Citations

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.