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1.  Macrophage Mitochondrial Oxidative Stress Promotes Atherosclerosis and NF-κB-Mediated Inflammation in Macrophages 
Circulation research  2013;114(3):421-433.
Rationale
Mitochondrial oxidative stress (mitoOS) has been shown to correlate with the progression of human atherosclerosis. However, definitive cell-type specific causation studies in vivo are lacking, and the molecular mechanisms of potential pro-atherogenic effects remain to be determined.
Objective
To assess the importance of macrophage mitoOS in atherogenesis and explore the underlying molecular mechanisms.
Methods & Results
We first validated Western-type diet-fed Ldlr-/- mice as a model of human mitoOS-atherosclerosis association by showing that a marker of mitoOS in lesional macrophages, non-nuclear oxidative DNA damage, correlates with aortic root lesion development. To investigate the importance of macrophage-mitoOS, we used a genetic engineering strategy in which the OS suppressor catalase was ectopically expressed in mitochondria (mCAT) in macrophages. MitoOS in lesional macrophages was successfully suppressed in these mice, and this led to a significant reduction in aortic root lesional area. The mCAT lesions had less monocyte-derived cells, less Ly6chi monocyte infiltration into lesions, and lower levels of the monocyte chemotactic protein-1 (MCP-1). The decrease in lesional MCP-1 was associated with suppression of other markers of inflammation and with decreased phosphorylation of RelA (NF-κB p65), indicating decreased activation of the pro-inflammatory NF-κB pathway. Using models of mitoOS in cultured macrophages, we showed that mCAT suppressed MCP-1 expression by decreasing activation of the Iκ-kinase-RelA NF-κB pathway.
Conclusions
MitoOS in lesional macrophages amplifies atherosclerotic lesion development by promoting NF-κB-mediated entry of monocytes and other inflammatory processes. In view of the mitoOS-atherosclerosis link in human atheromata, these findings reveal a potentially new therapeutic target to prevent the progression of atherosclerosis.
doi:10.1161/CIRCRESAHA.114.302153
PMCID: PMC3946745  PMID: 24297735
Mitochondrial oxidative stress; atherosclerosis; macrophage; reactive oxygen species (ROS); NF-κB
2.  Molecular mechanisms underlying genotype-dependent responses to dietary restriction 
Aging cell  2013;12(6):10.1111/acel.12130.
Summary
Dietary restriction (DR) increases lifespan and attenuates age-related phenotypes in many organisms; however, the effect of DR on longevity of individuals in genetically heterogeneous populations is not well characterized. Here we describe a large-scale effort to define molecular mechanisms that underlie genotype-specific responses to DR. The effect of DR on lifespan was determined for 166 single-gene deletion strains in Saccharomyces cerevisiae. Resulting changes in mean lifespan ranged from a reduction of 79% to an increase of 103%. Vacuolar pH homeostasis, superoxide dismutase activity, and mitochondrial proteostasis were found to be strong determinants of the response to DR. Proteomic analysis of cells deficient in prohibitins revealed induction of a mitochondrial unfolded protein response (mtUPR) which has not previously been described in yeast. Mitochondrial proteotoxic stress in prohibitin mutants was suppressed by DR via reduced cytoplasmic mRNA translation. A similar relationship between prohibitins, the mtUPR, and longevity was also observed in Caenorhabditis elegans. These observations define conserved molecular processes that underlie genotype-dependent effects of DR that may be important modulators of DR in higher organisms.
doi:10.1111/acel.12130
PMCID: PMC3838465  PMID: 23837470
aging; replicative lifespan; longevity; yeast; dietary restriction; mitochondria; mitochondrial unfolded protein response
3.  Clonal expansions and short telomeres are associated with neoplasia in early-onset, but not late-onset, ulcerative colitis 
Inflammatory bowel diseases  2013;19(12):10.1097/MIB.0b013e3182a87640.
BACKGROUND
Patients with ulcerative colitis (UC) are at risk of developing colorectal cancer. We have previously reported that cancer progression is associated with the presence of clonal expansions and shorter telomeres in non-dysplastic mucosa. We sought to validate these findings in an independent case-control study.
METHODS
This study included 33 patients with UC: 14 Progressors (patients with high-grade dysplasia or cancer) and 19 Non-Progressors. For each patient, a mean of 5 non-dysplastic biopsies from proximal, mid, and distal colon were assessed for clonal expansions, as determined by clonal length altering mutations in polyguanine tracts, and telomere length, as measured by Quantitative-PCR. Both parameters were compared with individual clinico-pathological characteristics.
RESULTS
Clonal expansions and shorter telomeres were more frequent in non-dysplastic biopsies from UC Progressors than Non-Progressors, but only for patients with early-onset of UC (diagnosis at less than 50 years of age). Late-onset Progressor patients had very few or no clonal expansions and longer telomeres. A few Non-Progressors exhibited clonal expansions, which were associated with older age and shorter telomeres. In Progressors, clonal expansions were associated with proximity to dysplasia. The mean percentage of clonally expanded mutations distinguished early-onset Progressors from Non-Progressors with 100% sensitivity and 80% specificity.
CONCLUSIONS
Early-onset Progressors develop cancer in a field of clonally expanded epithelium with shorter telomeres. The detection of these clones in a few random non-dysplastic colon biopsies is a promising cancer biomarker in early-onset UC. Curiously, late-onset UC patients appear to develop cancer without the involvement of such fields.
doi:10.1097/MIB.0b013e3182a87640
PMCID: PMC3885330  PMID: 24097228
inflammatory bowel disease; preneoplasia; field effect; cancer biomarker; age of onset
4.  MRE11-Deficiency Associated with Improved Long-Term Disease Free Survival and Overall Survival in a Subset of Stage III Colon Cancer Patients in Randomized CALGB 89803 Trial 
PLoS ONE  2014;9(10):e108483.
Purpose
Colon cancers deficient in mismatch repair (MMR) may exhibit diminished expression of the DNA repair gene, MRE11, as a consequence of contraction of a T11 mononucleotide tract. This study investigated MRE11 status and its association with prognosis, survival and drug response in patients with stage III colon cancer.
Patients and Methods
Cancer and Leukemia Group B 89803 (Alliance) randomly assigned 1,264 patients with stage III colon cancer to postoperative weekly adjuvant bolus 5-fluorouracil/leucovorin (FU/LV) or irinotecan+FU/LV (IFL), with 8 year follow-up. Tumors from these patients were analyzed to determine stability of a T11 tract in the MRE11 gene. The primary endpoint was overall survival (OS), and a secondary endpoint was disease-free survival (DFS). Non-proportional hazards were addressed using time-dependent covariates in Cox analyses.
Results
Of 625 tumor cases examined, 70 (11.2%) exhibited contraction at the T11 tract in one or both MRE11 alleles and were thus predicted to be deficient in MRE11 (dMRE11). In pooled treatment analyses, dMRE11 patients showed initially reduced DFS and OS but improved long-term DFS and OS compared with patients with an intact MRE11 T11 tract. In the subgroup of dMRE11 patients treated with IFL, an unexplained early increase in mortality but better long-term DFS than IFL-treated pMRE11 patients was observed.
Conclusions
Analysis of this relatively small number of patients and events showed that the dMRE11 marker predicts better prognosis independent of treatment in the long-term. In subgroup analyses, dMRE11 patients treated with irinotecan exhibited unexplained short-term mortality. MRE11 status is readily assayed and may therefore prove to be a useful prognostic marker, provided that the results reported here for a relatively small number of patients can be generalized in independent analyses of larger numbers of samples.
Trial Registration
ClinicalTrials.gov NCT00003835
doi:10.1371/journal.pone.0108483
PMCID: PMC4195600  PMID: 25310185
5.  Global Proteomics and Pathway Analysis of Pressure-overload Induced Heart Failure and Its Attenuation by Mitochondrial Targeted Peptides 
Circulation. Heart failure  2013;6(5):10.1161/CIRCHEARTFAILURE.113.000406.
Background
We investigated the protective effects of mitochondrial-targeted antioxidant and protective peptides, SS31 and SS20, on cardiac function, proteomic remodeling and signaling pathways.
Methods and Results
We applied an improved label-free shotgun proteomics approach to evaluate the global proteomics changes in transverse aortic constriction (TAC) induced heart failure, and the associated signaling pathway changes using Ingenuity Pathway Analysis (IPA). We found 538 proteins significantly changed after TAC, which mapped to 53 pathways. The top pathways were in the categories of actin cytoskeleton, mitochondrial function, intermediate metabolism, glycolysis / gluconeogenesis and citrate cycle. Concomitant treatment with SS31 ameliorated the congestive heart failure phenotypes and mitochondrial damage induced by TAC, in parallel with global attenuation of mitochondrial proteome changes, with an average of 84% protection of mitochondrial and 69% of non-mitochondrial protein changes. This included significant amelioration of All the IPA pathways noted above. SS20 had only modest effects on heart failure and this tracked with only partial attenuation of global proteomics changes; furthermore, while actin cytoskeleton pathways were significantly protected in SS20, mitochondrial and metabolic pathways essentially were not.
Conclusions
This study elucidates the signaling pathways significantly changed in pressure-overload induced heart failure. The global attenuation of TAC-induced proteomic alterations by the mitochondrial targeted peptide SS-31 suggests that perturbed mitochondrial function may be an upstream signal to many of pathway alterations in TAC and supports the potential clinical application of mitochondrial-targeted peptide drugs for the treatment heart failure.
doi:10.1161/CIRCHEARTFAILURE.113.000406
PMCID: PMC3856238  PMID: 23935006
heart failure; mitochondria; proteomics; signal transduction
6.  Mitochondria and Tumor Progression in Ulcerative Colitis 
Background
The role of mitochondria in cancer is poorly understood. Ulcerative colitis (UC) is an inflammatory bowel disease that predisposes to colorectal cancer and is an excellent model to study tumor progression. Our goal was to characterize mitochondrial alterations in UC tumorigenesis.
Methods
Nondysplastic colon biopsies from UC patients with high-grade dysplasia or cancer (progressors; n = 9) and UC patients dysplasia free (nonprogressors; n = 9) were immunostained for cytochrome C oxidase (COX), a component of the electron transport chain, and were quantified by multispectral imaging. For six additional progressors, nondysplastic and dysplastic biopsies were stained for COX and additional mitochondrial proteins including PGC1α, the master regulator of mitochondrial biogenesis. Mitochondrial DNA (mtDNA) copy number was determined by quantitative polymerase chain reaction. Generalized estimating equations with two-sided tests were used to account for correlation of measurements within individuals.
Results
Nondysplastic biopsies of UC progressors showed statistically significant COX loss compared with UC nonprogressors by generalized estimating equation (−18.5 units, 95% confidence interval = −12.1 to −24.9; P < .001). COX intensity progressively decreased with proximity to dysplasia and was the lowest in adjacent to dysplasia and dysplastic epithelium. Surprisingly, COX intensity was statistically significantly increased in cancers. This bimodal pattern was observed for other mitochondrial proteins, including PGC1α, and was confirmed by mtDNA copy number.
Conclusions
Mitochondrial loss precedes the development of dysplasia, and it could be used to detect and potentially predict cancer. Cancer cells restore mitochondria, suggesting that mitochondria are needed for further proliferation. This bimodal pattern might be driven by transcriptional regulation of mitochondrial biogenesis by PGC1α.
doi:10.1093/jnci/djt167
PMCID: PMC3748006  PMID: 23852949
7.  mTOR Inhibition Alleviates Mitochondrial Disease in a Mouse Model of Leigh Syndrome 
Science (New York, N.Y.)  2013;342(6165):1524-1528.
Mitochondrial dysfunction contributes to numerous health problems, including neurological and muscular degeneration, cardiomyopathies, cancer, diabetes, and pathologies of aging. Severe mitochondrial defects can result in childhood disorders such as Leigh syndrome, for which there are no effective therapies. We found that rapamycin, a specific inhibitor of the mechanistic target of rapamycin (mTOR) signaling pathway, robustly enhances survival and attenuates disease progression in a mouse model of Leigh syndrome. Administration of rapamycin to these mice, which are deficient in the mitochondrial respiratory chain subunit Ndufs4 [NADH dehydrogenase (ubiquinone) Fe-S protein 4], delays onset of neurological symptoms, reduces neuroinflammation, and prevents brain lesions. Although the precise mechanism of rescue remains to be determined, rapamycin induces a metabolic shift toward amino acid catabolism and away from glycolysis, alleviating the buildup of glycolytic intermediates. This therapeutic strategy may prove relevant for a broad range of mitochondrial diseases.
doi:10.1126/science.1244360
PMCID: PMC4055856  PMID: 24231806
8.  Independent and Combined Effects of Dietary Weight Loss and Exercise on Leukocyte Telomere Length in Postmenopausal Women 
Obesity (Silver Spring, Md.)  2013;21(12):10.1002/oby.20509.
Objective
Investigate the effects of 12 months of dietary weight loss and/or aerobic exercise on leukocyte telomere length in postmenopausal women.
Design and Methods
439 overweight or obese women (50–75 y) were randomized to: i) dietary weight loss (N=118); ii) aerobic exercise (N=117), iii) diet + exercise (N=117), or iv) control (N=87). The diet intervention was a group-based program with a 10% weight loss goal. The exercise intervention was 45 mins/day, 5 days/week of moderate-to-vigorous aerobic activity. Fasting blood samples were taken at baseline and 12 months. DNA was extracted from isolated leukocytes and telomere length was measured by quantitative-polymerase chain reaction (qPCR). Mean changes were compared between groups (intent-to-treat) using generalized estimating equations.
Results
Baseline telomere length was inversely associated with age (r=−0.12 p<0.01) and positively associated with maximal oxygen uptake (r=0.11, p=0.03), but not with BMI or %body fat. Change in telomere length was inversely correlated with baseline telomere length (r=−0.47, p<0.0001). No significant difference in leukocyte telomere length was detected in any intervention group compared to controls, nor was the magnitude of weight loss associated with telomere length at 12 months.
Conclusions
Twelve-months of dietary weight loss and exercise did not change telomere length in postmenopausal women.
doi:10.1002/oby.20509
PMCID: PMC3786031  PMID: 23640743
caloric restriction; physical activity; lifestyle; ageing; chromosomes
9.  Altered proteome turnover and remodeling by short-term caloric restriction or rapamycin rejuvenate the aging heart 
Aging Cell  2014;13(3):529-539.
Chronic caloric restriction (CR) and rapamycin inhibit the mechanistic target of rapamycin (mTOR) signaling, thereby regulating metabolism and suppressing protein synthesis. Caloric restriction or rapamycin extends murine lifespan and ameliorates many aging-associated disorders; however, the beneficial effects of shorter treatment on cardiac aging are not as well understood. Using a recently developed deuterated-leucine labeling method, we investigated the effect of short-term (10 weeks) CR or rapamycin on the proteomics turnover and remodeling of the aging mouse heart. Functionally, we observed that short-term CR and rapamycin both reversed the pre-existing age-dependent cardiac hypertrophy and diastolic dysfunction. There was no significant change in the cardiac global proteome (823 proteins) turnover with age, with a median half-life 9.1 days in the 5-month-old hearts and 8.8 days in the 27-month-old hearts. However, proteome half-lives of old hearts significantly increased after short-term CR (30%) or rapamycin (12%). This was accompanied by attenuation of age-dependent protein oxidative damage and ubiquitination. Quantitative proteomics and pathway analysis revealed an age-dependent decreased abundance of proteins involved in mitochondrial function, electron transport chain, citric acid cycle, and fatty acid metabolism as well as increased abundance of proteins involved in glycolysis and oxidative stress response. This age-dependent cardiac proteome remodeling was significantly reversed by short-term CR or rapamycin, demonstrating a concordance with the beneficial effect on cardiac physiology. The metabolic shift induced by rapamycin was confirmed by metabolomic analysis.
doi:10.1111/acel.12203
PMCID: PMC4040127  PMID: 24612461
caloric restriction; cardiac aging; dynamics; proteomics; rapamycin
10.  Preserving Youth: Does Rapamycin Deliver? 
Science translational medicine  2013;5(211):211fs40.
Research suggests that the drug rapamycin slows mammalian aging, but a provocative new study has gained attention by claiming to show it does not.
doi:10.1126/scitranslmed.3007316
PMCID: PMC4019780  PMID: 24225941
11.  Mitochondrial oxidative stress in aging and healthspan 
The free radical theory of aging proposes that reactive oxygen species (ROS)-induced accumulation of damage to cellular macromolecules is a primary driving force of aging and a major determinant of lifespan. Although this theory is one of the most popular explanations for the cause of aging, several experimental rodent models of antioxidant manipulation have failed to affect lifespan. Moreover, antioxidant supplementation clinical trials have been largely disappointing. The mitochondrial theory of aging specifies more particularly that mitochondria are both the primary sources of ROS and the primary targets of ROS damage. In addition to effects on lifespan and aging, mitochondrial ROS have been shown to play a central role in healthspan of many vital organ systems. In this article we review the evidence supporting the role of mitochondrial oxidative stress, mitochondrial damage and dysfunction in aging and healthspan, including cardiac aging, age-dependent cardiovascular diseases, skeletal muscle aging, neurodegenerative diseases, insulin resistance and diabetes as well as age-related cancers. The crosstalk of mitochondrial ROS, redox, and other cellular signaling is briefly presented. Potential therapeutic strategies to improve mitochondrial function in aging and healthspan are reviewed, with a focus on mitochondrial protective drugs, such as the mitochondrial antioxidants MitoQ, SkQ1, and the mitochondrial protective peptide SS-31.
doi:10.1186/2046-2395-3-6
PMCID: PMC4013820  PMID: 24860647
Mitochondria; Oxidative stress; Aging; Healthspan
12.  Mitochondria and Cardiovascular Aging 
Circulation research  2012;110(8):10.1161/CIRCRESAHA.111.246140.
Old age is a major risk factor for cardiovascular diseases. Several lines of evidence in experimental animal models have indicated the central role of mitochondria both in lifespan determination and cardiovascular aging. In this article we review the evidence supporting the role of mitochondrial oxidative stress, mitochondrial damage and biogenesis as well as the crosstalk between mitochondria and cellular signaling in cardiac and vascular aging. Intrinsic cardiac aging in the murine model closely recapitulates age-related cardiac changes in humans (left ventricular hypertrophy, fibrosis and diastolic dysfunction), while the phenotype of vascular aging include endothelial dysfunction, reduced vascular elasticity and chronic vascular inflammation. Both cardiac and vascular aging involve neurohormonal signaling (e.g. renin-angiotensin, adrenergic, insulin-IGF1 signaling) and cell-autonomous mechanisms. The potential therapeutic strategies to improve mitochondrial function in aging and cardiovascular diseases are also discussed, with a focus on mitochondrial-targeted antioxidants, calorie restriction, calorie restriction mimetics and exercise training.
doi:10.1161/CIRCRESAHA.111.246140
PMCID: PMC3867977  PMID: 22499901
13.  Measuring telomere length for the early detection of precursor lesions of esophageal squamous cell carcinoma 
BMC Cancer  2013;13:578.
Background
Esophageal cancer is the sixth leading cause of cancer death worldwide; current early detection screening tests are inadequate. Esophageal balloon cytology successfully retrieves exfoliated and scraped superficial esophageal epithelial cells, but cytologic reading of these cells has poor sensitivity and specificity for detecting esophageal squamous dysplasia (ESD), the precursor lesion of esophageal squamous cell carcinoma (ESCC). Measuring telomere length, a marker for chromosomal instability, may improve the utility of balloon cytology for detecting ESD and early ESCC.
Methods
We examined balloon cytology specimens from 89 asymptomatic cases of ESD (37 low-grade and 52 high-grade) and 92 age- and sex-matched normal controls from an esophageal cancer early detection screening study. All subjects also underwent endoscopy and biopsy, and ESD was diagnosed histopathologically. DNA was extracted from the balloon cytology cells, and telomere length was measured by quantitative PCR. A receiver operating characteristic (ROC) curve was plotted for telomere length as a diagnostic marker for high-grade dysplasia.
Results
Telomere lengths were comparable among the low- and high-grade dysplasia cases and controls, with means of 0.96, 0.96, and 0.92, respectively. The area under the ROC curve was 0.55 for telomere length as a diagnostic marker for high-grade dysplasia. Further adjustment for subject characteristics, including sex, age, smoking, drinking, hypertension, and body mass index did not improve the use of telomere length as a marker for ESD.
Conclusions
Telomere length of esophageal balloon cytology cells was not associated with ESCC precursor lesions. Therefore, telomere length shows little promise as an early detection marker for ESCC in esophageal balloon samples.
doi:10.1186/1471-2407-13-578
PMCID: PMC3882883  PMID: 24308314
Esophageal squamous cell carcinoma; Esophageal squamous dysplasia; Early detection; Screening; Balloon cytology; Telomeres
14.  Mitochondrial oxidative stress mediates Angiotensin II-induced cardiac hypertrophy and Gαq overexpression-induced heart failure 
Circulation research  2011;108(7):837-846.
Rationale
Mitochondrial dysfunction has been implicated in several cardiovascular diseases; however, the roles of mitochondrial oxidative stress and DNA damage in hypertensive cardiomyopathy are not well understood.
Objective
We evaluated the contribution of mitochondrial reactive oxygen species (ROS) to cardiac hypertrophy and failure by using genetic mouse models overexpressing catalase targeted to mitochondria and to peroxisomes.
Methods and Results
Angiotensin II increases mitochondrial ROS in cardiomyocytes, concomitant with increased mitochondrial protein carbonyls, mitochondrial DNA deletions, increased autophagy and signaling for mitochondrial biogenesis in hearts of Angiotensin II treated mice. The causal role of mitochondrial ROS in Angiotensin II-induced cardiomyopathy is shown by the observation that mice that overexpress catalase targeted to mitochondria, but not mice that overexpress wild-type peroxisomal catalase, are resistant to cardiac hypertrophy, fibrosis and mitochondrial damage induced by Angiotensin II, as well as heart failure induced by overexpression of Gαq. Furthermore, primary damage to mitochondrial DNA, induced by zidovudine administration or homozygous mutation of mitochondrial polymerase gamma, is also shown to contribute directly to the development of cardiac hypertrophy, fibrosis and failure.
Conclusions
These data indicate the critical role of mitochondrial ROS in cardiac hypertrophy and failure and support the potential use of mitochondrial-targeted antioxidants for prevention and treatment of hypertensive cardiomyopathy.
doi:10.1161/CIRCRESAHA.110.232306
PMCID: PMC3785241  PMID: 21311045
mitochondria; reactive oxygen species; angiotensin; cardiomyopathy; heart failure
15.  Mitochondrial targeted antioxidant peptide ameliorates hypertensive cardiomyopathy 
Objectives
We investigated the effect of reducing mitochondrial oxidative stress by the mitochondrial-targeted antioxidant peptide SS-31 in hypertensive cardiomyopathy.
Background
Oxidative stress has been implicated in hypertensive cardiovascular diseases. Mitochondria and NADPH oxidase have been proposed as primary sites of reactive oxygen species (ROS) generation.
Methods
The mitochondrial targeted antioxidant peptide SS-31 was used to determine the role of mitochondrial oxidative stress in Angiotensin II (Ang)-induced cardiomyopathy, as well as in Gαq overexpressing mice with heart failure.
Results
Angiotensin II induces mitochondrial ROS in neonatal cardiomyocytes, which is prevented by SS-31, but not the non-targeted antioxidant N-acetyl cysteine (NAC). Continuous administration of Ang for 4 weeks in mice significantly increased both systolic and diastolic blood pressure, and this was not affected by SS-31 treatment. Ang was associated with upregulation of NADPH oxidase 4 (NOX4) expression, increased cardiac mitochondrial protein oxidative damage and induced the signaling for mitochondrial biogenesis. Reducing mitochondrial ROS by SS-31 substantially attenuated Ang-induced NOX4 upregulation, mitochondrial oxidative damage, upregulation of mitochondrial biogenesis, phosphorylation of p38 MAP kinase, and prevented apoptosis, concomitant with amelioration of Ang induced cardiac hypertrophy, diastolic dysfunction, and fibrosis, despite the absence of blood pressure lowering effect. NAC did not show any beneficial effect. SS-31 administration for 4 weeks also partially rescued the heart failure phenotype of Gαq overexpressing mice.
Conclusions
Mitochondrial targeted peptide SS-31 ameliorates cardiomyopathy resulting from prolonged Ang stimulation as well as Gαq overexpression, suggesting its potential clinical application for target organ protection in hypertensive cardiovascular diseases.
doi:10.1016/j.jacc.2010.12.044
PMCID: PMC3742010  PMID: 21620606
mitochondria; hypertension; cardiomyopathy
16.  Pan-colonic field defects are detected by CGH in the colons of UC patients with dysplasia/cancer 
Cancer Letters  2012;320(2):180-188.
BAC arrays were used to evaluate genomic instability along the colon of patients with ulcerative colitis (UC). Genomic instability increases with disease progression and biopsies more proximal to dysplasia showed increased instability. Pan-colonic field copy number gain or loss involving small (< 1 Mb) regions were detected in most patients and were particularly apparent in the UC progressor patients who had dysplasia or cancer. Chromosomal copy gains or losses affecting large regions were mainly restricted to dysplastic biopsies. Areas of significant chromosomal losses were detected in the UC progressors on chromosomes 2q36, 3q25, 3p21, 4q34, 4p16.2, 15q22, and 16p13 (p-value ≤ 0.04). These results extend our understanding of the dynamic nature of pan-colonic genomic instability in this disease.
doi:10.1016/j.canlet.2012.02.031
PMCID: PMC3406733  PMID: 22387989
ulcerative colitis; genomic instability; BAC array
17.  mTOR is a key modulator of ageing and age-related disease 
Nature  2013;493(7432):338-345.
Many experts in the biology of ageing believe that pharmacological interventions to slow ageing are a matter of ‘when’ rather than ‘if’. A leading target for such interventions is the nutrient response pathway defined by the mechanistic target of rapamycin (mTOR). Inhibition of this pathway extends lifespan in model organisms and confers protection against a growing list of age-related pathologies. Characterized inhibitors of this pathway are already clinically approved, and others are under development. Although adverse side effects currently preclude use in otherwise healthy individuals, drugs that target the mTOR pathway could one day become widely used to slow ageing and reduce age-related pathologies in humans.
doi:10.1038/nature11861
PMCID: PMC3687363  PMID: 23325216
18.  Mitochondria-targeted catalase reduces abnormal APP processing, amyloid β production and BACE1 in a mouse model of Alzheimer's disease: implications for neuroprotection and lifespan extension 
Human Molecular Genetics  2012;21(13):2973-2990.
The purpose of this study was to investigate the protective effects of the mitochondria-targeted antioxidant catalase (MCAT) and lifespan extension in mice that express amyloid beta (Aβ). Using immunoblotting and immunostaining analyses, we measured the production of full-length amyloid precursor protein (APP), soluble APPα, C-terminal fragments CTF99 and CTF83, monomeric and oligomeric Aβ, Aβ deposits and beta site amyloid precursor protein cleaving enzyme 1 (BACE1), in different stages of disease progression in MCAT/AβPP and AβPP mice. Using quantitative reverse transcriptase polymerase chain reaction and immunostaining analyses, we studied the expression of catalase, BACE1, the Alzheimer's disease (AD) markers, synaptophysin, APP, neprilysin, insulin-degrading enzyme and transthyretin in MCAT, AβPP, MCAT/AβPP and wild-type (WT) mice. Using the high pressure liquid chromatography analysis of 8-hydroxy-2-deoxyguanosine, we measured oxidative DNA damage in the cerebral cortical tissues from MCAT, AβPP, MCAT/AβPP and WT mice. We found that the AβPP transgenic mice that carried the human MCAT gene lived 5 months longer than did the AβPP mice. We also found that the overexpression of MCAT in the brain sections from the MCAT/AβPP transgenic mice significantly correlated with a reduction in the levels of full-length APP, CTF99, BACE1, Aβ levels (40 and 42), Aβ deposits and oxidative DNA damage relative to the brain sections from the AβPP mice. Interestingly, we found significantly increased levels of soluble APPα and CTF83 in the MCAT/AβPP mice, relative to the AβPP mice. These data provide direct evidence that oxidative stress plays a primary role in AD etiopathology and that in MCAT mice express Aβ, MCAT prevents abnormal APP processing, reduces Aβ levels and enhances Aβ-degrading enzymes in mice at different ages, corresponding to different stages of disease progression. These findings indicate that mitochondria-targeted molecules may be an effective therapeutic approach to treat patients with AD.
doi:10.1093/hmg/dds128
PMCID: PMC3373244  PMID: 22492996
19.  Ex-vivo imaging of excised tissue using vital dyes and confocal microscopy 
Current Protocols in Cytometry  2012;CHAPTER:Unit9.39.
Vital dyes routinely used for staining cultured cells can also be used to stain and image live tissue slices ex-vivo. Staining tissue with vital dyes allows researchers to collect structural and functional data simultaneously and can be used for qualitative or quantitative fluorescent image collection. The protocols presented here are useful for structural and functional analysis of viable properties of cells in intact tissue slices, allowing for the collection of data in a structurally relevant environment. With these protocols, vital dyes can be applied as a research tool to disease processes and properties of tissue not amenable to cell culture based studies.
doi:10.1002/0471142956.cy0939s61
PMCID: PMC3401092  PMID: 22752953
20.  Cardiac Aging: From Molecular Mechanisms to Significance in Human Health and Disease 
Antioxidants & Redox Signaling  2012;16(12):1492-1526.
Abstract
Cardiovascular diseases (CVDs) are the major causes of death in the western world. The incidence of cardiovascular disease as well as the rate of cardiovascular mortality and morbidity increase exponentially in the elderly population, suggesting that age per se is a major risk factor of CVDs. The physiologic changes of human cardiac aging mainly include left ventricular hypertrophy, diastolic dysfunction, valvular degeneration, increased cardiac fibrosis, increased prevalence of atrial fibrillation, and decreased maximal exercise capacity. Many of these changes are closely recapitulated in animal models commonly used in an aging study, including rodents, flies, and monkeys. The application of genetically modified aged mice has provided direct evidence of several critical molecular mechanisms involved in cardiac aging, such as mitochondrial oxidative stress, insulin/insulin-like growth factor/PI3K pathway, adrenergic and renin angiotensin II signaling, and nutrient signaling pathways. This article also reviews the central role of mitochondrial oxidative stress in CVDs and the plausible mechanisms underlying the progression toward heart failure in the susceptible aging hearts. Finally, the understanding of the molecular mechanisms of cardiac aging may support the potential clinical application of several “anti-aging” strategies that treat CVDs and improve healthy cardiac aging.
I. Introduction
II. Aging and Epidemiology of CVDs
III. Physiology of Cardiac Aging
A. Ventricular changes
B. Valvular changes
IV. Animal Models of Cardiac Aging
A. Rodents
B. Drosophila
C. Canines
D. Nonhuman primates
V. Mitochondria and the Free Radical Theory of Aging
A. ROS and aging
B. Pleiotropy of ROS
C. Mitochondrial hormesis in aging
D. Mitochondrial turnover in aging
VI. Molecular Mechanisms of Cardiac Aging
A. Mitochondrial oxidative stress in cardiac aging
B. Neurohormonal regulation of cardiac aging
1. Renin-angiotensin system in cardiac aging
2. B-adrenergic signaling
3. Insulin/insulin-like growth factor 1/PI3K signaling
4. Natriuretic peptides signaling
C. Nutrient signaling in cardiac aging
D. Cardiac stem cell aging and telomeres
VII. Aging, Oxidative Stress, and CVDs
A. Oxidative stress and mitochondria in CVDs
1. The central role of mitochondrial oxidative stress and redox status in hypertension and heart failure
2. The role of mitochondria and oxidative stress in IR injury
B. Mechanisms of progression to heart failure in the aged hypertrophic heart
1. Increased cardiomyocyte death
2. ECM remodeling
3. Alteration of calcium handling proteins
4. Hypoxic response and angiogenesis
5. Mitochondrial dysfunction and abnormalities in energetics
VIII. Exercise, Cardiovascular Risks, and Cardiac Aging
IX. Emerging “Anti-Aging” Interventional Strategies for Cardiac Aging and CVDs
A. Dietary restriction
B. Antioxidant interventions
1. Nontargeted antioxidants
2. Mitochondrial-targeted antioxidants
a. TPP+conjugated antioxidants
b. Szeto-schiller peptides
C. Resveratrol and SIRTs activators
X. Conclusion and Future Directions
doi:10.1089/ars.2011.4179
PMCID: PMC3329953  PMID: 22229339
21.  Rapamycin Reverses Elevated mTORC1 Signaling in Lamin A/C–Deficient Mice, Rescues Cardiac and Skeletal Muscle Function, and Extends Survival 
Science translational medicine  2012;4(144):144ra103.
Mutations in LMNA, the gene that encodes A-type lamins, cause multiple diseases including dystrophies of the skeletal muscle and fat, dilated cardiomyopathy, and progeria-like syndromes (collectively termed laminopathies). Reduced A-type lamin function, however, is most commonly associated with skeletal muscle dystrophy and dilated cardiomyopathy rather than lipodystrophy or progeria. The mechanisms underlying these diseases are only beginning to be unraveled. We report that mice deficient in Lmna, which corresponds to the human gene LMNA, have enhanced mTORC1 (mammalian target of rapamycin complex 1) signaling specifically in tissues linked to pathology, namely, cardiac and skeletal muscle. Pharmacologic reversal of elevated mTORC1 signaling by rapamycin improves cardiac and skeletal muscle function and enhances survival in mice lacking A-type lamins. At the cellular level, rapamycin decreases the number of myocytes with abnormal desmin accumulation and decreases the amount of desmin in both muscle and cardiac tissue of Lmna–/– mice. In addition, inhibition of mTORC1 signaling with rapamycin improves defective autophagic-mediated degradation in Lmna–/– mice. Together, these findings point to aberrant mTORC1 signaling as a mechanistic component of laminopathies associated with reduced A-type lamin function and offer a potential therapeutic approach, namely, the use of rapamycin-related mTORC1 inhibitors.
doi:10.1126/scitranslmed.3003802
PMCID: PMC3613228  PMID: 22837538
22.  Mitochondrial proteome remodelling in pressure overload-induced heart failure: the role of mitochondrial oxidative stress 
Cardiovascular Research  2011;93(1):79-88.
Aims
We investigate the role of mitochondrial oxidative stress in mitochondrial proteome remodelling using mouse models of heart failure induced by pressure overload.
Methods and results
We demonstrate that mice overexpressing catalase targeted to mitochondria (mCAT) attenuate pressure overload-induced heart failure. An improved method of label-free unbiased analysis of the mitochondrial proteome was applied to the mouse model of heart failure induced by transverse aortic constriction (TAC). A total of 425 mitochondrial proteins were compared between wild-type and mCAT mice receiving TAC or sham surgery. The changes in the mitochondrial proteome in heart failure included decreased abundance of proteins involved in fatty acid metabolism, an increased abundance of proteins in glycolysis, apoptosis, mitochondrial unfolded protein response and proteolysis, transcription and translational control, and developmental processes as well as responses to stimuli. Overexpression of mCAT better preserved proteins involved in fatty acid metabolism and attenuated the increases in apoptotic and proteolytic enzymes. Interestingly, gene ontology analysis also showed that monosaccharide metabolic processes and protein folding/proteolysis were only overrepresented in mCAT but not in wild-type mice in response to TAC.
Conclusion
This is the first study to demonstrate that scavenging mitochondrial reactive oxygen species (ROS) by mCAT not only attenuates most of the mitochondrial proteome changes in heart failure, but also induces a subset of unique alterations. These changes represent processes that are adaptive to the increased work and metabolic requirements of pressure overload, but which are normally inhibited by overproduction of mitochondrial ROS.
doi:10.1093/cvr/cvr274
PMCID: PMC3243039  PMID: 22012956
Mitochondria; Oxidative stress; Proteome; Pressure overload; Cardiomyopathy
23.  A novel radial water tread maze tracks age-related cognitive decline in mice 
Pathobiology of Aging & Age Related Diseases  2013;3:10.3402/pba.v3i0.20679.
There is currently no treatment and cure for age-related dementia and cognitive impairment in humans. Mice suffer from age-related cognitive decline just as people do, but assessment is challenging because of cumbersome and at times stressful performance tasks. We developed a novel radial water tread (RWT) maze and tested male C57BL/6 (B6) and C57BL/6 x Balb/c F1 (CB6F1) mice at ages 4, 12, 20, and 28 months. B6 mice showed a consistent learning experience and memory retention that gradually decreased with age. CB6F1 mice showed a moderate learning experience in the 4 and 12 month groups, which was not evident in the 20 and 28 month groups. In conclusion, CB6F1 mice showed more severe age-related cognitive impairment compared to B6 mice and might be a suitable model for intervention studies. In addition, the RWT maze has a number of operational advantages compared to currently accepted tasks and can be used to assess age-related cognition impairment in B6 and CB6F1 mice as early as 12 months of age.
doi:10.3402/pba.v3i0.20679
PMCID: PMC3791354  PMID: 24106580
memory impairment; aging; water tread radial maze; mouse cognition
24.  Correction: Cardiomyocyte-Specific Expression of Lamin A Improves Cardiac Function in Lmna−/− Mice 
PLoS ONE  2012;7(9):10.1371/annotation/92be6b32-d8e7-44c2-80a9-21097ad27965.
doi:10.1371/annotation/92be6b32-d8e7-44c2-80a9-21097ad27965
PMCID: PMC3465171
25.  Cardiomyocyte-Specific Expression of Lamin A Improves Cardiac Function in Lmna−/− Mice 
PLoS ONE  2012;7(8):e42918.
Lmna−/− mice display multiple tissue defects and die by 6–8 weeks of age reportedly from dilated cardiomyopathy with associated conduction defects. We sought to determine whether restoration of lamin A in cardiomyocytes improves cardiac function and extends the survival of Lmna−/− mice. We observed increased total desmin protein levels and disorganization of the cytoplasmic desmin network in ∼20% of Lmna−/− ventricular myocytes, rescued in a cell-autonomous manner in Lmna−/− mice expressing a cardiac-specific lamin A transgene (Lmna−/−; Tg). Lmna−/−; Tg mice displayed significantly increased contractility and preservation of myocardial performance compared to Lmna−/− mice. Lmna−/−; Tg mice attenuated ERK1/2 phosphorylation relative to Lmna−/− mice, potentially underlying the improved localization of connexin43 to the intercalated disc. Electrocardiographic recordings from Lmna−/− mice revealed arrhythmic events and increased frequency of PR interval prolongation, which is partially rescued in Lmna−/−; Tg mice. These findings support our observation that Lmna−/−; Tg mice have a 12% median extension in lifespan compared to Lmna−/− mice. While significant, Lmna−/−; Tg mice only have modest improvement in cardiac function and survival likely stemming from the observation that only 40% of Lmna−/−; Tg cardiomyocytes have detectable lamin A expression. Cardiomyocyte-specific restoration of lamin A in Lmna−/− mice improves heart-specific pathology and extends lifespan, demonstrating that the cardiac pathology of Lmna−/− mice limits survival. The expression of lamin A is sufficient to rescue certain cellular defects associated with loss of A-type lamins in cardiomyocytes in a cell-autonomous fashion.
doi:10.1371/journal.pone.0042918
PMCID: PMC3419749  PMID: 22905185

Results 1-25 (63)