Imran N. Mungrue

Cardiomyocyte overexpression of iNOS in mice results in peroxynitrite generation, heart block, and sudden death

Increased inducible nitric oxide synthase (iNOS) expression is a component of the immune response and has been demonstrated in cardiomyocytes in septic shock, myocarditis, transplant rejection, ischemia, and dilated cardiomyopathy. To explore whether the consequences of such expression are adaptive or pathogenic, we have generated a transgenic mouse model conditionally targeting the expression of a human iNOS cDNA to myocardium. Chronic cardiac-specific upregulation of iNOS in transgenic mice led to increased production of peroxynitrite. This was associated with a mild inflammatory cell infiltrate, cardiac fibrosis, hypertrophy, and dilatation. While iNOS-overexpressing mice infrequently developed overt heart failure, they displayed a high incidence of sudden cardiac death due to bradyarrhythmia. This dramatic cardiac phenotype was rescued by specific attenuation of transgene activity. These data implicate cardiomyocyte iNOS overexpression as sufficient to cause cardiomyopathy, bradyarrhythmia, and sudden cardiac death.

THIOREDOXIN-INTERACTING PROTEIN: A CRITICAL LINK BETWEEN GLUCOSE TOXICITY AND BETA CELL APOPTOSIS

OBJECTIVE—In diabetes, glucose toxicity affects different organ systems, including pancreatic islets where it leads to β-cell apoptosis, but the mechanisms are not fully understood. Recently, we identified thioredoxin-interacting protein (TXNIP) as a proapoptotic β-cell factor that is induced by glucose, raising the possibility that TXNIP may play a role in β-cell glucose toxicity. RESEARCH DESIGN AND METHODS—To assess the effects of glucose on TXNIP expression and apoptosis and define the role of TXNIP, we used INS-1 β-cells; primary mouse islets; obese, diabetic BTBR.ob mice; and a unique mouse model of TXNIP deficiency (HcB-19) that harbors a natural nonsense mutation in the TXNIP gene. RESULTS—Incubation of INS-1 cells at 25 mmol/l glucose for 24 h led to an 18-fold increase in TXNIP protein, as assessed by immunoblotting. This was accompanied by increased apoptosis, as demonstrated by a 12-fold induction of cleaved caspase-3. Overexpression of TXNIP revealed that TXNIP induces the intrinsic mitochondrial pathway of apoptosis. Islets of diabetic BTBR.ob mice also demonstrated increased TXNIP and apoptosis as did isolated wild-type islets incubated at high glucose. In contrast, TXNIP-deficient HcB-19 islets were protected against glucose-induced apoptosis as measured by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and caspase-3, indicating that TXNIP is a required causal link between glucose toxicity and β-cell death. CONCLUSIONS—These findings shed new light onto the molecular mechanisms of β-cell glucose toxicity and apoptosis, demonstrate that TXNIP induction plays a critical role in this vicious cycle, and suggest that inhibition of TXNIP may represent a novel approach to reduce glucotoxic β-cell loss.

Thioredoxin-interacting protein deficiency induces Akt/Bcl-xL signaling and pancreatic beta-cell mass and protects against diabetes

Pancreatic beta‐cell loss through apoptosis represents a key factor in the pathogenesis of diabetes;however, no effective approaches to block this process and preserve endogenous beta‐cell mass are currently available. To study the role of thioredoxin‐interacting protein (TXNIP), a proapoptotic beta‐cell factor we recently identified, we used HcB‐19 (TXNIP nonsense mutation) and beta‐cell‐specific TXNIP knockout (bTKO) mice. Interestingly, HcB‐19 mice demonstrate increased adiposity, but have lower blood glucose levels and increased pancreatic beta‐cell mass (as assessed by morphometry). Moreover, HcB‐19 mice are resistant to streptozotocin‐induced diabetes. When intercrossed with obese, insulin‐resistant, and diabetic mice, double‐mutant BTBRlepob/obtxniphcb/hcb are even more obese, but are protected against diabetes and beta‐cell apoptosis, resulting in a 3‐fold increase in beta‐cell mass. Beta‐cell‐specific TXNIP deletion also enhanced beta‐cell mass (P< 0.005) and protected against diabetes, and terminal deoxynucleotidyl transferase‐mediated nick end labeling (TUNEL) revealed a ~50‐fold reduction in beta‐cell apoptosis in streptozotocin‐treated bTKO mice. We further discovered that TXNIP deficiency induces Akt/Bcl‐xL signaling and inhibits mitochondrial beta‐cell death, suggesting that these mechanisms may mediate the beta‐cell protective effects of TXNIP deficiency. These results suggest that lowering beta‐cell TXNIP expression could serve as a novel strategy for the treatment of type 1 and type 2 diabetes by promoting endogenous beta‐cell survival.—Chen, J., Hui, S. T., Couto, F. M., Mungrue, I. N., Davis, D. B., Attie, A. D., Lusis, A. J., Davis, R. A., Shalev, A. Thioredoxin‐interacting protein deficiency induces Akt/ Bcl‐xL signaling and pancreatic beta‐cell mass and protects against diabetes. FASEB J. 22, 3581–3594 (2008)

CHAC1/MGC4504 Is a Novel Proapoptotic Component of the Unfolded Protein Response, Downstream of the ATF4-ATF3-CHOP Cascade

To understand pathways mediating the inflammatory responses of human aortic endothelial cells to oxidized phospholipids, we previously used a combination of genetics and genomics to model a coexpression network encompassing >1000 genes. CHAC1 (cation transport regulator-like protein 1), a novel gene regulated by ox-PAPC (oxidized 1-palmitoyl-2-arachidonyl-sn-3-glycero-phosphorylcholine), was identified in a co-regulated group of genes enriched for components of the ATF4 (activating transcription factor 4) arm of the unfolded protein response pathway. Herein, we characterize the role of CHAC1 and validate the network model. We first define the activation of CHAC1 mRNA by chemical unfolded protein response-inducers, but not other cell stressors. We then define activation of CHAC1 by the ATF4-ATF3-CHOP (C/EBP homologous protein), and not parallel XBP1 (X box-binding protein 1) or ATF6 pathways, using siRNA and/or overexpression plasmids. To examine the subset of genes downstream of CHAC1, we used expression microarray analysis to identify a list of 227 differentially regulated genes. We validated the activation of TNFRSF6B (tumor necrosis factor receptor superfamily, member 6b), a FASL decoy receptor, in cells treated with CHAC1 small interfering RNA. Finally, we showed that CHAC1 overexpression enhanced apoptosis, while CHAC1 small interfering RNA suppressed apoptosis, as determined by TUNEL, PARP (poly(ADP-ribose) polymerase) cleavage, and AIF (apoptosis-inducing factor) nuclear translocation.

nNOS at a glance: implications for brain and brawn

Nitric oxide (NO), formed enzymatically from L-arginine, functions as an endogenous signaling molecule in numerous organs and tissues throughout the animal and plant kingdoms. The first NO synthase (NOS) was isolated from mammalian brain and named neuronal NOS (nNOS, aka: NOS1) owing to its

Network for Activation of Human Endothelial Cells by Oxidized Phospholipids: A Critical Role of Heme Oxygenase 1

Rationale: Oxidized palmitoyl arachidonyl phosphatidylcholine (Ox-PAPC) accumulates in atherosclerotic lesions, is proatherogenic, and influences the expression of more than 1000 genes in endothelial cells. Objective: To elucidate the major pathways involved in Ox-PAPC action, we conducted a systems analysis of endothelial cell gene expression after exposure to Ox-PAPC. Methods and Results: We used the variable responses of primary endothelial cells from 149 individuals exposed to Ox-PAPC to construct a network that consisted of 11 groups of genes, or modules. Modules were enriched for a broad range of Gene Ontology pathways, some of which have not been identified previously as major Ox-PAPC targets. Further validating our method of network construction, modules were consistent with relationships established by cell biology studies of Ox-PAPC effects on endothelial cells. This network provides novel hypotheses about molecular interactions, as well as candidate molecular regulators of inflammation and atherosclerosis. We validated several hypotheses based on network connections and genomic association. Our network analysis predicted that the hub gene CHAC1 (cation transport regulator homolog 1) was regulated by the ATF4 (activating transcription factor 4) arm of the unfolded protein response pathway, and here we showed that ATF4 directly activates an element in the CHAC1 promoter. We showed that variation in basal levels of heme oxygenase 1 (HMOX1) contribute to the response to Ox-PAPC, consistent with its position as a hub in our network. We also identified G-protein–coupled receptor 39 (GPR39) as a regulator of HMOX1 levels and showed that it modulates the promoter activity of HMOX1. We further showed that OKL38/OSGN1 (oxidative stress–induced growth inhibitor), the hub gene in the blue module, is a key regulator of both inflammatory and antiinflammatory molecules. Conclusions: Our systems genetics approach has provided a broad view of the pathways involved in the response of endothelial cells to Ox-PAPC and also identified novel regulatory mechanisms.

Conditional Expression of a Dominant-Negative c-Myb in Vascular Smooth Muscle Cells Inhibits Arterial Remodeling After Injury

Abstract— Inhibiting activity of the c-Myb transcription factor attenuates G1 to S phase cell cycle transitions in vascular smooth muscle cells (SMCs) in vitro. To determine the effects of arterial SMC-specific expression of a dominant-negative c-Myb molecule (Myb-Engrailed) on vascular remodeling in vivo, we performed carotid artery wire-denudation in 2 independent lines of binary transgenic mice with SM22&agr; promoter-defined Doxycycline-suppressible expression of Myb-Engrailed. Adult mice with arterial SMC-specific expression of Myb-Engrailed were overtly normal in appearance and did not display any changes in cardiovascular structure or physiology. However, bromodeoxyuridine-defined arterial SMC proliferation, neointima formation, medial hyperplasia, and arterial remodeling were markedly decreased in mice expressing arterial SMC-restricted Myb-Engrailed after arterial injury. These data suggest that c-Myb activity in arterial SMCs is not essential for arterial structure or function during development, but is involved in the proliferation of arterial SMCs as occurs in vascular pathology, and that the expression of a dominant-negative c-Myb can dramatically reduce adverse arterial remodeling in an in vivo model of restenosis. As such, this model represents a novel tissue-specific strategy for the potential gene therapy of diseases characterized by arterial SMC proliferation.

Effect of 9p21.3 Coronary Artery Disease Locus Neighboring Genes on Atherosclerosis in Mice

Background— The human 9p21.3 chromosome locus has been shown to be an independent risk factor for atherosclerosis in multiple large-scale genome-wide association studies, but the underlying mechanism remains unknown. We set out to investigate the potential role of the 9p21.3 locus neighboring genes, including Mtap, the 2 isoforms of Cdkn2a, p16Ink4a and p19Arf, and Cdkn2b, in atherosclerosis using knockout mice models. Methods and Results— Gene-targeted mice for neighboring genes, including Mtap, Cdkn2a, p19Arf, and Cdkn2b, were each bred to mice carrying the human APO*E3 Leiden transgene that sensitizes the mice for atherosclerotic lesions through elevated plasma cholesterol. We found that the mice heterozygous for Mtap developed larger lesions compared with wild-type mice (49623±21650 versus 18899±9604 &mgr;m2 per section [mean±SD]; P=0.01), with morphology similar to that of wild-type mice. The Mtap heterozygous mice demonstrated changes in metabolic and methylation profiles and CD4+ cell counts. The Cdkn2a knockout mice had smaller lesions compared with wild-type and heterozygous mice, and there were no significant differences in lesion size in p19Arf and Cdkn2b mutants compared with wild type. We observed extensive, tissue-specific compensatory regulation of the Cdkn2a and Cdkn2b genes among the various knockout mice, making the effects on atherosclerosis difficult to interpret. Conclusions— Mtap plays a protective role against atherosclerosis, whereas Cdkn2a appears to be modestly proatherogenic. However, no relation was found between the 9p21 genotype and the transcription of 9p21 neighboring genes in primary human aortic vascular cells in vitro. There is extensive compensatory regulation in the highly conserved 9p21 orthologous region in mice.The 9p21.3 region of the genome has been identified as the locus with strongest association to coronary artery disease (CAD) and myocardial infarction (MI) in multiple independent large scale genome-wide association studies (GWAS).1-3 The locus is within a 58kb region that is devoid of protein coding genes, suggestive of a regulatory function (Figure 1). Interestingly, the neighboring genes in the region include well-known tumor suppressor genes, including CDKN2A and CDKN2B.4-6 The CDKN2A locus encodes a cyclin-dependent protein kinase (CDK) inhibitory protein (CKI), p16INK4A, and a p53-regulatory protein, p19ARF. The CDKN2B gene encodes another CKI, p15INK4B. Another gene in the region is methylthioadenosine phosphorylase (MTAP), which encodes a ubiquitously expressed metabolic enzyme S-methyl-5′-thioadenosine phosphorylase7 that processes the polyamine biosynthesis byproduct in the methionine salvage pathway. Loss or inactivation of MTAP has frequently been observed in a number of different human tumors, and it has been shown to have a tumor suppressive role in a mice model.8 Figure 1 The landscape of the 9p21.3 region Multiple studies demonstrated a potential role for cell cycle regulatory mechanisms in atherosclerosis progression. Previously, the master tumor suppressor gene p53 has been implicated in the development of atherosclerosis in apolipoprotein E (ApoE)-null mice9, 10, affecting both cell proliferation and apoptosis within the atheroma. Another tumor suppressor gene, p21Waf1, was also shown to increase the atheroma size in ApoE-null mice11, whereas the tumor suppressor p27Kip1 was shown to protect against atherosclerosis.12 Correlations of the 9p21 locus SNP genotype to differential expression of the neighboring genes have been observed in several studies with inconsistent findings.13-15 A knockout (KO) mouse model involving the entire region orthologous to the 9p21.3 CAD locus showed significant decreases in the expressed levels of Cdkn2a and Cdkn2b, and increased proliferation of primary smooth muscle cells (SMC) and mouse embryonic fibroblasts (MEF), although an effect on atherosclerosis in vivo was not demonstrated.16 Mice deficient in the p19Arf gene were found to have increased atherosclerotic lesions in an ApoE null background with significant attenuation of apoptosis in lesions as well as in cultured primary macrophages and vascular smooth muscle cells.17 However, to date no observation regarding atherosclerotic phenotype has been made involving the other neighboring genes. We set out to survey the 9p21.3 orthologous region using knockout mice models to systematically address the role of the neighboring protein-coding genes in atherosclerosis. We chose the APOE*3 Leiden sensitizing model because it is dominant, simplifying the construction of the models, and also because it exhibits relatively modest elevations of cholesterol, more realistically modeling the human disease than other widely used models.

Human CHAC1 Protein Degrades Glutathione, and mRNA Induction Is Regulated by the Transcription Factors ATF4 and ATF3 and a Bipartite ATF/CRE Regulatory Element

Background: CHAC1 is associated with the stress response in atherosclerosis. Results: ATF4, ATF3, and CEBPβ regulate CHAC1 transcription. Human CHAC1 protein overexpression depletes glutathione. Conclusion: CHAC1 is induced following multiple cell stress signals and leads to depletion of glutathione. Significance: CHAC1 may be an essential link between stress signaling and the oxidative status of the cell, contributing to multiple diseases. Using an unbiased systems genetics approach, we previously predicted a role for CHAC1 in the endoplasmic reticulum stress pathway, linked functionally to activating transcription factor 4 (ATF4) following treatment with oxidized phospholipids, a model for atherosclerosis. Mouse and yeast CHAC1 homologs have been shown to degrade glutathione in yeast and a cell-free system. In this report, we further defined the ATF4-CHAC1 interaction by cloning the human CHAC1 promoter upstream of a luciferase reporter system for in vitro assays in HEK293 and U2OS cells. Mutation and deletion analyses defined two major cis DNA elements necessary and sufficient for CHAC1 promoter-driven luciferase transcription under conditions of ER stress or ATF4 coexpression: the −267 ATF/cAMP response element (CRE) site and a novel −248 ATF/CRE modifier (ACM) element. We also examined the ability of the CHAC1 ATF/CRE and ACM sequences to bind ATF4 and ATF3 using immunoblot-EMSA and confirmed ATF4, ATF3, and CCAAT/enhancer-binding protein β binding at the human CHAC1 promoter in the proximity of the ATF/CRE and ACM using ChIP. To further validate the function of CHAC1 in a human cell model, we measured glutathione levels in HEK293 cells with enhanced CHAC1 expression. Overexpression of CHAC1 led to a robust depletion of glutathione, which was alleviated in a CHAC1 catalytic mutant. These results suggest an important role for CHAC1 in oxidative stress and apoptosis with implications for human health and disease.

ABCC6 Localizes to the Mitochondria-Associated Membrane

Rationale: Mutations of the orphan transporter ABCC6 (ATP-binding cassette, subfamily C, member 6) cause the connective tissue disorder pseudoxanthoma elasticum. ABCC6 was thought to be located on the plasma membrane of liver and kidney cells. Objective: Mouse systems genetics and bioinformatics suggested that ABCC6 deficiency affects mitochondrial gene expression. We therefore tested whether ABCC6 associates with mitochondria. Methods and Results: We found ABCC6 in crude mitochondrial fractions and subsequently pinpointed its localization to the purified mitochondria-associated membrane fraction. Cell-surface biotinylation in hepatocytes confirmed that ABCC6 is intracellular. Abcc6-knockout mice demonstrated mitochondrial abnormalities and decreased respiration reserve capacity. Conclusions: Our finding that ABCC6 localizes to the mitochondria-associated membrane has implications for its mechanism of action in normal and diseased states.