Sirtuin 6 Protects Against Oxidative Stress and Vascular Dysfunction in Mice.

Objective: Sirtuin deacetylases are major regulators of organismal aging, and while depletion of sirtuin 6 (SIRT6) in mice results in a profound progeroid phenotype, the role of SIRT6 in the regulation of vasomotor function is unknown. Thus, our objective was to test the hypothesis that reductions in SIRT6 elicit endothelial dysfunction in young, genetically altered mice. Results and Approach: We used young (3 month old), littermate-matched, SIRT6 wild-type (WT), and SIRT6 heterozygous (HET) mice. SIRT6 expression (qRT-PCR) was reduced by 50% in HET mice. Carotid vessel responses to acetylcholine, sodium nitroprusside, U46619, and serotonin were examined in isolated organ chamber baths. Relaxation in response to acetylcholine (ACH) was impaired in HET mice compared to littermate-matched WT controls (67 ± 3% versus 76 ± 3%, respectively; p < 0.05), while responses to sodium nitroprusside were unchanged. Short-term incubation of carotid rings with the NAD(P)H oxidase inhibitor, apocynin, significantly improved in vessels from HET mice but not their WT littermates. Peak tension generated in response to either U46619 or serotonin was significantly blunted in HET mice compared to their WT littermates. Conclusion: These data suggest that SIRT6 is a key regulator of vasomotor function in conduit vessels. More specifically, we propose that SIRT6 serves as a tonic suppressor of NAD(P)H oxidase expression and activation, as inhibition of NAD(P)H oxidase improved endothelial function in SIRT6 haploinsufficient mice. Collectively, SIRT6 activation and/or histone acetyltransferase inhibition may be useful therapeutic approaches to reduce endothelial dysfunction and combat age-associated cardiovascular disease.

Effects of Altering Mitochondrial Antioxidant Capacity on Molecular and Phenotypic Drivers of Fibrocalcific Aortic Valve Stenosis.

Background: While a small number of studies suggest that oxidative stress has an influential role in fibrocalcific aortic valve disease (FCAVD), the roles of specific antioxidant enzymes in progression of this disease remain poorly understood. Here, we focused on selectively altering mitochondrial-derived oxidative stress-which has been shown to alter progression of a myriad of age-associated diseases-on the progression of molecular and phenotypic drivers of FCAVD. Methods: We generated low-density lipoprotein receptor-deficient, Apolipoprotein B100-only mice (LA) that were either haploinsufficient for MnSOD (LA-MnSOD +/-) or genetically overexpressing MnSOD (LA-MnSOD Tg/0). After 6 months of Western diet feeding, mice underwent echocardiography to assess valvular and cardiac function and tissues were harvested. Quantitative-RT PCR, immunohistochemistry, and histopathology were used to measure changes in molecular pathways related to oxidative stress, calcification, and fibrosis. Results: While reductions in MnSOD increased oxidative stress, there was not an overt phenotypic effect of MnSOD deficiency on valvular and cardiac function in LA-MnSOD +/- mice. While markers of canonical bone morphogenetic protein signaling tended to increase in valve tissue from LA-MnSOD +/- (e.g., p-SMAD1/5/8 and osterix), we did not observe statistically significant increases in osteogenic signaling. We did, however, observe highly significant reductions in expression of osteopontin, which were associated with significant increases in calcium burden in LA-MnSOD +/- mice. Reciprocally, genetically increasing MnSOD did not preserve valve function in LA-MnSOD Tg/0, but we did observe slight reductions in p-SMAD1/5/8 levels compared to their non-transgenic littermates. Interestingly, overexpression of MnSOD dramatically increased expression of osteopontin in valve tissue from LA-MnSOD Tg/0 mice, but was not sufficient to attenuate calcium burden when compared to their LA-MnSOD 0/0 littermates. Conclusions: Collectively, this study demonstrates that maintenance of mitochondrial antioxidant capacity is important in preventing accelerated disease progression in a mouse model of FCAVD, but that effectively altering mitochondrial antioxidant capacity as a monotherapeutic approach to slow key histopathological and molecular drivers of FCAVD remains biologically and therapeutically challenging.

MnSOD protects against vascular calcification independent of changes in vascular function in hypercholesterolemic mice.

BACKGROUND AND AIMS: The overall goal of this study was to determine the effects of MnSOD-deficiency on vascular structure and function in hypercholesterolemic mice. Previous work suggested that increases in mitochondrial-derived reactive oxygen species (ROS) can exacerbate vascular dysfunction and atherosclerosis. It remains unknown, however, how MnSOD-deficiency and local compensatory mechanisms impact atherosclerotic plaque composition. METHODS AND RESULTS: We used a hypercholesterolemic mouse model (ldlr-/-/ApoB100/100; LA), either wild-type for MnSOD (LA-MnSOD+/+) or MnSOD-haploinsufficient (LA-MnSOD+/-), that was fed a western diet for either 3 or 6 months. Consistent with previous reports, reductions of MnSOD did not significantly worsen hypercholesterolemia-induced endothelial dysfunction in the aorta. Critically, dramatic impairment of vascular function with Nox2 inhibition or catalase pretreatment suggested the presence of a significant NO-independent vasodilatory mechanism in LA-MnSOD+/- mice (e.g. H2O2). Despite remarkably well-preserved overall vascular relaxation, loss of mitochondrial antioxidant capacity in LA-MnSOD+/- mice significantly increased osteogenic signalling and vascular calcification compared to the LA-MnSOD+/+ littermates. CONCLUSIONS: Collectively, these data are the first to suggest that loss of mitochondrial antioxidant capacity in hypercholesterolemic mice results in dramatic upregulation of NADPH oxidase-derived H2O2. While this appears to be adaptive in the context of preserving overall endothelium-dependent relaxation and vascular function, these increases in ROS appear to be remarkably maladaptive and deleterious in the context of vascular calcification.

Chronic senolytic treatment alleviates established vasomotor dysfunction in aged or atherosclerotic mice.

While reports suggest a single dose of senolytics may improve vasomotor function, the structural and functional impact of long-term senolytic treatment is unknown. To determine whether long-term senolytic treatment improves vasomotor function, vascular stiffness, and intimal plaque size and composition in aged or hypercholesterolemic mice with established disease. Senolytic treatment (intermittent treatment with Dasatinib + Quercetin via oral gavage) resulted in significant reductions in senescent cell markers (TAF(+) cells) in the medial layer of aorta from aged and hypercholesterolemic mice, but not in intimal atherosclerotic plaques. While senolytic treatment significantly improved vasomotor function (isolated organ chamber baths) in both groups of mice, this was due to increases in nitric oxide bioavailability in aged mice and increases in sensitivity to NO donors in hypercholesterolemic mice. Genetic clearance of senescent cells in aged normocholesterolemic INK-ATTAC mice phenocopied changes elicited by D+Q. Senolytics tended to reduce aortic calcification (alizarin red) and osteogenic signaling (qRT-PCR, immunohistochemistry) in aged mice, but both were significantly reduced by senolytic treatment in hypercholesterolemic mice. Intimal plaque fibrosis (picrosirius red) was not changed appreciably by chronic senolytic treatment. This is the first study to demonstrate that chronic clearance of senescent cells improves established vascular phenotypes associated with aging and chronic hypercholesterolemia, and may be a viable therapeutic intervention to reduce morbidity and mortality from cardiovascular diseases.

Nonbiased Molecular Screening Identifies Novel Molecular Regulators of Fibrogenic and Proliferative Signaling in Myxomatous Mitral Valve Disease.

BACKGROUND: Pathological processes underlying myxomatous mitral valve degeneration (MMVD) remain poorly understood. We sought to identify novel mechanisms contributing to the development of this condition. METHODS AND RESULTS: Microarrays were used to measure gene expression in 11 myxomatous and 11 nonmyxomatous human mitral valves. Differential gene expression (thresholds P<0.05; fold-change >1.5) and pathway activation (Ingenuity) were confirmed using quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry. Contributions of bone morphogenetic protein 4 and transforming growth factor (TGF)-ß2 to differential gene expression were evaluated in vitro. Contributions of angiotensin II to differential pathway activation were examined in mice in vivo. A total of 2602 genes were differentially expressed between myxomatous and nonmyxomatous valves. Canonical TGF-ß signaling was increased in MMVD because of increased ligand expression and derepression of SMA mothers against decapentaplegic 2/3 signaling and was confirmed with quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry. Myxomatous valves demonstrated activation of canonical bone morphogenetic protein and Wnt/ß-catenin signaling and upregulation of their common target runt-related transcription factor 2. Our data set provided transcriptional and immunohistochemical evidence for activated immune cell infiltration. In vitro treatment of mitral valve interstitial cells with TGF-ß2 increased ß-catenin signaling at mRNA and protein levels, suggesting interactions between TGF-ß2 and Wnt signaling. In vivo infusion of mice with angiotensin II recaptured several changes in signaling pathways characteristic of human MMVD. CONCLUSIONS: These data support a new disease framework whereby activation of TGF-ß2, bone morphogenetic protein 4, Wnt/ß-catenin, or immune signaling plays major roles in the pathogenesis of MMVD. We propose these pathways act in a context-dependent manner to drive phenotypic changes that fundamentally differ from those observed in aortic valve disease and open novel avenues guiding future research into the pathogenesis of MMVD.

Transcriptional and phenotypic changes in aorta and aortic valve with aging and MnSOD deficiency in mice.

The purpose of this study was to characterize changes in antioxidant and age-related gene expression in aorta and aortic valve with aging, and test the hypothesis that increased mitochondrial oxidative stress accelerates age-related endothelial and aortic valve dysfunction. Wild-type (MnSOD(+/+)) and manganese SOD heterozygous haploinsufficient (MnSOD(+/-)) mice were studied at 3 and 18 mo of age. In aorta from wild-type mice, antioxidant expression was preserved, although there were age-associated increases in Nox2 expression. Haploinsufficiency of MnSOD did not alter antioxidant expression in aorta, but increased expression of Nox2. When compared with that of aorta, age-associated reductions in antioxidant expression were larger in aortic valves from wild-type and MnSOD haploinsufficient mice, although Nox2 expression was unchanged. Similarly, sirtuin expression was relatively well-preserved in aorta from both genotypes, whereas expression of SIRT1, SIRT2, SIRT3, SIRT4, and SIRT6 were significantly reduced in the aortic valve. Expression of p16(ink4a), a marker of cellular senescence, was profoundly increased in both aorta and aortic valve from MnSOD(+/+) and MnSOD(+/-) mice. Functionally, we observed comparable age-associated reductions in endothelial function in aorta from both MnSOD(+/+) and MnSOD(+/-) mice. Interestingly, inhibition of NAD(P)H oxidase with apocynin or gp91ds-tat improved endothelial function in MnSOD(+/+) mice but significantly impaired endothelial function in MnSOD(+/-) mice at both ages. Aortic valve function was not impaired by aging or MnSOD haploinsufficiency. Changes in antioxidant and sirtuin gene expression with aging differ dramatically between aorta and aortic valve. Furthermore, although MnSOD does not result in overt cardiovascular dysfunction with aging, compensatory transcriptional responses to MnSOD deficiency appear to be tissue specific.

TGF-ß signalling and reactive oxygen species drive fibrosis and matrix remodelling in myxomatous mitral valves.

AIMS: Myxomatous mitral valve disease (MMVD) is associated with leaflet thickening, fibrosis, matrix remodelling, and leaflet prolapse. Molecular mechanisms contributing to MMVD, however, remain poorly understood. We tested the hypothesis that increased transforming growth factor-ß (TGF-ß) signalling and reactive oxygen species (ROS) are major contributors to pro-fibrotic gene expression in human and mouse mitral valves. METHODS AND RESULTS: Using qRT-PCR, we found that increased expression of TGF-ß1 in mitral valves from humans with MMVD (n = 24) was associated with increased expression of connective tissue growth factor (CTGF) and matrix metalloproteinase 2 (MMP2). Increased levels of phospho-SMAD2/3 (western blotting) and expression of SMAD-specific E3 ubiquitin-protein ligases (SMURF) 1 and 2 (qRT-PCR) suggested that TGF-ß1 signalling occurred through canonical signalling cascades. Oxidative stress (dihydroethidium staining) was increased in human MMVD tissue and associated with increases in NAD(P)H oxidase catalytic subunits (Nox) 2 and 4, occurring despite increases in superoxide dismutase 1 (SOD1). In mitral valves from SOD1-deficient mice, expression of CTGF, MMP2, Nox2, and Nox4 was significantly increased, suggesting that ROS can independently activate pro-fibrotic and matrix remodelling gene expression patterns. Furthermore, treatment of mouse mitral valve interstitial cells with cell permeable antioxidants attenuated TGF-ß1-induced pro-fibrotic and matrix remodelling gene expression in vitro. CONCLUSION: Activation of canonical TGF-ß signalling is a major contributor to fibrosis and matrix remodelling in MMVD, and is amplified by increases in oxidative stress. Treatments aimed at reducing TGF-ß activation and oxidative stress in early MMVD may slow progression of MMVD.

Original research in the classroom: why do zebrafish spawn in the morning?

As part of an upper level undergraduate developmental biology course at the University of Minnesota Duluth, we developed a unit in which students carried out original research as part of a cooperative class project. Students had the opportunity to gain experience in the scientific method from experimental design all of the way through to the preparation of publication on their research that included text, figures, and tables. This kind of inquiry-based learning has been shown to have many benefits for students, including increased long-term learning and a better understanding of the process of scientific discovery. In our project, students designed experiments to explore why zebrafish typically spawn in the first few hours after the lights come on in the morning. The results of our experiments suggest that spawning still occurs when the dark-to-light transition is altered or absent. This is consistent with the work of others that demonstrates that rhythmic spawning behavior is regulated by an endogenous circadian clock. Our successes and failures carrying out original research as part of an undergraduate course should contribute to the growing approaches for using zebrafish to bring the excitement of experimental science to the classroom.

Sophorolipids and their derivatives are lethal against human pancreatic cancer cells.

INTRODUCTION: We have previously demonstrated that sophorolipids, a class of easily chemoenzymatically modifiable glycolipids, possess anti-inflammatory effects in vitro and in vivo. Since glycolipids have been shown to have anticancer activity, we investigated the effects of sophorolipids and their derivatives against pancreatic cancer. MATERIALS AND METHODS: Human pancreatic carcinoma cells were treated with increasing concentrations of sophorolipid natural mixture or select derivatives (ethyl ester, methyl ester, ethyl ester monoacetate, ethyl ester diacetate, acidic sophorolipid [AS], lactonic sophorolipid diacetate [LSD]) for 24 h and assessed for cell necrosis (cytotoxicity-lactate dehydrogenase release). Controls consisted of cells treated with media or vehicle alone and sophorolipid treatment of peripheral blood mononuclear cells. RESULTS: Sophorolipids demonstrated anticancer activity against human pancreatic carcinoma cells. Natural mixture mediated consistent cytotoxicity at all doses tested (20 +/- 4%). However, methyl ester derivative mediated much greater levels of cytotoxicity (63 +/- 5%) compared with other derivatives (ethyl ester diacetate, 36 +/- 6%, ethyl ester monoacetate, 18 +/- 7%; P < 0.05). In contrast, LSD- and AS-mediated toxicity was inversely proportional with dose (LSD, 40.3% at 0.5 mg/mL, 3.4% at 2.0 mg/mL; AS, 49% at 0.5 mg/mL, 0% at 2.0 mg/mL). Sophorolipid treatment did not affect peripheral blood mononuclear cells at all doses tested. CONCLUSIONS: These results suggest that sophorolipids and select derivatives may be effective in treating human pancreatic cancer. Furthermore select derivatives may use different mechanisms toward this end. The ability to chemoenzymatically modify sophorolipids can provide effective lead compounds toward the treatment of pancreatic cancer.