Miller, Jordan D.

Background: Calcific aortic valve stenosis (CAVS) is characterized by the progressive accrual of aortic valvular calcium (AVC), resulting in impaired valvular compliance and mechanics and impaired cardiac function leading to heart failure (HF). In a phase 2 trial of 23 patients with moderate CAVS, ataciguat (ATA), a soluble guanylate cyclase activator, relative to placebo slowed the deposition of AVC, decreases in aortic valve area (AVA; by continuity equation), and increases in diastolic dysfunction and left ventricular (LV) mass. ATA may have other favorable effects on ventricular, valvular, and/or vascular pathophysiology relevant to patients with CAVS. Hypothesis: We hypothesized that slowing the rate of AVC deposition is associated with improvements in valvular compliance and in measures of cardiac structure and function. Methods: A phase 2 study randomized patients with moderate CAVS 1:1 to receive ATA 200 mg/day or placebo for up to 12 months (NCT02481258). The primary endpoint was change in AVC assessed by cardiac CT, and measures of aortic valve function and cardiac structure and function were also assessed. AVA was calculated by the Modified Gorlin (Hakki) equation to additionally assess valvular compliance. Correlations between AVC, AVA, and other measures of cardiac structure and function were assessed by exploratory linear mixed models. Results: Changes in AVC from baseline were negatively correlated with changes in AVA (slope = −0.0002 [95% CI −0.0004, 0.0001]; Figure 1), such that patients with the least AVC deposition had minimal changes in AVA. Reciprocally, changes from baseline in AVA were positively correlated with changes in cardiac output (CO; slope = 0.16 [95% CI 0.13, 0.18]; Figure 2). Treatment with ATA, compared with placebo, was associated with improvements in systolic function. Changes in AVC deposition correlated with changes in CO (slope = −0.0007 [95% CI −0.0021, 0.0008]; Figure 3), and measured improvements in CO were generally more observed in patients with less of an increase in AVC, which were more frequently observed in those treated with ATA compared with placebo. Conclusions: These data suggest that slowing the rate of AVC deposition with ATA may result in improvements in CO through improved myocardial function and valvular compliance. Larger controlled trials are needed to assess such favorable myocardial/valvular effects that may help to preserve functional capacity and slow progression to HF.

Background Myxomatous mitral valve disease (MMVD) is a degenerative disorder marked by excess tissue fibrosis and matrix remodeling, leading to leaflet prolapse. While recent studies linked transforming growth factor beta (TGF-β) activation to MMVD development and progression, upstream regulators of this and other modulatory/causal pathways remain largely unexplored. Objectives In this study, we utilized high-throughput sequencing to conduct unbiased analyses of mRNA and microRNA (miRNA) levels in myxomatous and healthy mitral valve tissues, aiming to uncover novel molecular mechanisms involved in MMVD. Methods We defined differentially expressed mRNAs and miRNAs transcripts displaying a fold-change > 1.5 and P < .05. Pathway analysis was performed using Ingenuity Pathway Analysis and DAVID, with key findings validated via qRT-PCR. A total of 2378 transcripts were differentially expressed between myxomatous and normal valves. Established pathways, including TGF-β signaling, were confirmed as major contributors to MMVD. Additionally, Random Forests with Boruta Feature Selection identified transcripts with a 95% likelihood of importance, and subsequent pathway analysis on this subset of genes revealed unique signaling pathways. Results Most notably, circadian rhythm disruption emerged as a novel, highly ranked pathway in MMVD. Key miRNAs, such as miR-1, miR-133a, and miR-217 were highlighted as highly relevant, with miRNA–mRNA interactions displaying distinct molecular signatures predictive of MMVD. Conclusions Collectively, this study represents the first comprehensive analysis of both miRNA and mRNA expression in MMVD, revealing both established and novel disease-associated pathways. The discovery of circadian rhythm disturbances and new regulatory miRNAs suggests promising directions for further research and potential therapeutic targets for nonsurgical treatment strategies in patients with MMVD.

Introduction: Previous findings suggest that sirtuin 6 (SIRT6), a nuclear histone deacetylase, is involved in fundamental processes that regulate aging, which include DNA damage repair and maintenance of genomic stability. It remains unclear, however, whether SIRT6 overexpression can mitigate age-related vascular dysfunction due to multiple mechanisms of posttranslational enzyme inactivation (oxidation, ubiquitination-dependent degradation, etc). We hypothesized that overexpression of SIRT6, will improve both endothelium-dependent relaxation and smooth muscle contractile function and improve molecular mechanisms underlying these functions in healthy aged mice. Methods: We used normocholesterolemic mice that were either wild-type for SIRT6 (WT) or overexpressing one copy of SIRT6 (TG) and studied them at either 3 or 18 months of age. We assessed molecular changes in SIRT6 expression, cellular senescence, and vascular contractile protein expression using qPCR, and aortic rings in isolated organ bath chambers to measure endothelium-dependent relaxation to acetylcholine (ACH), endothelium-independent relaxation to sodium nitroprusside (SNP), and contractile responses to serotonin (5HT) and PGF 2α . Results: As expected, overexpression of SIRT6 in TG mice resulted in ~3-fold mRNA expression increase compared to WT mice in aorta. In line with our previous reports, SIRT6 mRNA levels significantly increased with age in aorta. CDKN2A, a cellular senescence marker, increased with aging in WT mice. Surprisingly, SIRT6 overexpression did not attenuate this increase, with TG mice tending to have higher CDKN2A expression than WT at 18m. ACTA2, a contractile protein marker, had modest reductions with age in WT mice, and surprisingly, overexpression of SIRT6 did not alter ACTA2 levels compared to WT at either time point. Finally, overexpression of SIRT6 did not mitigate age-related changes in vascular relaxation or contractile function. Conclusion: Collectively, these data at the surface level suggest that overexpressing SIRT6 does not protect against conduit vessel dysfunction in aged mice and is associated with paradoxical increases in senescent cell markers. It remains elusive whether there are other molecular modulators of aging that offset or alter the activity of SIRT6 with its genetic overexpression. Therefore, future studies investigating molecular determinants of SIRT6 bioactivity and its tissue/context dependence with aging are warranted.

Calcific aortic valve disease (CAVD) is a highly active process, and upregulation of osteogenic signaling is thought to be a major driver to initiation and progression of disease. While previous reports by our group and others demonstrated dysregulation of antioxidants and pro-oxidants in calcified aortic valves, it remains unclear if loss of antioxidant is a causal event driving valvular calcification and dysfunction. We hypothesized that MnSOD deficiency—an essential mitochondrial antioxidant—would result in accelerated reductions of cusp separation distance, increased osteogenic signaling, and augmented calcium deposition in a hypercholesterolemic mouse model of CAVD. We used ldlr -/- / ApoB 100/100 (LA) mice that were either wild-type (LA-MnSOD +/+ ) or MnSOD-deficient (LA-MnSOD +/- ), fed a Western Diet for 3 or 6 months. We used qRT-PCR and immunohistochemistry to measure changes in pro-osteogenic signaling, histopathological analyses of calcium burden (Alizarin Red), and echocardiography to assess aortic valve function. BMP2—a pro-osteogenic marker—increased over time with Western diet feeding, though MnSOD-deficiency did not exacerbate these increases. mRNA levels of pro-osteogenic markers (Runx2, Msx2) were also unchanged with either time on diet or with experimental reduction of MnSOD. Surprisingly, protein levels of late- stage osteoblast marker, osterix, were modestly decreased at the three month time point in in LA-MnSOD +/- mice compared to age-matched wild-type littermates. Consistent with our hypothesis, however, loss of mitochondrial antioxidant capacity substantially increased osterix protein levels and calcium burden at the 6 month time point compared to age-matched wild-type littermates. Functionally, loss of mitochondrial antioxidant capacity did not alter aortic valve cusp separation distance at both 3 and 6 month time points. Collectively, these data suggest that loss of MnSOD does not alter disease initiation but instead augments osteogenic signaling and valvular calcification in established aortic valve disease. Future studies aimed at targeting mitochondrial oxidative stress could elucidate novel strategies to slow progression of established CAVD.

Marfan Syndrome (MFS) is often associated with progressive aortic root dilation, ultimately leading to aortic aneurysm and dissection. While recent work showed increased transforming growth factor beta (TGF-β) signaling contributes to aneurysm formation in aorta, efficacious therapeutic targets remain elusive. Consistent with prior reports of progeroid phenotypes in a subset of Marfan patients we previously demonstrated increased cellular senescence in a MFS mouse model. Here, we sought to determine whether administration of senolytic drugs reduces cellular senescent markers, TGF-β signaling, and aortic root dilation in aged MFS mice by assessing aortic root dimensions by echocardiography and gene expression levels by quantitative real time PCR at 15 months of age following treatment with vehicle (VEH) or the senolytic compound Fisetin for one month. As expected, aortic sinus dimensions were increased dramatically in fibrillin-1 mutant mice compared to wild-type controls and were associated with increased expression of TGF-β1, the senescence marker CDKN2A, the matrix remodeling enzyme MMP2, and the osteogenic and proliferative gene RUNX2. Consistent with previous data from our group, Fisetin treatment dramatically reduced CDKN2A expression in marfanoid mice compared to VEH, which was associated with reductions in TGF-β1, MMP2, and RUNX2. Phenotypically, we observed modest reductions in aortic sinus dimensions compared to VEH, but no substantive change in ascending aortic dilatation. Collectively, these data suggest that senescent cells are significant contributors to molecular changes occurring in MFS and clearance of senescent cells with Fisetin slows the progression aortic root-dilation in fibrillin-1 mutant mice.

Perivascular adipose tissue releases inflammatory paracrine factors that accelerate development of vascular dysfunction, atherosclerosis, and large vessel diseases such as abdominal aortic aneurysm. Our group has shown that chronic hypercholesterolemia and chronological aging both significantly increase senescent cell burden in vascular and endothelial cell populations of large arteries, but the effects of chronic hypercholesterolemia on senescent cell burden in perivascular adipose tissue—as well as the biological impact of reducing those senescent cells using pharmacological or genetic interventions—is poorly understood. Here, we sought to investigate the effects of chronic, intermittent senescent cell clearance by treating mice with Fisetin—a flavenoid shown to be a potent senolytic in multiple tissue types—and its potential synergies with risk factor mitigation. In brief, “Reversa” mice (ldlr-deficient, apoB100-only mice in which lipids can be lowered with a genetic switch) were fed a Western diet for 6 months, at which point they were randomized to one of three groups for 2 additional months: 1) continued progression on Western Diet (WD), 2) risk factor mitigation/lipid lowering with a genetic switch (LL) or 3) weekly, intermittent treatment with Fisetin + lipid lowering (FIS+LL). Compared to WD mice, LL mice had modest reductions in perivascular adipose inflammatory markers such as IL-6, CCL2, and CD-68, as well as the pro-oxidant Nox2; however, LL mice experienced no impact on expression of the pro-inflammatory cytokine TNFalpha. Interestingly, while intermittent Fisetin treatment and lipid lowering in the FIS+LL group had a significant additive effect on reducing IL-6, Nox2, and CD68, these changes were associated with modest reductions in expression of p16 ink4a or p21 (both of which are markers of senescent cell burden). Collectively, these data suggest that Fisetin is likely to modestly reduce senescent cell burden in perivascular fat, while effectively synergizing with lipid lowering to reduce a subset of inflammatory markers in this tissue, which may ultimately have implications for management of multiple age-associated vascular diseases.

Activation of soluble guanylate cyclase (sGC), a key downstream target of nitric oxide (NO) signaling, is reduced in valve tissue from patients with fibrocalcific aortic valve stenosis (FCAVS). We hypothesized that restoring NO signaling pathway using Ataciguat (ATA)—an activator of oxidized sGC—would slow progression of aortic valve calcification. To determine whether Ataciguat attenuates bone morphogenetic protein (BMP2) signaling in vitro, we treated mouse aortic valve interstitial cells with exogenous BMP2(200ng/ml) with or without ATA(10nm), and demonstrated that ATA significantly reduced canonical BMP2 signaling by more than 50% (p< 0.01). To determine whether Ataciguat slows progression of FCAVS in vivo , ldlr -/- /apoB 100/100 mice were assigned to one of two groups:1) 9 months of western diet feeding (WD), or 2) 6 months of WD followed by 3 months of WD + ATA (5mg/kg ATA). In this model, Ataciguat attenuated BMP signaling in aortic valve tissue (p < 0.05), reduced aortic valve calcification (p < 0.05), and prevented progression of aortic valve dysfunction (p < 0.05). Finally, to determine whether ATA slows progression of calcification in humans, we treated patients with mild-to-moderate FCAVS with placebo or ATA (200 mg/day) for 6 months. In this Phase II clinical trial, ATA significantly slowed progression of aortic valve calcification (computed tomography imaging; p = 0.05) and also tended to slow progressive reductions in aortic valve area and valvular stenosis. Collectively, we believe this is the first study to demonstrate the pre-clinical and clinical utility of pharmacologically stimulating sGC to attenuate osteogenic signaling and slow progression of aortic valve disease in mice and humans, and creates a unique opportunity for larger trials focused on the effectiveness of this strategy to delay or prevent surgery in FCAVS.

While the strong connection between oxidative stress and atherosclerosis has many to hypothesize that antioxidant therapy may be viable therapeutic target in combating progression of atherosclerosis, numerous clinical trials of antioxidant interventions that scavenge multiple reactive oxygen species have been negative. Here, we test the hypothesis that overexpression of catalase—an enzyme which selectively degrades H 2 O 2 —plays a protective role against the initiation and expansion of calcific lesions in advanced atherosclerotic plaques. In vitro, we used aortic smooth muscle cells (SMC) from Ldlr -/- ApoB 100/100 (LA) mice that were either wild-type for human catalase (LA-Cat 0/0 ) or overexpressing human catalase (LA-Cat Tg/0 ). After treatment with BMP2 or TNFα for 24 hours, we found that catalase-overexpressing VSMCs had modest reductions in TNFα-induced inflammatory markers but paradoxical increases in mRNA levels of BMP2-induced osterix and the SMAD-specific target ID1. To determine if these effects were conserved in vivo, we analyzed aortic tissue from LA mice fed a Western diet for 3 or 12 months. Surprisingly—and unlike our cell-based experiments—aorta from LA-Cat Tg/0 tended to have higher expression of inflammatory markers compared to their LA-Cat 0/0 littermates. Similarly, expression of osterix was increased in LA-Cat Tg/0 mice compare to their LA-Cat 0/0 littermates, which was also associated with an increase in intimal plaque calcification. Collectively, these data strongly suggest that systemically overexpression of catalase (a close parallel to systemic administration of a selective H 2 O 2 -scavenging antioxidant) does not result in molecular, histopathological, or functional benefits in large arteries from hypercholesterolemic mice. Future studies aimed at characterizing the complex interplay between reactive oxygen species and specific intracellular signaling pathways are an essential next step to understanding the role of endogenous reactive oxygen species in the pathogenesis of atherosclerotic vascular disease.

Sirtuin-6 (SIRT6) is a protein deacetylase that functions in several pathways related to aging, as complete loss of SIRT6 in mice elicits multiple age-associated phenotypes and premature death. While increasing age is a major risk factor for development of calcific aortic valve disease (CAVD), it is unclear whether loss of SIRT6 accelerates aortic valve calcification. Here, we report the role of SIRT6 in a subset of aortic valve interstitial cell progenitors (neural crest-derived VICs) by crossing mice carrying Cre recombinase under the control of a PAX3 promoter with mice harboring a SIRT6 gene that can be conditionally inactivated (SIRT6 FL/+ ). We hypothesized that tissue-specific reductions of SIRT6 would increase calcification, increase pro-osteogenic signaling, and impair aortic valve function. We examined mice of the genotypes Cre-/SIRT6 FL/+ or Cre+/ SIRT6 FL/+ at two different time points: young (3 months) and old (14-24 months). First, we used qRT-PCR to demonstrate that SIRT6 was effectively reduced in the aortic valve from PAX3-cre/SIRT6 FL/FL mice. Although reductions in SIRT6 did not change aortic valve calcium (Alizarin Red) in young mice, reducing SIRT6 increased valvular calcium levels in old mice compared to their WT littermates. Interestingly, protein levels of osterix were substantially increased in young, SIRT6-deficient mice compared to WT littermates but were paradoxically decreased in old SIRT6-deficient mice. Regardless of age, histopathological, or molecular changes, we did not observe detectable changes in cusp separation distance at any time point. Collectively, these data suggest that, contrary to our hypothesis, depletion of SIRT6 in neural crest cells—which are key progenitors of valve interstitial cells—is not sufficient to impair aortic valve function. Importantly, this finding suggests that age-associated reductions in SIRT6 may be a permissive context for development of CAVD but relies heavily on the presence of additional risk factors.

Key Points Activin A is implicated in profibrotic and prosenescent kidney injury and correlates with kidney injury markers in animals and humans. Follistatin, through activin A antagonism, reduces senescence burden, macrophage infiltration, and proinflammatory pathway activation in murine diabetic kidney disease. Follistatin and other antagonists of activin A signaling pathways may be promising, novel therapeutics for diabetic kidney disease. Background Circulating activin A, an inflammatory mediator implicated in profibrotic kidney injury and cellular senescence-induced adipose tissue dysfunction, is increased in human diabetic kidney disease (DKD) and directly correlates with kidney dysfunction. We tested the hypothesis that activin A increases kidney injury, senescent cell abundance, and macrophage infiltration in DKD and antagonism through follistatin (FS) therapy diminishes these effects. Methods An accelerated nephropathy type 2 diabetes (db/db) mouse model was generated by implantation of angiotensin II-loaded osmotic minipumps resulting in increased albuminuria and glomerular and tubular injury. Kidney repair effects of FS (5 µ g intraperitoneal; two doses) were assessed through markers of kidney injury, fibrosis, inflammation, cellular senescence, and macrophage infiltration. In vitro studies examined antiactivin effects of FS on high glucose-exposed human monocytes, renal fibroblasts, and renal tubule epithelial cells. Results Activin A antagonism with FS reduced senescence (p19), proinflammatory (including senescence-associated secretory phenotype), and profibrotic markers including activin A. FS improved kidney morphology, restored podocyte markers (nephrin and Wilms tumor-1), and reduced kidney injury biomarkers, albuminuria and kidney fibrosis. FS decreased kidney macrophage and leukocyte infiltration and absent in melanoma 2 inflammasome activation. FS seemed to suppress inflammation through the toll-like receptor-4/NF kappa-light-chain-enhancer of activated B cells pathway in vivo further supported in human macrophages in vitro . In addition, FS reduced hyperglycemia-induced renal fibroblast activation and renal tubule epithelial cell senescence in vitro . Conclusions Activin A is a mediator of kidney injury through macrophage-associated inflammation in murine DKD. FS acts through senomorphic activities which inhibit profibrotic, proinflammatory, and prosenescence signaling by activin A. Hence, antiactivin targeting may aid in the development of a promising, novel therapeutic for DKD.