Miller, Jordan D.

Recent work by other investigators have shown that SIRT3 is an important regulator of endothelial function in obese patients or animals, as reductions in SIRT3 drive increased mitochondrial ROS (mtROS) production. The role of SIRT3 in atherosclerotic mice, however, remains unclear. Therefore, we hypothesized genetic inactivation of SIRT3 in hypercholesterolemic mice will impair vascular function, reduce antioxidant gene expression, and increase pro‐osteogenic signaling. We used Ldlr −⁄− /ApoB 100/100 mice that were either wild‐type (LA‐SIRT3 +/+ ) or null for SIRT3 (LA‐SIRT3 −⁄− ). After 12 weeks of western diet feeding, we assessed endothelial function of aorta using isolated organ bath chambers and measured changes in gene expression using qRT‐PCR. Consistent with our hypothesis, loss of SIRT3 significantly impaired relaxation to acetylcholine (Ach) (LA‐SIRT3 −⁄− : 48.2%±3.0, LA‐SIRT3 +/+ : 57.6%±1.7). Incubation with Mito‐Tempol significantly improved endothelial‐dependent relaxation in LA‐SIRT3 −⁄− mice (57.0%±2.9), but not in LA‐SIRT3 +/+ mice. Interestingly, losses of SIRT3 did not alter expression of MnSOD, ecSOD, or CuZnSOD. Gene expression of the late‐stage osteoblast marker, osterix, tended to be increased in LA‐SIRT3 −⁄− mice compared to wild‐type mice (p = 0.06). Collectively, losses of SIRT3 impair endothelial function and tend to increase osteogenic signaling in severe hypercholesterolemia. Importantly, these functional and molecular changes and may result from increased mtROS, which may serve as a viable therapeutic target to slow pathological processes during progression of atherosclerosis. Support or Funding Information NHLBI R01‐HL111121

Excess mitochondrial reactive oxygen species can drive endothelial dysfunction and vascular calcification in atherosclerosis. Recently, SIRT3 has been linked to the activity of the ROS‐degrading enzyme MnSOD through regulation of protein acetylation levels. It remains unclear, however, whether SIRT3 protects against endothelial dysfunction and osteogenic signaling in hypercholesterolemia. We hypothesized, that reduction of SIRT3 will impair endothelial function, decrease mitochondrial antioxidant capacity, and increase osteogenic signaling in hypercholesterolemic mice. We used Ldlr −/− /ApoB 100/100 (LA) mice that were either wild‐type (LA‐SIRT3 +/+ ) or null for SIRT3 (LA‐SIRT3 −/− ). After 12 months of western diet feeding, we assessed endothelial function in aorta using isolated organ chamber baths and measured changes in gene expression using qRT‐PCR. As expected, SIRT3 gene expression was significantly reduced in LA‐SIRT3 +/− mice compared to LA‐SIRT3 +/+ mice, and undetectable in LA‐SIRT3 −/− . Surprisingly, endothelial‐dependent relaxation to acetylcholine (MR Ach ) was unchanged between LA‐SIRT3 +/+ and LA‐SIRT3 −/− (37.0±4.4%, 38.2±4.4%). Endothelial‐independent relaxation (sodium nitroprusside) was also unchanged. MnSOD expression was reduced in LA‐SIRT3 −/− compared to LA‐SIRT3 +/+ (0.80 ± 0.02 vs 1.0±0.08, p < 0.05). In contrast, the late‐stage osteoblast marker, osterix, tended to increase in LA‐SIRT3 −/− (1.3 fold) but failed to reach statistical significance. Collectively, these results suggest that losses of SIRT3 do not impact endothelial function in advanced atherosclerosis, but may contribute to augmentation of osteogenic signaling and accelerated progression of vascular calcification. Support or Funding Information NHLBI

Background While recent work from our laboratory showed that DNA (cytosine‐5)‐methyltransferase 3 beta (DNMT3b) is significantly increased in tissue from humans with calcific aortic valve stenosis and is associated with increases in global genomic methylation, it is unknown whether the net effects of these changes are protective or deleterious. The objective of this study was to test the hypothesis that genetic reduction of DNMT3b allows for increased anti‐osteogenic gene expression, and consequently results in suppression of osteogenic signaling and slower progression of valvular stenosis. Methods We used Ldlr −/− /ApoB 100/100 (LA) mice that were wild‐type for DNMT3b (LA‐DNMT3b +/+ ) or deficient in DNMT3b (LA‐DNMT3b +/− ) and fed a western diet for six months. Results Using quantitative real‐time RT‐PCR, we confirmed that expression of DNMT3b was reduced by ~50% in valves from haploinsufficient mice. In line with our hypothesis, reductions in DNMT3b were associated with increases in expression of FABP4 (which opposes osteogenesis) and Smad6 (which opposes canonical BMP signaling). Similarly, expression of the osteogenic genes Msx2 and Spp1 were substantially reduced in DNMT3b‐deficient mice. Critically, aortic valve cusp separation distance was significantly greater in DNMT3b‐deficient mice, demonstrating slower progression of valvular dysfunction. No other gross abnormalities were noted in the mice. Conclusions Collectively, these data suggest that selectively reducing DNMT3b activity may be a viable therapeutic approach to slowing progression of calcific aortic valve stenosis in humans. Support or Funding Information Grant Funding Source: Supported by NHLBI

Aging is the leading predictor of acquired cardiovascular disease (CVD), and emerging evidence suggests cellular senescence plays a causal role in development of clinically-significant CVD. While we previously reported that senescent cell elimination delays or prevents progression of several aging phenotypes in aging mice, the responsible types of senescent cells contributing to cardiovascular dysfunction in vivo are not yet elucidated. To test the hypothesis that senescent endothelial cells are a major contributor to cardiovascular dysfunction, we developed Tie2-Cre; p16 Ink4a - LOX-ATTAC mice, which allows for specific elimination of p16 Ink4a+ senescent endothelial cells upon administration of AP20187 (which only affects cells carrying the conditionally-expressed ATTAC construct). Using a mouse model of diabetic vascular disease (40% fat diet for 12 weeks , HFD), we found that biweekly selective removal of senescent endothelial cells alleviated HFD-induced endothelial dysfunction in aorta (assessed ex vivo using isolated organ baths). Additionally, ejection fraction (assessed using in vivo echocardiography) was preserved in AP20187-treated mice (driving ATTAC-dependent removal of senescent endothelial cells) compared to their vehicle-treated controls (76.6%±4.2, 63.2%±4.2). In addition to improvement in cardiovascular function, metabolic parameters (HbA1c and fasting glucose levels) were improved by senescent endothelial cell elimination. Collectively, these data suggest that elimination of p16 Ink4a+ senescent endothelial cells may be a viable therapeutic strategy for alleviation of metabolic disease-induced cardiovascular dysfunction.

BACKGROUND: Dilated cardiomyopathy (DCM) is a major cause of heart failure and carries a high mortality rate. Myocardial recovery in DCM-related heart failure patients is highly variable, with some patients having little or no response to standard drug therapy. A genome-wide association study may agnostically identify biomarkers and provide novel insight into the biology of myocardial recovery in DCM. METHODS: A genome-wide association study for change in left ventricular ejection fraction was performed in 686 White subjects with recent-onset DCM who received standard pharmacotherapy. Genome-wide association study signals were subsequently functionally validated and studied in relevant cellular models to understand molecular mechanisms that may have contributed to the change in left ventricular ejection fraction. RESULTS: The genome-wide association study identified a highly suggestive locus that mapped to the 5′-flanking region of the CDCP1 (CUB [complement C1r/C1s, Uegf, and Bmp1] domain containing protein 1) gene (rs6773435; P =7.12×10 −7 ). The variant allele was associated with improved cardiac function and decreased CDCP1 transcription. CDCP1 expression was significantly upregulated in human cardiac fibroblasts (HCFs) in response to the PDGF (platelet-derived growth factor) signaling, and knockdown of CDCP1 significantly repressed HCF proliferation and decreased AKT (protein kinase B) phosphorylation. Transcriptomic profiling after CDCP1 knockdown in HCFs supported the conclusion that CDCP1 regulates HCF proliferation and mitosis. In addition, CDCP1 knockdown in HCFs resulted in significantly decreased expression of soluble ST2 (suppression of tumorigenicity-2), a prognostic biomarker for heart failure and inductor of cardiac fibrosis. CONCLUSIONS: CDCP1 may play an important role in myocardial recovery in recent-onset DCM and mediates its effect primarily by attenuating cardiac fibrosis.

Background: Plaque fibrosis and re-differentiation of vascular smooth muscle cells (VSMCs) from a contractile to a “synthetic” phenotype are hallmarks of advanced atherosclerosis. While in vitro studies demonstrated a clear role of oxidative stress in the loss of VSMC contractile phenotypes, the roles of specific antioxidant mechanisms and their therapeutic utility remain unclear. Here we test the hypothesis that overexpression of catalase protects against excess plaque fibrosis and losses in contractile phenotypes in vivo . Methods: We used littermate-matched, hypercholesteremic (ldlr -/- /ApoB 100/100 ; LA) mice lacking a transgenic construct (LA-CAT 0/0 ) or harboring a single copy of the human catalase gene (LA-CAT Tg/0 ), which were fed a Western Diet for 3 or 12 months. Isolated organ bath chambers, histopathological analyses, and quantitative RT-PCR were used to assess vasomotor function, structure, and molecular changes in aorta. Results: Oxidative stress increased with disease progression in aortic tissue from LA-CAT 0/0 mice and was modestly reduced in LA-CAT Tg/0 mice. Collagen deposition and fiber thickness increased with disease progression in LA-CAT 0/0 mice, and these increases in fiber thickness were attenuated in LA-CAT Tg/0 mice at 12 months and more pronounced in male mice. In parallel, we observed favorable rightward shifts in the vascular stress-strain relationship in LA-CAT Tg/0 mice that were more pronounced in aged male mice. Interestingly, these structural changes were associated with improved contractile responses to either PGF 2α or serotonin in male LA-CAT Tg/0 mice at 12 months. Consistent with these changes, the contractile marker calponin-1 was profoundly reduced with increasing age in LA-CAT 0/0 mice, with these changes being partially attenuated in aging, male LA-CAT Tg/0 mice. In vitro, treatment of VSMCs with PEG-catalase attenuated BMP2-dependent contractile protein expression. Conclusion: These results demonstrate that moderate overexpression of catalase can attenuate deleterious fibrotic and contractile changes associated with prolonged progression of atherosclerosis. Future mechanistic investigation exploring the context-dependence of these changes (e.g., sex dependence) is warranted.

Introduction: While increased hydrogen peroxide (H 2 O 2 ) accelerates atherosclerotic vascular disease, the role of catalase---which selectively metabolizes H 2 O 2 in lysosomes—is unclear. Given the established role of H 2 O 2 in accelerating osteogenic vascular calcification and vascular smooth muscle cell de-differentiation, we sought to determine the effects of reducing catalase in vivo on functional and molecular changes in aorta in early and late-stage atherosclerosis. Hypothesis: Haploinsufficiency of catalase will worsen vascular contractile function and promote ectopic osteogenesis in early and late-stage atherosclerosis. Methods: We used ldlr -/- /ApoB 100/100 (LA) mice, which were wild-type or heterozygous for catalase (LA-Cat +/+ or LA-Cat +/- , respectively) and fed a western diet for 3 or 12 months. Vasomotor function was assessed using an isolated organ chamber system. Gene expression of VSMC contractile proteins and osteogenic markers was quantified using qRT-PCR. Results: Endothelial function worsened over time in both strains of mice and was modestly worsened in LA-Cat +/- mice. Vascular contraction to PGF 2α was reduced with aging in LA-Cat +/+ mice, and unaffected by catalase deficiency at 3 months. Surprisingly, contractile tension in response to PGF 2α was increased in LA-Cat +/- at 12 months, with this effect being most pronounced in female mice. Consistent with previous literature, expression of VSMC contractile genes (smooth muscle α-actin and calponin-1) was decreased in all groups following 12 months of diet (p<0.0001), and expression of osteogenic genes (BMP2, Runx2, and osterix) increased with aging (p<0.05). Surprisingly, and in contrast to our contractile function data, these time-dependent changes in gene expression tended to be augmented in LA-Cat +/- mice compared to LA-Cat +/+ littermates. Conclusions: Our data suggest that systemic inactivation of one copy of catalase increases contractile function in aorta from aged, hypercholesterolemic mice while paradoxically increasing osteogenic signaling. Future investigation into the role of specific antioxidant enzymes in driving functional and structural changes in advanced vascular disease will be critical to informing therapeutic strategies in this domain.

Introduction: Senescent cell accumulation and senescence-associated secretory phenotype (SASP) are putative major mechanisms of tissue aging, and growing evidence suggests that pharmacological clearance of senescent cells slows progression of multiple age-related diseases. Here, we hypothesized that treating with a known senolytic, Fisetin (FIS), would reduce molecular drivers of fibrosis and calcification and ultimately slow disease progression of fibrocalcific aortic valve stenosis (FCAVS) in mice when initiated in mild/moderate stages of disease. Methods: Male and female Ldlr -/- / ApoB 100/100 mice were fed a western diet for 9 months and assigned to one of two treatment groups: FIS or vehicle, with treatment beginning at 6 months of diet. Results: Treatment with FIS reduced CDKN2A (p < 0.01) and SASP marker TNFα (p < 0.002) in both sexes, and consistent with our hypothesis was associated with reduced expression of the osteogenic markers RUNX2 (p < 0.021) and SPP1 (p < 0.006) in valve tissue from female mice. In contrast, expression of WNT5A was reduced in valves only from male mice (p < 0.004). In line with these molecular changes, valvular calcium burden (alizarin red staining) also tended to decrease in both sexes. Interestingly, TGFβ1 expression was significantly reduced (p < 0.018) in both sexes and was associated with reductions in collagen 3A1. These molecular changes in fibrogenic signaling manifested as a shift towards thinner collagen fibers only in male animals (p < 0.05). Functionally, slower progression of valvular dysfunction (declines in aortic valve cusp separation distance assessed with high-resolution echocardiography) was observed predominantly in females (p < 0.002). Conclusions: In conclusion, these data suggest that long-term, intermittent clearance of senescent cells robustly attenuates osteogenic and fibrogenic signaling in mild/moderate FCAVS, although the molecular, histopathological, and functional consequences of senolytic therapy may occur in a sex-specific manner. Future studies evaluating preclinical efficacy of senolytics should account for tissue and sex specificity and warrant longer-term treatment to assess the functional consequences of the biological and histopathological changes observed here.

Abstract Dynamic measures of resilience—the ability to resist and recover from a challenge—may be informative of the rate of aging before overt manifestations such as chronic disease, disability, and frailty. From this perspective mid-life resilience may predict longevity and late-life health. To test this hypothesis, we developed simple, reproducible, clinically relevant challenges, and outcome measures of physical resilience that revealed differences between and within age groups of genetically heterogeneous mice, and then examined associations between mid-life resilience and both lifespan and late-life measures of physiological function. We demonstrate that time to recovery from isoflurane anesthesia and weight change following a regimen of chemotherapy significantly differed among young, middle-aged, and older mice, and were more variable in older mice. Females that recovered faster than the median time from anesthesia (more resilient) at 12 months of age lived 8% longer than their counterparts, while more resilient males in mid-life exhibited better cardiac (fractional shortening and left ventricular volumes) and metabolic (glucose tolerance) function at 24 months of age. Moreover, female mice with less than the median weight loss at Day 3 of the cisplatin challenge lived 8% longer than those that lost more weight. In contrast, females who had more weight loss between Days 15 and 20 were relatively protected against early death. These data suggest that measures of physical resilience in mid-life may provide information about individual differences in aging, lifespan, and key parameters of late-life health.

Senescent cell accumulation has been observed in age-associated diseases including cardiovascular diseases. Senescent cells lack proliferative capacity and secrete senescence-associated secretory phenotype (SASP) factors that may cause or worsen many cardiovascular diseases. Therapies targeting senescent cells, especially senolytic drugs that selectively induce senescent cell removal, have been shown to delay, prevent, alleviate, or treat multiple age-associated diseases in preclinical models. Some senolytic clinical trials have already been completed or are underway for a number of diseases and geriatric syndromes. Understanding how cellular senescence affects the various cell types in the cardiovascular system, such as endothelial cells, vascular smooth muscle cells, fibroblasts, immune cells, progenitor cells, and cardiomyocytes, is important to facilitate translation of senotherapeutics into clinical interventions. This review highlights: (1) the characteristics of senescent cells and their involvement in cardiovascular diseases, focusing on the aforementioned cardiovascular cell types, (2) evidence about senolytic drugs and other senotherapeutics, and (3) the future path and clinical potential of senotherapeutics for cardiovascular diseases.