Thalji, Nassir M

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.

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)

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.

Increased BMP signaling and markers of osteogenic differentiation are strongly associated with regions of valvular calcification in aortic valves from humans with end-stage calcific aortic valve stenosis. While oxidative stress is also markedly elevated in these regions, it is unknown whether increasing antioxidant capacity can reduce pro-osteogenic signaling and valvular calcification. Therefore, we hypothesized that overexpression of manganese superoxide dismutase (MnSOD) can reduce pro-osteogenic signaling in the aortic valves from hypercholesterolemic mice, ultimately resulting in reduced valvular calcification and improved valvular function. We used Ldlr -/- /ApoB 100/100 (LA) mice that were wild-type for MnSOD (LA-MnSOD 0/0 ) or overexpressing MnSOD (LA-MnSOD Tg/0 ) and fed a western diet (TD88137) for six months. We first measured mRNA levels of the BMP ligands BMP2 and BMP4 (qRT-PCR), both of which were unchanged between LA-MnSOD 0/0 and LA-MNSOD Tg/0 mice. While there was a small but significant increase in Runx2 mRNA levels in LA-MnSOD Tg/0 mice compared to wild-type littermates (1.22±0.09, 1.0±0.09, qRT-PCR, p < 0.05.) gene expression levels of late stage osteoblast marker Sp7 were reduced by approximately 50% in LA-MnSOD Tg/0 compared to wild-type littermates (0.53±0.15, 1.0±0.24, qRT-PCR, p < 0.05). Sp7 protein levels (immunohistochemistry) were also significantly reduced in LA-MnSOD Tg/0 compared to wild-type littermates (164.5±12.3 RFU vs 259.6±10.4 RFU, p < 0.05). Critically, aortic valve calcification (Alizarin Red) was also significantly reduced in LA-MnSOD Tg/0 mice compared to their LA-MnSOD 0/0 littermates. Functionally, aortic valve cusp separation distance (high resolution echocardiography) was slightly improved in LA-MnSOD Tg/0 mice compared to LA-MnSOD 0/0 mice (0.98±0.04 mm vs 0.91±0.03 mm, p = n.s.). Collectively, these data suggest that increasing mitochondrial antioxidant capacity can reduce pro-osteogenic signaling and attenuate aortic valve calcification, and may be a potential therapeutic target to slow the progression of valvular calcification and dysfunction in patients with calcific aortic valve stenosis.

Increases in local angiotensin II (ANGII) signaling have been implicated in a number of diseases, including atherosclerosis, hypertension, and aneurysm. Endothelial dysfunction is strongly associated with increased ANGII signaling and the pathogenesis of these diseases. While increases in Runx2 have been implicated in cardiovascular calcification and vascular stiffening in diabetes, little is known regarding the role of Runx2 in the regulation of endothelial function. In the present study, we hypothesized that Runx2 is a key mediator of impaired vascular function and increased vascular wall stiffness provoked by ANGII. Wild-type mice (WT) and littermates that were deficient in one copy of Runx2 (HET) were implanted with osmotic minipumps containing saline or a pressor dose of ANGII (1000 ng/kg/min). Blood pressure was measured non-invasively using tail-cuff methods (CODA) and mice were sacrificed following 14 days of treatment. Changes in endothelium-dependent relaxation (relaxation to acetylcholine, R ACH ) and passive pressure-diameter relationships were measured in vitro (Living Systems arteriograph). In WT mice, treatment with ANGII for 14 days resulted in profound impairment in R ACH (85 ± 4% vs 47 ± 14% p<0.05) and slight reductions in vascular diameter at high distending pressures (~10% reduction in maximal diameter). While Runx2 deficiency did not alter R ACH or vascular distensibility/compliance in saline-treated mice, loss of one copy of Runx2 in ANGII-treated mice dramatically improved R ACH compared to their wild-type, ANGII-treated littermates. Interestingly, impairments in the pressure-diameter relationship with ANGII infusion were not affected by loss of one copy of Runx2. Critically, reductions in Runx2 did not significantly alter the pressor response to ANGII. In conclusion, our data suggest that Runx2 may play an integral role in the pathogenesis of ANGII-induced endothelial dysfunction, with these changes occurring independently of alterations in extracellular matrix remodeling and vascular stiffness. These data suggest that Runx2 may play a key, context-dependent role mediating vascular responses to ANGII.

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.

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.

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.

Activation of NADPH oxidase is a major contributor to hypertension, reactive oxygen species generation, endothelial dysfunction and increased vascular distensibility in angiotensin II (AngII)‐induced hypertension. Since exercise has been associated with increased antioxidant enzyme expression in blood vessels, we hypothesized that exercise training would improve endothelial function and vascular distensibility in mice with AngII‐induced hypertension. C57BL/6J mice received saline or AngII (1000 ng/kg/min) via osmotic minipumps, and were assigned to sedentary or treadmill exercise groups (13 m/min for 30 minutes, 5 days/week) for 14 days. Endothelium‐dependent relaxation in response to acetylcholine and arterial distensibility were assessed in carotid arteries using a pressurized arteriograph system. Gene expression was examined by qRT‐PCR. In AngII‐treated sedentary mice, endothelium‐dependent relaxation was impaired, while distensibility was higher than in saline‐infused sedentary mice. Compared to AngII‐infused sedentary mice, however, endothelium‐dependent relaxation was partially improved, and distensibility normalized in AngII‐infused exercised mice. In response to exercise, mRNA levels of SOD1 and SOD2 in aorta were increased, while mRNA levels of NOX2 were reduced. We conclude that exercise training can dramatically improve endothelial dysfunction and reverse derangements in carotid distensibility following prolonged AngII‐induced hypertension. Additional work examining mechanisms underlying our observed transcriptional changes will be critical to development of novel therapeutic targets.