A call to reform the taxonomy of human disease.

A coordinated effort to incorporate advances in the understanding of the molecular and genomic variations in common diseases, such as hypertension, into their diagnosis and treatment could transform drug development and medicine.

Site-specific antiatherogenic effect of the antioxidant ebselen in the diabetic apolipoprotein E-deficient mouse.

OBJECTIVE: Recently we showed that lack of the antioxidant enzyme glutathione peroxidase-1 (GPx1) accelerates atherosclerosis and upregulates proatherogenic pathways in diabetic apoE/GPx1-deficient double-knockout mice, thereby establishing GPx1 as an important therapeutic target. In vivo studies now investigate ebselen, a seleno-organic GPx1-mimetic, for its potential to reduce diabetes-associated atherosclerosis. METHODS AND RESULTS: Lesions were significantly increased in diabetic apoE(-/-) aortas (P<0.001) compared with nondiabetic controls after 20 weeks of diabetes. Ebselen-gavage significantly reduced total aortic lesions (P<0.001), with significant regional reductions in the arch (P<0.001), thoracic (P<0.001), and abdominal regions (P<0.05), but not within the aortic sinus of diabetic apoE(-/-) mice. These reductions were accompanied by significantly lower nitrotyrosine and Nox2 levels, reduced proatherogenic cellularity (macrophages and SMCs), and reduced expression of the proatherogenic mediator RAGE. Within the aortic sinus, ebselen reduced nitrotyrosine, Nox2, and VEGF levels but had no effect on RAGE. Studies in HAECs show that ebselen abrogates H(2)O(2)-induced increases in P-IKK, P-JNK, TNF-alpha, and Nox2. CONCLUSIONS: Ebselen reduces atherosclerotic lesions in most regions of diabetic apoE(-/-) aorta, except within the aortic sinus, suggesting its effectiveness as a potential antiatherogenic therapy in diabetic-macrovascular disease. Ebselen may elicit its effect via modulation of transcription factors such as NF-kappaB and AP-1.

Lack of the antioxidant enzyme glutathione peroxidase-1 accelerates atherosclerosis in diabetic apolipoprotein E-deficient mice.

BACKGROUND: Recent clinical studies have suggested a major protective role for the antioxidant enzyme glutathione peroxidase-1 (GPx1) in diabetes-associated atherosclerosis. We induced diabetes in mice deficient for both GPx1 and apolipoprotein E (ApoE) to determine whether this is merely an association or whether GPx1 has a direct effect on diabetes-associated atherosclerosis. METHODS AND RESULTS: ApoE-deficient (ApoE-/-) and ApoE/GPx1 double-knockout (ApoE-/- GPx1-/-) mice were made diabetic with streptozotocin and aortic lesion formation, and atherogenic pathways were assessed after 10 and 20 weeks of diabetes. Aortic proinflammatory and profibrotic markers were determined by both quantitative reverse-transcription polymerase chain reaction analysis after 10 weeks of diabetes and immunohistochemical analysis after 10 and 20 weeks of diabetes. Sham-injected nondiabetic counterparts served as controls. Atherosclerotic lesions within the aortic sinus region, as well as arch, thoracic, and abdominal lesions, were significantly increased in diabetic ApoE-/- GPx1-/- aortas compared with diabetic ApoE-/- aortas. This increase was accompanied by increased macrophages, alpha-smooth muscle actin, receptors for advanced glycation end products, and various proinflammatory (vascular cell adhesion molecule-1) and profibrotic (vascular endothelial growth factor and connective tissue growth factor) markers. Quantitative reverse-transcription polymerase chain reaction analysis showed increased expression of receptors for advanced glycation end products (RAGE), vascular cell adhesion molecule-1, vascular endothelial growth factor, and connective tissue growth factor. Nitrotyrosine levels were significantly increased in diabetic ApoE-/- GPx1-/- mouse aortas. These findings were observed despite upregulation of other antioxidants. CONCLUSIONS: Lack of functional GPx1 accelerates diabetes-associated atherosclerosis via upregulation of proinflammatory and profibrotic pathways in ApoE-/- mice. Our study provides evidence of a protective role for GPx1 and establishes GPx1 as an important antiatherogenic therapeutic target in patients with or at risk of diabetic macrovascular disease.

Targeting of the ETS factor GABPalpha disrupts neuromuscular junction synaptic function.

The GA-binding protein (GABP) transcription factor has been shown in vitro to regulate the expression of the neuromuscular proteins utrophin, acetylcholine esterase, and acetylcholine receptor subunits delta and epsilon through the N-box promoter motif (5'-CCGGAA-3'), but its in vivo function remains unknown. A single point mutation within the N-box of the gene encoding the acetylcholine receptor epsilon subunit has been identified in several patients suffering from postsynaptic congenital myasthenic syndrome, implicating the GA-binding protein in neuromuscular function and disease. Since conventional gene targeting results in an embryonic-lethal phenotype, we used conditional targeting to investigate the role of GABPalpha in neuromuscular junction and skeletal muscle development. The diaphragm and soleus muscles from mutant mice display alterations in morphology and distribution of acetylcholine receptor clusters at the neuromuscular junction and neurotransmission properties consistent with reduced receptor function. Furthermore, we confirmed decreased expression of the acetylcholine receptor epsilon subunit and increased expression of the gamma subunit in skeletal muscle tissues. Therefore, the GABP transcription factor aids in the structural formation and function of neuromuscular junctions by regulating the expression of postsynaptic genes.

Retinal light damage: structural and functional effects of the antioxidant glutathione peroxidase-1.

PURPOSE: The role of the antioxidant enzyme glutathione peroxidase-1 (GPx1) in protecting the retina against photo-oxidative damage was investigated in GPx1-deficient and wild-type mice. METHOD: Albino GPx1-deficient and age-matched wild-type mice were examined. Baseline electroretinograms (ERGs) were recorded. Thereafter, mice were exposed to intense light for 12 hours. After a 24-hour recovery in darkness, post-light-insult ERGs were recorded and compared with baseline. Structural effects of light insult were evaluated by retinal histology. Antioxidant expression was investigated by quantitative reverse transcription-PCR (qRT-PCR). RESULTS: Light insult significantly affected ERG responses, with reduced a- and b-wave amplitudes. Structurally, photoreceptor layers were predominantly affected. As expected, GPx1 expression was negligible in GPx1-deficient mice but was upregulated in wild-type mice in response to light insult. Similarly, hemeoxygenase-1 and thioredoxin-1 expression increased significantly in wild-type retinas after light exposure. Catalase, GPx isoforms (GPx2 to -4), peroxiredoxin-6, glutaredoxin-1, and thioredoxin-2 expression was unaffected by GPx1 deficiency and light insult, whereas significant increases in glutaredoxin-2 occurred in non-light-exposed (baseline) GPx1-deficient retinas. Compared with baseline wild-type retinas, lipid peroxidation (TBARS assay), an indicator of oxidative stress, was elevated in baseline GPx1-deficient retinas. Unexpectedly, the light insult induced diminution of retinal function, in terms of ERG amplitude, and structural damage was significantly greater in wild-type than in with GPx1-deficient retinas. CONCLUSIONS: The data showing increased oxidative damage in baseline GPx-deficient retina give rise to the hypothesis that increased oxidative stress provides a "preconditioning" environment in which protective mechanisms paradoxically render GPx1-deficient retinas less vulnerable to light-induced oxidative damage. This study identified glutaredoxin-2 as a potential candidate.

Lack of the antioxidant glutathione peroxidase-1 does not increase atherosclerosis in C57BL/J6 mice fed a high-fat diet.

Oxidative stress is thought to contribute to the initiation and progression of atherosclerosis. As glutathione peroxidase-1 (Gpx1) is an antioxidant enzyme that detoxifies lipid hydroperoxides, we tested the impact of Gpx1 deficiency on atherosclerotic processes and antioxidant enzyme expression in mice fed a high-fat diet (HFD). After 12 weeks of HFD, atherosclerotic lesions at the aortic sinus were of similar size in control and Gpx1-deficient mice. However, after 20 weeks of HFD, lesion size increased further in control but not in Gpx1-deficient mice, even though plasma and aortic wall markers of oxidative damage did not differ between groups. In control mice, the expression of Gpx1 increased and that of Gpx3 decreased at the aortic sinus after 20 weeks of HFD, with no change in the expression of Gpx2, Gpx4, catalase, peroxiredoxin-6, glutaredoxin-1 and -2, or thioredoxin-1 and -2. By comparison, in Gpx1-deficient mice, the expression of antioxidant genes was unaltered except for a decrease in glutaredoxin-1 and an increase in glutaredoxin-2. These changes were associated with increased expression of the proinflammatory marker monocyte chemoattractant protein-1 in control mice but not in Gpx1-deficient mice. In summary, a specific deficiency in Gpx1 was not accompanied by an increase in markers of oxidative damage or increased atherosclerosis in a murine model of HFD-induced atherogenesis.

Kidney expression of glutathione peroxidase-1 is not protective against streptozotocin-induced diabetic nephropathy.

In many diseases, including progressive renal disorders, tissue injury and pathological intracellular signaling events are dependent on oxidative stress. Glutathione peroxidase-1 (Gpx1) is an antioxidant enzyme that is highly expressed in the kidney and removes peroxides and peroxynitrite that can cause renal damage. Therefore, we examined whether this abundant renal antioxidant enzyme limits renal damage during the development of type 1 diabetic nephropathy. Wild-type (Gpx1+/+) and deficient (Gpx1-/-) mice were made diabetic by intraperitoneal injection of streptozotocin (100 mg/kg) on 2 consecutive days. Diabetic Gpx1+/+ and -/- mice with equivalent blood glucose levels (23 +/- 4 mM) were selected and examined after 4 mo of diabetes. Compared with normal mice, diabetic Gpx1+/+ and -/- mice had a two- to threefold increase in urine albumin excretion at 2 and 4 mo of diabetes. At 4 mo, diabetic Gpx1+/+ and -/- mice had equivalent levels of oxidative renal injury (increased kidney reactive oxygen species, kidney lipid peroxidation, urine isoprostanes, kidney deposition of advanced glycoxidation, and nitrosylation end products) and a similar degree of glomerular damage (hypertrophy, hypercellularity, sclerosis), tubular injury (apoptosis and vimentin expression), and renal fibrosis (myofibroblasts, collagen, TGF-beta excretion). A lack of Gpx1 was not compensated for by increased levels of catalase or other Gpx isoforms in diabetic kidneys. Contrary to expectations, this study showed that the high level of Gpx1 expressed in the kidney is not protective against the development of renal oxidative stress and nephropathy in a model of type 1 diabetes.

Identification and expression analysis of alternative transcripts of the mouse GA-binding protein (Gabp) subunits alpha and beta1.

The erythroblast transformation specific (ETS) transcription factor GA-binding protein (Gabp) is widely expressed and acts on a diverse range of target genes, including nuclear-encoded mitochondrial proteins and neuromuscular-specific genes. The GABPalpha subunit contains an ETS DNA binding domain and the beta subunit contains a nuclear localization signal (NLS) and transactivation domain. Here, we show coincident expression of Gabpalpha and beta1 throughout mouse embryogenesis, consistent with the gene products functioning in a complex. We have also identified 2 alternatively spliced, tissue-specific exons 1 (5' untranslated regions) of mouse Gabpalpha and 4 alternative 3' polyadenylation signals that, in combination, result in 12 transcripts for Gabpalpha. These alternative transcripts are suggested to have altered stability, subcellular localization and/or translation efficiency. Further, we identified nine differentially expressed splice variants of mouse Gabpbeta1 that encode beta protein forms lacking functional domains, suggesting a dominant negative function. Together, alternative transcripts of Gabpalpha and beta1 provide a mechanism for tissue-specific regulation of Gabp activity.

Elf5 is essential for early embryogenesis and mammary gland development during pregnancy and lactation.

Elf5 is an epithelial-specific ETS factor. Embryos with a null mutation in the Elf5 gene died before embryonic day 7.5, indicating that Elf5 is essential during mouse embryogenesis. Elf5 is also required for proliferation and differentiation of mouse mammary alveolar epithelial cells during pregnancy and lactation. The loss of one functional allele led to complete developmental arrest of the mammary gland in pregnant Elf5 heterozygous mice. A quantitative mRNA expression study and Western blot analysis revealed that decreased expression of Elf5 correlated with the downregulation of milk proteins in Elf5(+/-) mammary glands. Mammary gland transplants into Rag(-/-) mice demonstrated that Elf5(+/-) mammary alveolar buds failed to develop in an Elf5(+/+) mammary fat pad during pregnancy, demonstrating an epithelial cell autonomous defect. Elf5 expression was reduced in Prolactin receptor (Prlr) heterozygous mammary glands, which phenocopy Elf5(+/-) glands, suggesting that Elf5 and Prlr are in the same pathway. Our data demonstrate that Elf5 is essential for developmental processes in the embryo and in the mammary gland during pregnancy.

Neonatal calyceal dilation and renal fibrosis resulting from loss of Adamts-1 in mouse kidney is due to a developmental dysgenesis.

BACKGROUND: A disintegrin and metalloproteinase with thrombospondin motifs 1, Adamts-1, is important for the development and function of the kidney. Mice lacking this protein present with renal lesions comprising enlarged calyces, and reduced cortex and medulla layers. Our current findings are consistent with the defect occurring due to a developmental dysgenesis. METHODS: We generated Adamts-1 null mice, and further investigated their kidney phenotype in a time course study ranging from E18.5 to 12 months of age. Immunohistochemistry was used to assess the localization of type IV collagen, TGF-beta and F4/80-positive macrophages in the kidneys of Adamts-1 null mice compared to wild-type control animals. The expression of Adamts-1 mRNA was determined in metanephric kidney explants by in situ hybridization. RESULTS: Adamts-1 null mice have a gross kidney defect. At day 18.5 of gestation, the Adamts-1 null kidney has a normal appearance but at birth when the kidney begins to function, the defect becomes evident. During development of the kidney Adamts-1 expression was specifically detected in the developing loops of Henle, as well as in the proximal and distal convoluted tubules. Expression was not detected in the ureter, ureteric bud or its derivatives as had been previously suggested. At 6 months and 1 year of age, the Adamts-1 null mice displayed interstitial fibrosis in the cortical and medullary regions of the kidney. At 1 year of age, the Adamts-1 null mice displayed mild interstitial matrix expansion associated with increased collagen type IV expression, without apparent tubular dilatation, compared to wild-type animals. Immunohistochemical analysis demonstrated TGF-beta protein localized to infiltrating macrophages and glomeruli of Adamts-1 null mice. CONCLUSIONS: Adamts-1 is required for the normal development of the kidney. The defect observed in its absence results from a dysgenic malformation affecting the medulla that becomes apparent at birth, once the kidneys start to function.

Tissue-specific overexpression of the HSA21 gene GABPalpha: implications for DS.

The ETS transcription factor GABPalpha is encoded by a gene on HSA21 and interacts with an ankyrin repeat-containing beta subunit to form the GABP complex. GABP regulates expression of genes involved in mitochondrial respiration and neuromuscular signalling. When GABPalpha mRNA is overexpressed in human DS fibroblast cell lines, or by tranfection in NIH3T3 cells, no increase in protein level is detected. However, increased Gabpalpha gene dosage in the Ts65Dn segmental trisomy mouse model of DS (DS) results in elevated Gabpalpha protein levels in brain and skeletal muscle only. These findings suggest that GABPalpha protein levels are tightly regulated in a tissue-specific manner, and consequently GABP may play a role in DS pathologies in tissues where GABPalpha protein levels are elevated.

The ETS transcription factor GABPalpha is essential for early embryogenesis.

The ETS transcription factor complex GABP consists of the GABPalpha protein, containing an ETS DNA binding domain, and an unrelated GABPbeta protein, containing a transactivation domain and nuclear localization signal. GABP has been shown in vitro to regulate the expression of nuclear genes involved in mitochondrial respiration and neuromuscular signaling. We investigated the in vivo function of GABP by generating a null mutation in the murine Gabpalpha gene. Embryos homozygous for the null Gabpalpha allele die prior to implantation, consistent with the broad expression of Gabpalpha throughout embryogenesis and in embryonic stem cells. Gabpalpha(+/-) mice demonstrated no detectable phenotype and unaltered protein levels in the panel of tissues examined. This indicates that Gabpalpha protein levels are tightly regulated to protect cells from the effects of loss of Gabp complex function. These results show that Gabpalpha function is essential and is not compensated for by other ETS transcription factors in the mouse, and they are consistent with a specific requirement for Gabp expression for the maintenance of target genes involved in essential mitochondrial cellular functions during early cleavage events of the embryo.

Fibroblasts derived from Gpx1 knockout mice display senescent-like features and are susceptible to H2O2-mediated cell death.

The Free Radical Theory of Aging proposes that reactive oxygen species (ROS) contribute to the pathophysiology of aging. Our previous data highlight the importance of antioxidant enzymes, superoxide dismutase 1 (Sod1) and glutathione peroxidase 1 (Gpx1), in regulating this process. Previously, we demonstrated that a perturbation in the Sod1-to-Gpx1 ratio, as a consequence of Sod1 overexpression, leads to senescence-like changes. We proposed that this was mediated via the Sod1 dismutation product H2O2, because H2O2 induced similar changes in control cells. However, it has been suggested that H2O2 production, via Sod1 dismutation, is rate-limited by the availability of the substrate O2*-, and therefore age-related changes may occur as a result of other functions of Sod1. In this study, we test this notion in fibroblasts derived from Gpx1 null mutant mice (Gpx1-/-) that have elevated H2O2 as a consequence of the lack of its removal by Gpx1. We demonstrate senescence-like changes in Gpx1-/- fibroblasts that include (1) reduced proliferative capacity, DNA synthesis, and responsiveness to EGF and serum; (2) elevated levels of Cip1; (3) increased NF-kappaB activation; and (4) morphological features of senescent cells. Gpx1-/- fibroblasts also demonstrate a dose-dependent susceptibility to H2O2-induced apoptosis. Our findings suggest that Gpx1 is protective against both ROS-mediated senescence-like changes and oxidant-mediated cell death.

An imbalance in antioxidant defense affects cellular function: the pathophysiological consequences of a reduction in antioxidant defense in the glutathione peroxidase-1 (Gpx1) knockout mouse.

Aerobic cells are subjected to damaging reactive oxygen species (ROS) as a consequence of oxidative metabolism and/or exposure to environmental toxins. Antioxidants limit this damage, yet peroxidative events occur when oxidant stress increases. This arises due to increased radical formation or decreased antioxidative defenses. The two-step enzymatic antioxidant pathway limits damage to important biomolecules by neutralising superoxides to water. However, an imbalance in this pathway (increased first-step antioxidants relative to second-step antioxidants) has been proposed as etiological in numerous pathologies. This review presents evidence that a shift in favor of hydrogen peroxide and/or lipid peroxides has pathophysiological consequences. The involvement of antioxidant genes in the regulation of redox status, and ultimately cellular homeostasis, is explored in murine transgenic and knockout models. The investigations of Sod1 transgenic cell-lines and mice, as well as Gpx1 knockout mice (both models favor H(2)O(2) accumulation), are presented. Although in most instances accumulation of H(2)O(2) affects cellular function and leads to exacerbated pathology, this is not always the case. This review highlights those instances where, for example, increased Sod1 levels are beneficial, and indicates a role for superoxide radicals in pathogenesis. Studies of Gpx1 knockout mice (an important second-step antioxidant) lead us to conclude that Gpx1 functions as the primary protection against acute oxidative stress, particularly in neuropathological situations such as stroke and cold-induced head trauma, where high levels of ROS occur during reperfusion or in response to injury. In summary, these studies clearly highlight the importance of limiting ROS-induced cellular damage by maintaining a balanced enzymatic antioxidant pathway.

Positive and negative regulation of the gamma-secretase activity by nicastrin in a murine model.

Nicastrin is a component of the gamma-secretase complex that has been shown to adhere to presenilin-1 (PS1), Notch, and APP. Here we demonstrate that Nicastrin-deficient mice showed a phenotype that is indistinguishable from PS1/PS2 double knock-out mice, whereas heterozygotes were healthy and viable. Fibroblasts derived from Nicastrin-deficient embryos were unable to generate amyloid beta-peptide and failed to release the intracellular domain of APP- or Notch1-Gal4-VP16 fusion proteins. Additionally, C- and N-terminal fragments of PS1 and the C-terminal fragments of PS2 were not detectable in Nicastrin-null fibroblasts, whereas full-length PS1 accumulated in null fibroblasts, indicating that Nicastrin is required for the endoproteolytic processing of presenilins. Interestingly, cells derived from Nicastrin heterozygotes produced relatively higher levels of amyloid beta-peptide whether the source was endogenous mouse or transfected human APP. These data demonstrate that Nicastrin is essential for the gamma-secretase cleavage of APP and Notch in mammalian cells and that Nicastrin has both positive and negative functions in the regulation of gamma-secretase activity.

Glutathione peroxidase-1 contributes to the neuroprotection seen in the superoxide dismutase-1 transgenic mouse in response to ischemia/reperfusion injury.

The authors hypothesized that glutathione peroxidase-1 (Gpx-1) contributes to the neuroprotection seen in the superoxide dismutase-1 transgenic (Sod-1 tg) mouse. To investigate this hypothesis, they crossed the Gpx-1 -/- mouse with the Sod-1 tg and subjected the cross to a mouse model of ischemia/reperfusion. Two hours of focal cerebral ischemia followed by 24 hours of reperfusion was induced via intraluminal suture. The Sod-1 tg/Gpx-1 -/- cross exhibited no neuroprotection when infarct volume was measured; indeed, infarct volume increased in the Sod-1 tg/Gpx-1 -/- cross compared with the wild-type mouse. Our results suggest that Gpx-1 plays an important regulatory role in the protection of neural cells in response to ischemia/reperfusion injury.