Hagler, Michael A

In Marfan syndrome, mutations in the fibrillin gene are known to result in aortic dilatation and aneurysm formation, which is associated with impaired endothelial function and increased vascular stiffness. Previous studies suggested these phenotypes may be partly attributable to overactivation of TGF β signaling, which leads to increased reactive oxygen species production, extracellular matrix production, and increased matrix metalloproteinase activity in aortic wall. To our knowledge, however, there are no studies comprehensively evaluating the relationships between conduit vessel vasomotor function, vascular stiffness, and aortic dilatation in young and aged models of Marfan syndrome. Thus, the aim of this study was to test the hypothesis that carotid arterial vasomotor dysfunction and increased vascular stiffness precede aortic dilatation in fibrillin mutant mice. We used wild‐type (+/+) or fibrillin mutant (+/C1037G) mice fed a normal chow diet for 3 months (young) or 14 months (old), measured changes in vasomotor function and vascular stiffness in carotid arterial segments using pressurized arteriography, aortic dimensions using high resolution echocardiography, gene expression using qRT‐PCR, and protein levels using immunohistochemistry. Consistent with our hypothesis, young +/C1037G mice had impaired endothelium‐dependent relaxation to acetylcholine (ACH) compared to +/+ littermates, but did not exhibit changes in vascular stiffness. In contrast, old +/C1037G mice did not have significant changes in vasomotor responses to ACH compared to +/+ mice, but displayed markedly increased vascular stiffness compared to their wild‐type littermates. Aortic dimensions were significantly increased in young +/C1037G mice compared to their +/+ littermates, and these changes were exaggerated further with aging. Consistent with previous reports, we found increased TGF beta‐1 expression and canonical TGF beta signaling (p‐Smad2/3 levels) in +/C1037G mice. Interestingly, we also observed increased expression of Runx2 with aging in +/+ mice with aging, and expression of Runx2 was substantially increased in +/C1037G mice compared to their +/+ littermates at both time points. Collectively, our data show that vasomotor function and vascular stiffness in carotid arterial segments change independent of changes in aortic root diameter, suggesting significant heterogeneity in the mechanical and molecular responses to fibrillin deficiency throughout the vascular system. Support or Funding Information NHLBI (R01‐111121)

The structural protein, fibrillin, is important for maintaining the strength and reliability of connective tissue found in blood vessels and heart valves. Mutations in fibrillin‐1 result in Marfan syndrome and leads to left‐sided heart valve regurgitation, aortic root dilatation and aortic aneurysm. While previous studies have focused predominantly on young fibrillin‐mutant mice, hemodynamically significant disease generally does not emerge in patients with fibrillin mutations until later in life. Thus, this study aims to evaluate molecular and functional changes in heart valves from aged fibrillin mutant mice. Wild‐type (+/+) or fibrillin mutant (+/C1037G) mice were fed normal chow diet for 3 months (young) or 14 months (old). Heart valve function, aortic dimensions, and cardiac function were evaluated using high‐resolution echocardiography, and gene expression was measured using qRT‐PCR. Consistent with previous reports, we found no evidence of mitral regurgitation in young mice of either genotype. In old +/C1037G mice, however, we did find evidence of mitral regurgitation (~5%). In contrast, aortic valve regurgitation was evident in a relatively large proportion of +/C1037G mice at both ages (~20% at both time points), and was not detected in any +/+ animals. Interestingly, left ventricular dimensions were not associated with the presence of mitral regurgitation, but aortic root dilatation was present in all mice with aortic valve regurgitation. To gain insight into the molecular underpinnings of these phenotypic changes, we measured expression of IL‐10 (an anti‐inflammatory gene) and Runx2 (a pro‐proliferative gene that drives matrix remodeling), both of which are genes previously implicated in the pathogenesis of mitral and aortic valve disease. In young animals, expression of IL‐10 in mitral valves tended to be increased in +/C1037G mice compared to +/+ littermates. With aging, IL‐10 increased substantially in mitral valves from in +/+ mice, and increased further in +/C1037G littermates. Similar trends were observed in expression of Runx2 in mitral valves from both genotypes of mice. While IL‐10 followed a similar pattern in aortic valve tissue from young and old mice of both genotypes, Runx2 was not significantly increased across any of the groups. Collectively, while our data suggest that there are substantial, potentially adaptive molecular changes that occur during development of regurgitant valvular diseases, aortic dilatation may be a key early factor driving development of aortic regurgitation. Future experimental work aimed at determining the relative mechanical and molecular contributions to development of heart valve regurgitation will be critical to development of novel therapeutic strategies in patients with syndromes secondary to fibrillin mutations. Support or Funding Information NHLBI (R01‐HL111121)

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

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.

Increases in vascular calcification (VC) and fibrosis are a near ubiquitous finding with aging. Here, we sought to test the hypothesis that Metformin (MET)—an AMPK activator entering large clinical trials that is thought to attenuate multimorbidity associated with aging—attenuates key molecular processes known to drive vascular calcification (VC) and fibrogenic signaling in vascular smooth muscle cells (VSMCs) in vitro. Given recent reports highlighting the importance of mitochondria and sirtuins with aging, we also tested the hypothesis that MET-driven reductions in osteogenic signaling are associated with upregulation of SIRT3. We used exogenous BMP2 (100 ng/ml) and TGFβ1 (10 ng/ml) as osteogenic and fibrogenic stimuli, respectively. Consistent with prior work from our lab, treatment with BMP2 for 24 hours robustly increased expression of the osteogenic genes Msx2 (3.9±1.5 fold-change, FC), Runx2 (3.5±1.2 FC), and osterix (17.2±5.2 FC) in VSMCs. Critically, co-treatment with Metformin dramatically reduced expression of these genes by 68 ± 22%, 58 ±20%, and 63 ± 4%, respectively. Subsequent experiments treating cells with exogenous TGFβ1 resulted in significant induction of the fibrogenic marker/known TGFβ1 target versican (1.7±0.4 FC), an effect that was largely attenuated by co-treatment with MET (58±3%). Treatment with MET did not, however, impact TGFβ1-induced increases in CTGF or fibronectin. Treatment with either BMP2 or TGFβ1 resulted in significant increases in mRNA levels of SIRT3 (2.5 ±0.11 and 1.4 ±0.17 FC, respectively), whereas treatment with MET resulted in modest and less consistent increases in SIRT3 mRNA (1.3 ± 0.2 FC). Interestingly, however, co-treatment with BMP2+MET or TGFβ1+MET resulted in uniform suppression of BMP2/ TGFβ1-induced increases in SIRT3 (54±19% and 25±12%, respectively). Collectively, these data suggest that treatment with MET may be a viable strategy to attenuate age-associated increases in osteogenic and fibrogenic signaling in conduit vessels, and suggest further in vivo investigation into therapeutic efficacy is warranted.

We recently reported activation of TGF-β superfamily pathways and activation of Wnt/β-catenin signaling in the pathogenesis of myxomatous mitral valve disease (MMVD), which may converge upon increased Runx2 expression. We also demonstrated that chronic in vivo administration of angiotensin II (AngII) recaptures various molecular aberrations characteristic of human disease -including increased TGF-β signaling and activation of Runx2- in mouse mitral valves. Here, we sought to determine whether Runx2 haploinsufficiency can attenuate molecular and/or phenotypic effects of AngII infusion in mouse mitral valves. We implanted littermate matched Runx2+/+ and Runx2+/- mice with osmotic minipumps delivering AngII (1000ng/kg/min) or saline for 2 weeks. We measured changes in mRNA (qRT-PCR) and protein (immunohistochemistry) levels in mitral valves, and assessed mitral valve function by echocardiography. In Runx2+/+ mice, AngII elicited increased mRNA levels of TGF-β3 and the TGF-β target COL3A1, and increased staining for canonical (p-SMAD2/3) and non-canonical (p-AKT) TGF-β signaling proteins. Runx2 haploinsufficiency attenuated AngII-induced changes in COL3A1 expression and p-AKT staining, without impacting AngII-mediated alterations in TGF-β3 and p-SMAD2/3. Evaluating collagen thickness by picrosirius red staining and visualization under circularly polarized light, AngII infusion in Runx2+/+ mice was associated with increased collagen density at the base of posterior mitral valve leaflets, a change that was abrogated by Runx2 haploinsufficiency. Similarly, Runx2-haploinsufficiency was again protective when examining mRNA and protein levels of the proliferation marker KI67 in response to AngII. Echocardiography identified a trend towards increased mitral valve regurgitation following AngII infusion that was unaltered by Runx2 haploinsuffiency. Collectively, these data suggest that increases in Runx2 expression in the mitral valve may serve to orchestrate tissue fibrosis and cellular proliferation that are hallmarks of MMVD, and that future pharmacological strategies aimed at reducing activity of Runx2 in the mitral valve may yield viable non-surgical alternatives for the management of this condition.