Citations

Thrombin plays a major role in coagulation cascade and participates in regulation of vascular function through its effects on protease-activated receptors (PARs). The effect of thrombin inhibition on vascular and aortic valve function during prolonged hypercholesterolemia, however, remains unknown. In the present study, we tested the hypothesis that direct thrombin inhibition (DTI, dabigatran etexilate) slows progression of endothelial and aortic valve dysfunction in ldlr-deficient, apolipoprotein B100-only (LA) mice. Carotid arteries from LA mice were used for studies of endothelial function in isolated organ baths, and aortic valve function was evaluated using echocardiography. In placebo-treated mice, maximum relaxation to acetylcholine was reduced following 6 and 9 months of hypercholesterolemia (maximum relaxation to acetylcholine (MR ACH ) =75%± 3.7, and 67%± 3.6, respectively), but not affected by DTI (75%± 2.5, and 62%± 3.2, respectively). Relaxation to exogenous NO donors was also unaffected by DTI. Aortic valve function progressively deteriorated at 3, 6, and 9 months in placebo-treated mice (2.1 m/s± 0.04, 2.5 m/s± 0.1, and 2.9m/s ± 0.1, respectively), and was not affected by DTI (2.0m/s± 0.1, 2.5m/s± 0.1 and 3.0m/s ± 0.4, respectively). While PAR receptor expression was detected in aorta and aortic valve tissue, expression of SPHK1 (an indicator of PAR1 activity) was not altered by DTI. Expression of CTGF in aortic valves, however, tended to be reduced by DTI. In conclusion, our data suggest that DTI does not prevent vasomotor or aortic valve dysfunction in mice with prolonged hypercholesterolemia, but may attenuate pro-fibrotic signaling in aortic valves.

Introduction While progression of calcific aortic valve stenosis (CAVS) is known to be strongly associated with activation of pro-inflammatory and pro-osteogenic pathways, effective treatments for CAVS have remained elusive. TNF-α is a pleiotropic inflammatory cytokine and is upregulated in patients with CAVS. Hence, we hypothesized that TNF-α inhibition, using the neutralizing antibody infliximab, can slow the progression of CAVS in vivo . Methods We used Ldlr -/- Apob 100/100 mice, which develop robust CAVS in an age-dependent manner, as a model. Mice were fed a Western-type diet for 6 months, then randomized into one of two groups: 1) twice-weekly infliximab injections (10 mg/kg intraperitoneally; n=10), and 2) saline injections (n=9). Aortic valve function was evaluated using echocardiography, and gene expression in aortic valves was measured using quantitative real-time PCR. Data are presented as mean ± SEM. Results During the 3 months preceding treatment initiation, aortic valve cusp separation distance decreased progressively in both groups (reduction by 0.082±0.011 mm in control group, 0.095±0.016 mm in infliximab group; p =0.27). Three months after initiation of treatment, cusp separation continued to decrease in control mice (by 0.108±0.021 mm), while progression of valve dysfunction was slower by 42% in the infliximab group (reduction of 0.063±0.009 mm; p =0.03 vs control). Expression of the pro-inflammatory genes Vcam1 and Nos2 was significantly reduced in valves of the infliximab group (by 37% and 60%, respectively). Moreover, endothelium-dependent vasodilation was improved by infliximab (improvement in maximum relaxation to acetylcholine in infliximab group 18±6% compared to control). Conclusions This study is the first to report that in vivo inhibition of TNF-α slows progression of CAVS in mice with established valve disease. This beneficial effect was accompanied by reduction in pro-inflammatory gene expression and improvement of systemic endothelial function, suggesting infliximab may exert its effects in part by increasing nitric oxide bioavailability. The findings warrant further investigation into the role of TNF-α in initiation and progression of CAVS, as well as its potential as a therapeutic target in human CAVS.

Increases in mitochondrial reactive oxygen species (ROS) can contribute to endothelial dysfunction in conduit arteries by reducing nitric oxide bioavailability. SIRT3 is a protein deacetylase localized to mitochondria and regulates activity of numerous electron transport chain enzymes, and can be inactivated by increases in mitochondrial ROS. Whether reductions in SIRT3 are an independent contributor to endothelial dysfunction, however, are unknown. Therefore, we hypothesized that SIRT3 null mice (SIRT3 -/- ) will have impaired relaxation to acetylcholine (MR Ach ) compared to wild-type littermates (SIRT3 +/+ ), and that this difference will be exacerbated by Western diet feeding for 3 months (TD88137, 42% of kcal from fat, 0.2% cholesterol). Endothelial function was measured using isolated organ bath chambers, and endothelium-dependent (MR Ach ) and endothelium-independent (sodium nitroprusside, MR SNP ) relaxation were measured. In contrast to our hypothesis, MR ACH was virtually identical between SIRT3 +/+ and SIRT3 -/- fed a standard chow diet (MR ACH = 68±2% and 69±3%; respectively), and MR SNP also remained unchanged between the two genotypes. In groups fed a Western diet, however, endothelium-dependent relaxation was significantly impaired in SIRT3 -/- mice compared to SIRT3 +/+ littermates (MR ACH = 49±4% and 58±3%, respectively; p<0.05). MR SNP was not altered by either Western diet feeding or SIRT3 deficiency. Interestingly, impairments in MR Ach in SIRT3 -/- mice fed a Western diet were not rescued with acute incubation of apocynin (an NAD(P)H oxidase inhibitor) or mito-tempol (a mitochondria-targeted antioxidant), suggesting that chronic antioxidant and/or acetyltransferase inhibition may be required to improve vasomotor function in this model. Collectively, our data suggest that SIRT3 plays a major role in regulating endothelial function with dietary stress, and may be a useful therapeutic target to reduce cardiovascular risk.

Introduction: Myxomatous mitral valve disease (MMVD) is a degenerative condition characterized by tissue fibrosis and matrix remodeling which ultimately progresses to leaflet prolapse. While recent work has shown that transforming growth factor beta (TGF-β) signaling contributes to the pathogenesis of MMVD, upstream regulators of this and other pathways remain poorly understood. Hypothesis: We sought to use high-throughput RNA sequencing coupled with microRNA (miRNA) sequencing to identify novel molecular targets as well as upstream regulators contributing to MMVD. Methods: We conducted mRNA and miRNA sequencing on normal (n = 10) and myxomatous human mitral valve samples (n = 10). Differential expression was identified using linear modeling and parallel random forest analyses. Canonical pathways were identified by Ingenuity Pathway Analyses (IPA). Predicted miRNA targets were identified using TargetScanHuman 6.2. Results: We found 2784 mRNAs that were differentially expressed between normal and myxomatous mitral valves, which IPA largely categorized in to pro-fibrotic, matrix remodeling and cellular proliferation signaling. In miRNA sequenced from the same samples, 67 miRNAs were differentially-expressed between normal and myxomatous mitral valves. Increased expression of TGF-β ligands, collagen isoforms, and matrix metalloproteinases were associated with reductions in miRNAs predicted to target them. Conversely, mRNA levels of the “protective” genes TGFβ-induced factor homeobox 1, salt-inducible kinase 1, TIMP metallopeptidase inhibitor 4, and cyclin-dependent kinase inhibitor 1C mRNA levels were decreased in myxomatous tissue, and miRNAs predicted to target these genes (e.g., miR-656, miR-379-3p, miR-664a-3p, and miR-34c-5p) were significantly increased. Conclusions: Collectively, these data not only identify novel genes that are differentially regulated in MMVD, but also suggest miRNAs may play an active role in suppressing key protective molecules in MMVD. Thus, anti-mIRs therapy may be a viable therapeutic target to restore anti-fibrotic and anti-proliferative molecules in the valve and slow progression of MMVD. Future mechanistic studies will lay a critical foundation for translational work in these areas.

Increasing age is a major risk factor for calcific aortic valve disease (CAVD). Interestingly, SIRT6 knockout mice have a marked progeroid phenotype, and we recently reported that sirtuin enzyme expression is dramatically reduced with aging and that SIRT6 expression is reduced further in valves from patients with end-stage CAVD. It is unknown, however, whether experimentally reducing SIRT6 promotes osteogenic signaling in the valve and ultimately accelerates progression of CAVD. Thus, we used cultured mouse aortic valve interstitial cells and ldlr-deficient, apolipoprotein B100-only mice (LA) that were SIRT6 wild-type (LA-SIRT6 +/+ ) or heterozygous (LA-SIRT6 +/- ) and fed a Western diet for 3 or 12 months to determine the role of SIRT6 in valve calcification. In vitro , reduction of SIRT6 increased histone acetylation and significantly increased mRNA and protein levels of the osteogenic genes Runx2 and Sp7 in response to bone morphogenetic protein 2 (100ng/ml BMP2 for 18 hours), and siRNA knockdown of SIRT6 increased mRNA levels of Sp7 even in the absence of exogenous bone morphogens. Using high-resolution ultrasound to evaluate aortic valve function in vivo, we found that 3 month old LA-SIRT6 +/- mice did not have significant impairments in valve function compared to LA-SIRT6 +/+ mice. In contrast, 12 month old LA-SIRT6 +/- had dramatically worsened aortic valve dysfunction and stenosis compared to LA-SIRT6 +/+ mice, which was also associated with reductions in left ventricular ejection fraction. Collectively, our data strongly suggest SIRT6 plays a critical role in the tonic repression of osteogenic signaling in the aortic valve, and that age-related reductions in SIRT6 are likely to increase susceptibility to valve calcification in response to risk factors for CAVD such as hypercholesterolemia. Collectively, increasing activity of SIRT6 or reducing acetylation of its targets may serve as viable therapeutic strategies to slow progression of age-related valvular calcification and stenosis.

Endothelial cell senescence promotes inflammation and impairs endothelium-dependent vasodilation. While aging, hypercholesterolemia, and caloric excess are known to drive cellular senescence in part through activation of mTOR, it is unknown whether reducing mTOR signaling can improve vasomotor function in aging mice. Here, we used hypercholesterolemic mice (Ldlr -/- /apoB 100/100 , or LA) carrying intact copies of mTOR (LA-mTOR +/+ ) or were deficient in one copy of mTOR (LA- mTOR +/- ). Mice were fed Western Diet for 6 or 12 months, and vasomotor function in aorta was evaluated using isolated organ chamber baths. Maximum relaxation to acetylcholine (MR ACH ) was progressively impaired from 6 months to 12 months in LA-mTOR +/+ mice (55±4% and 30±4%, respectively). In contrast to our hypothesis, however, MR ACH was not significantly improved in LA-mTOR +/- mice at either time point (62±3% and 35±5%, respectively). Interestingly, a subgroup analysis showed significant improvement in MR ACH in male mice at the 6-month point (male LA-mTOR +/+ mice = 30 ± 9% vs male LA-mTOR +/- mice = 55±4%; p < 0.01), whereas vasomotor function in female mice was nearly identical across groups. There was no significant difference at the 12-month point (male LA-mTOR +/+ mice = 19 ± 3% vs male LA-mTOR +/- mice = 22 ± 5%). Relaxation to acetylcholine was attenuated by L-NAME at 6 months and 12 months (but identical between genotypes) and relaxation to nitroprusside was identical between strains at each time point, suggesting that changes in endothelial function were not masked by compensatory mechanisms (e.g., endothelium-derived hyperpolarizing factors). Furthermore, reduction of mTOR did not elicit changes in maximal tension generated in response to Prostaglandin F 2 α at either time point. Collectively, despite compelling evidence from in vitro model systems, genetic reduction of mTOR signaling in vivo does not appear to be sufficient to improve vasomotor function in our model of aging hypercholesterolemic mice. Our data do, however, shed light on a potential role of sex in dictating phenotypic changes in early to moderate stages of disease, which warrants further investigation.

Marfan syndrome (MFS) is a genetic disease with a mutation for the microfibrillar constituent protein fibrillin-1 being the most prevalent. MFS is often associated with progressive aortic root dilation, ultimately progressing to aortic aneurysm and dissection. While recent work has shown that increased angiotensin-II receptor type-1 and transforming growth factor beta (TGF-β) signaling contributes to aneurysm formation in aorta, efficacious therapeutic targets remain elusive. Given previous reports of progeriod phenotypes in a subset of Marfan patients, we sought to determine whether there are molecular changes that are consistent with accelerated aging in a mouse model of Marfan syndrome. In mice carrying a loss-of-function mutation in fibrillin-1, we assessed aortic root dimensions by echocardiography and gene expression levels of TGF-β1-3, runt related transcription factor 2 (RUNX2), and the cellular senescent marker CDKN2A by quantitative real time PCR at 3 and 14 months of age. As expected, aortic sinus dimensions did not change significantly with aging in wild type mice, but increased dramatically in fibrillin-1 mutant mice compared to wild-type littermate controls and with age (p < 0.05 for both). TGF-β1 ligand expression paralleled age and disease-dependent changes in aortic dimensions, however TGF-β2 and TGF-β3 mRNA levels did not. Aortic dilatation was associated with increased gene expression of RUNX2 with aging and in marfanoid mice. Interestingly, fibrillin-1 mutant mice demonstrated marked increases in expression of the anti-proliferative cell-cycle checkpoint protein CDKN2A at both time points, and correlated with changes in TGF-β1 (R 2 =0.54) and RUNX2 (R 2 =0.69) mRNA. CDNK2A gene expression patterns, however, demonstrated a poor correlation with expression of TGF-β2 and TGF-β3 (R 2 =0.04 and 0.06. respectively). Collectively, these data lend insight into novel mechanisms that may regulate development of aortic root dilation in patients MFS and are the first to implicate increased senescent cell burden in Marfan syndrome. Furthermore, we propose that clearance of senescent cells could be a viable therapeutic intervention to slow progression aortic root-dilation and aneurysm in patients with Marfan syndrome.

Increasing age is the greatest risk factor for development and progression of calcific aortic valve disease (CAVD), and mitochondrial dysfunction has been implicated in the pathogenesis of cardiovascular calcification. Previous findings by our group suggested a significant reduction in expression of multiple sirtuin (SIRT) isoforms in normocholesterolemic aged mouse aortic valves, however, it is unclear if losses in SIRT3—a major mitochondrial SIRT isoform—modulates progression of aortic valve calcification. Therefore, we hypothesized that loss of SIRT3 in a hypercholesterolemic mouse model of CAVD will result in augmented calcium burden in aortic valve, increased osteogenic signaling, and impaired aortic valve function. To test this we used mice that were Ldlr -/- /ApoB 100/100 mice that were either wild-type (LA-SIRT3 +/+ ) or null for SIRT3 (LA-SIRT3 -/- ) and fed a western diet (TD88137) for 12 months. Alizarin red was used to quantitate calcium burden, qRT-PCR was used to measure changes in mRNA levels, and high-resolution echocardiography was used to assess aortic valve function (cusp separation distance). In line with our hypothesis, we observed a substantial increase in calcium burden in LA-SIRT3 -/- mice compared to their LA-SIRT3 +/+ littermates(11.7±4.0, 4.8±1.8; respectively). Interestingly, expression of Runx2 and osterix—classic markers of osteogenic differentiation—were decreased in LA-SIRT3 -/- mice compared to their LA-SIRT3 +/+ littermates. Expression of Msx2, were markedly increased in aortic valve tissue from LA-SIRT3-deficient mice. Despite these histological and molecular changes, SIRT3 deletion did not alter cusp separation distance (LA-SIRT3 +/+ = 0.81±0.04; LA-SIRT3 -/- = 0.81±0.02). Collectively, this data suggest losses in SIRT3 can contribute to accelerated valve calcification through unclear molecular mechanisms, but these changes are not sufficient to drive reductions in cusp separation distance. Additional experiments delineating the histopathological and molecular sequelae of SIRT3 deletion will be critical to understanding its role in the pathogenesis of CAVD.

Marfan syndrome is a connective tissue disorder frequently driven by mutations in the fibrillin-1 gene (Fbn1). This results in multiple aberrant cardiovascular phenotypes, including aortic aneurysm and mitral valve prolapse. While the molecular changes underlying aortic aneurysm formation have been extensively studied, the molecular underpinnings of mitral valve prolapse in this syndrome remain poorly understood. Therefore, we hypothesized that smad2/3 phosphorylation would be significantly increased in both aorta and mitral valve from Fbn1 +/1037G mice with Marfan syndrome and associated with impaired anti-inflammatory signaling in both tissues. We used young (3 months) mice that were wild-type (Fbn1 +/+ ) or fibrillin-1 mutant (Fbn1 +/1037G ) and fed a normal chow diet. qRT-PCR was used to measure mRNA levels in aortic arch and mitral valve tissue, and fluorescent immunohistochemistry to evaluate changes in canonical TGFβ signaling. Consistent with previous reports, TGF-β1 mRNA levels were increased in Fbn1 +/1037G compared to wild-type mice in both aorta and mitral valve. Interestingly, while p-SMAD2/3 protein levels were increased in aorta from Fbn1 +/1037G mice, we were surprised to find that p-SMAD2/3 protein levels were decreased in mitral valve from Fbn1 +/1037G mice compared their wild-type littermates. Given the emerging role of inflammatory signaling in accelerated development of Marfan phenotypes, we sought to determine whether there were compensatory or maladaptive changes in IL10 (a known inhibitor of p-SMAD2/3). In aorta, IL10 mRNA levels were decreased in Fbn1 +/1037G compared to Fbn1 +/+ mice, suggesting a maladaptive response. In contrast, IL10 levels in mitral valve tissue were significantly increased in Fbn1 +/1037G mice compared to their wild-type littermates, suggesting a compensatory/protective response. Collectively, these data suggest that tissue-specific changes in IL10 levels may modulate p-SMAD2/3 signaling in aorta and mitral valve, which may be a key permissive step in the onset of Marfan-related phenotypes. Future work to experimentally determine the role of IL10 in the regulation of canonical and non-canonical TGFβ signaling and the penetrance of Marfan-associated phenotypes is warranted.

Previous studies have reported increases in oxidative stress due to reductions in antioxidant enzymes in calcified regions of stenotic human aortic valves. Whether reductions in antioxidant enzymes are adaptive, maladaptive, or an epiphenomenon in calcifying aortic valves remains unclear. Thus, the purpose of this study was to test whether overexpression of catalase reduces valvular calcification and slows progression of aortic valve stenosis in mice. Wild type (WT) mice with calcific aortic valve disease (ldlr-deficient, apoB100-only) were crossed with transgenic mice that overexpress human catalase (TG). All mice were fed a western diet (TD88137; Harlan Teklad). Functional echocardiogram data was collected and analyzed at early (3 months) and advanced (12 months) stages of valve disease, and valves were OCT embedded, sectioned, and stained for calcium levels (Alizarin Red). The transgenic valves show a slight increase in calcification compared to wild type counterparts. While there were no differences in valve function between genotypes at the early time point, cusp separation was decreased in the transgenic mice (0.74 ± 0.02 mm) compared to their wild-type littermates (0.84 ± 0.03 mm) at the 12 month time point (p < 0.05). In a subset of animals, valve calcification also tended to increase in transgenic mice at both time points compared to their wild-type littermates. In conclusion, overexpression of catalase resulted in a surprising finding of accelerated valvular calcification and stenosis. Interestingly, this finding aligns with the findings from several negative clinical trials testing antioxidant therapies in humans with atherosclerosis, suggests that the net impact of changing redox balance on disease progression are highly context dependent, and that increasing antioxidant capacity may not be a viable strategy to slow progression of calcific aortic valve disease in humans.