mAIA. Add-back experiments with TL1A confirmed that bone pathology was a DR3 specific phenomenon. Our in vitro osteoclast differentiation assays revealed a potential mechanism. We observed heightened DR3-specific osteoclastogenesis in mouse and human macrophage cultures. Osteoclasts are unique in their ability to resorb bone. Finally, we tested the therapeutic efficacy of DR3 antagonism using a TL1A blocking antibody. We found that anti-TL1A significantly reduced the incidence and severity of mCIA. Indeed arthritis was virtually ameliorated at endpoint; all disease activity scores were significantly reduced in anti-TL1A treated mice. Our study reveals the DR3/TL1A axis as an important trigger for adverse joint pathology during inflammatory arthritis and an important regulator of bone turnover. We provide proof-of-concept data which identifies DR3/TL1A antagonism as a potential target to modulate pathology in bone disease and inflammatory arthritis.

Deficits in Aversive but not in Safe Taste Memory in the APPswe/PS1dE9 mice

Age-related changes in taste memory were evaluated in APPswe/PS1dE9 transgenic (Tg) mice and age-matched wild type littermate controls (Wt). These Tg mice produce increasing amounts of amyloid-beta in the brain with age, develop significant amounts of plaques by 9 months of age, and provide an opportunity to study the effects of Alzheimer's disease-like amyloidosis on different aspects of taste memory. In groups of mice ranging from 15-16 months of age, the neophobic response and its attenuation were similar in Tg and Wt mice. However, conditioned taste aversion (CTA), which resulted from the association between a new taste and an artificially induced gastric malaise, was significantly reduced in the 15-16 month old Tg mice compared to the Wt mice, but not in the 3-4 or 7-8 month old mice. The extinction of CTA was normal in 3-4 month old Tg mice, but occurred more rapidly in the 7-8 and 15-16 months old Tg mice than in the age-matched controls. These results provide evidence of differences in the neuronal systems involved in the attenuation of neophobia and CTA and suggest that the progressive amyloidosis that takes place in APPswe/PS1dE9 mice selectively affects the aversion component of taste memory.

Retinoid metabolism and ALDH1A2 (RALDH2) expression are altered in the transgenic adenocarcinoma mouse prostate model

Retinoids, which include vitamin A (retinol) and metabolites such as retinoic acid, can inhibit tumor growth and reverse carcinogenesis in animal models of prostate cancer. We analyzed retinoid signaling and metabolism in the TRAMP (transgenic adenocarcinoma mouse prostate) model. We detected increased retinol and retinyl esters in prostates pooled from 24 to 36 week TRAMP transgenic positive mice compared to nontransgenic littermates by HPLC. We used quantitative RT-PCR to measure transcripts for genes involved in retinoid signaling and metabolism, including ALDH1A1, ALDH1A2, ALDH1A3, CYP26A1, LRAT, and RARβ 2, in prostate tissue from TRAMP positive (+) and age-matched littermate control mice ranging from 18 to 36 weeks. Transcript levels of ALDH1A1, a putative stem cell marker, were decreased in ventral and lateral lobes of prostates from TRAMP mice compared to age-matched, nontransgenic mice. ALDH1A2 (RALDH2) mRNA levels in dorsal and anterior lobes of TRAMP+ mice were lower than in age-matched (24 week) nontransgenic mice. We detected lower RARβ 2 mRNA levels in dorsal prostate lobes of 36 week TRAMP mice relative to nontransgenic mice. We detected high levels of ALDH1A2 protein in the cytoplasm and nucleus in nontransgenic murine prostate paraffin sections, and lower ALDH1A2 protein levels in all prostate lobes of TRAMP mice compared to nontransgenic mice by immunohistochemistry. We also detected much lower cytoplasmic ALDH1A2 protein levels in all human prostate cancer paraffin sections stained (19 total) relative to normal human prostate tissue on the same sections. Our data indicate that this reduction in ALDH1A2 protein is an early event in human prostate cancer.

Inhibitory Inputs to Hippocampal Interneurons Are Reorganized in Lis1 Mutant Mice

Epilepsy and brain malformation are commonly associated with excessive synaptic excitation and decreased synaptic inhibition of principal neurons. However, few studies have examined the state of synaptic inhibition of interneurons in an epileptic, malformed brain. We analyzed inhibitory inputs, mediated by gamma-aminobutyric acid (GABA), to hippocampal interneurons in a mouse model of type 1 lissencephaly, a neurological disorder linked with severe seizures and brain malformation. In the disorganized hippocampal area CA1 of Lis1(+/-) mice, we initially observed a selective displacement of fast-spiking, parvalbumin-positive basket-type interneurons from stratum oriens (SO) locations to s. radiatum and s. lacunosum-moleculare (R/LM). Next, we recorded spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs) onto visually identified interneurons located in SO or R/LM of Lis1(+/-) mice and age-matched littermate controls. We observed significant, layer-specific reorganizations in GABAergic inhibition of interneurons in Lis1 mutant mice. Spontaneous IPSC frequency onto SO interneurons was significantly increased in hippocampal slices from Lis1(+/-) mice, whereas mIPSC mean amplitude onto these interneurons was significantly decreased. In addition, the weighted decay times of sIPSCs and mIPSCs were significantly increased in R/LM interneurons. Taken together, these findings illustrate the extensive redistribution and reorganization of inhibitory connections between interneurons that can take place in a malformed brain.

A Novel Rat Model with Obesity-Associated Retinal Degeneration

A strong association between retinal degeneration and obesity has been shown in humans. However, the molecular basis of increased risk for retinal degeneration in obesity is unknown. Thus, an animal model with obesity and retinal degeneration would greatly aid the understanding of obesity-associated retinal degeneration. The retinal abnormalities in a novel rat model (WNIN-Ob) with spontaneously developed obesity are described. Histologic and immunohistochemical examination were performed on retinal sections of 2- to 12-month-old WNIN-Ob rats, and findings were compared with those of lean littermate controls. RNA from retinas of 12-month-old WNIN-Ob and lean littermate rats was used for microarray and qRT-PCR analysis. The WNIN-Ob rats developed severe obesity, with an onset at approximately 35 days. Evaluation of retinal morphology in 2- to 12-month-old WNIN-Ob and age-matched lean littermate controls revealed progressive retinal degeneration, with an onset between 4 to 6 months of age. Immunohistochemical analysis with anti-rhodopsin, anti-cone opsin, and PSD-95 antibodies further confirmed retinal degeneration, particularly rod cell loss and thinner outer plexiform layer, in the obese rat retina. Gene expression by microarray analysis and qRT-PCR established activation of stress response, tissue remodeling, impaired phototransduction, and photoreceptor degeneration in WNIN-Ob rat retina. WNIN-Ob rats develop increased stress in retinal tissue and progressive retinal degeneration after the onset of severe obesity. The WNIN-Ob rat is the first rat model to develop retinal degeneration after the onset of obesity. This novel rat model may be a valuable tool for investigating retinal degeneration associated with obesity in humans.

Cutting Edge: CD47 Controls the In Vivo Proliferation and Homeostasis of Peripheral CD4+CD25+Foxp3+ Regulatory T Cells That Express CD103

Peripheral CD103(+)Foxp3(+) regulatory T cells (Tregs) can develop both from conventional naive T cells upon cognate Ag delivery under tolerogenic conditions and from thymic-derived, expanded/differentiated natural Tregs. We here show that CD47 expression, a marker of self on hematopoietic cells, selectively regulated CD103(+)Foxp3(+) Treg homeostasis at the steady state. First, the proportion of effector/memory-like (CD44(high)CD62L(low)) CD103(+)Foxp3(+) Tregs rapidly augmented with age in CD47-deficient mice (CD47(-/-)) as compared with age-matched control littermates. Yet, the percentage of quiescent (CD44(low)CD62L(high)) CD103(-)Foxp3(+) Tregs remained stable. Second, the increased proliferation rate (BrdU incorporation) observed within the CD47(-/-)Foxp3(+) Treg subpopulation was restricted to those Tregs expressing CD103. Third, CD47(-/-) Tregs maintained a normal suppressive function in vitro and in vivo and their increased proportion in old mice led to a decline of Ag-specific T cell responses. Thus, sustained CD47 expression throughout life is critical to avoid an excessive expansion of CD103(+) Tregs that may overwhelmingly inhibit Ag-specific T cell responses.

Mechanical and Pharmacological Approaches to Investigate the Pathogenesis of Marfan Syndrome in the Abdominal Aorta

Background: Occurrence of disease complications in the abdominal aorta in Marfan syndrome, a connective tissue disorder caused by mutations in the gene encoding fibrillin-1, is relatively rare. We hypothesized that Marfan syndrome could affect the structure, vasomotor function and mechanical property of the abdominal aorta. Methods and Results: Abdominal aorta from mice at 3, 6, 9 and 12 months of age, heterozygous for the Fbn1 allele encoding a cysteine substitution (Fbn1^C1039G/+, Marfan mice, n = 50), were compared with those from age-matched control littermates (n = 50). Marfan abdominal aorta demonstrated pronounced elastic fiber degradation and disorganization, concomitant with an increased aortic stiffness during aging. In the isometric force measurement, vasoconstriction in response to membrane depolarization or phenylephrine stimulation was similar in both Marfan and control abdominal aorta. However, Marfan abdominal aorta was less sensitive to the inhibition of the phenylephrine-induced contraction by indomethacin and SQ-29548, during which the release of thromboxane A₂ was one half of that of the controls. Nevertheless, the protein expression of cyclooxygenase-1 and cyclooxygenase-2 detected by Western immunoblotting was not different between the 2 strains. Conclusions: We demonstrated that Marfan syndrome affected abdominal aorta with respect to matrix elastic fiber organization, aortic stiffness and release of thromboxane A₂.