Atorvastatin reduces calcification in valve interstitial cells via the NF-κB signalling pathway by promoting Atg5-mediated autophagy.

Aortic valve calcification (AVC) is a common cardiovascular disease and a risk factor for sudden death. However, the potential mechanisms and effective therapeutic drugs need to be explored. Atorvastatin is a statin that can effectively prevent cardiovascular events by lowering cholesterol levels. However, whether atorvastatin can inhibit AVC by reducing low-density lipoprotein (LDL) and its possible mechanism of action require further exploration. In the current study, we constructed an in vitro AVC model by inducing calcification of the valve interstitial cells. We found that atorvastatin significantly inhibited osteogenic differentiation, reduced the deposition of calcium nodules in valve interstitial cells, and enhanced autophagy in calcified valve interstitial cells, manifested by increased expression levels of the autophagy proteins Atg5 and LC3B-II/I and the formation of smooth autophagic flow. Atorvastatin inhibited the NF-κB signalling pathway and the expression of inflammatory factors mediated by NF-κB in calcified valve interstitial cells. The activation of the NF-κB signalling pathway led to the reversal of atorvastatin's effect on enhancing autophagy and alleviating valve interstitial cell calcification. In conclusion, atorvastatin inhibited the NF-κB signalling pathway by upregulating autophagy, thereby alleviating valve interstitial cell calcification, which was conducive to improving AVC.

Integrative genomic analyses identify candidate causal genes for calcific aortic valve stenosis involving tissue-specific regulation.

There is currently no medical therapy to prevent calcific aortic valve stenosis (CAVS). Multi-omics approaches could lead to the identification of novel molecular targets. Here, we perform a genome-wide association study (GWAS) meta-analysis including 14,819 cases among 941,863 participants of European ancestry. We report 32 genomic loci, among which 20 are novel. RNA sequencing of 500 human aortic valves highlights an enrichment in expression regulation at these loci and prioritizes candidate causal genes. Homozygous genotype for a risk variant near TWIST1, a gene involved in endothelial-mesenchymal transition, has a profound impact on aortic valve transcriptomics. We identify five genes outside of GWAS loci by combining a transcriptome-wide association study, colocalization, and Mendelian randomization analyses. Using cross-phenotype and phenome-wide approaches, we highlight the role of circulating lipoproteins, blood pressure and inflammation in the disease process. Our findings pave the way for the development of novel therapies for CAVS.

[DIAGNOSIS AND TREATMENT OF ELEVATED LIPOPROTEIN(A) IN ISRAEL: CONSENSUS STATEMENT FROM THE ISRAEL SOCIETY FOR RESEARCH, PREVENTION AND TREATMENT OF ATHEROSCLEROSIS AND ISRAEL SOCIETY FOR CLINICAL LABORATORY SCIENCES].

INTRODUCTION: Lipoprotein(a) [Lp(a)] is composed of 2 major protein components, a low-density lipoprotein (LDL) cholesterol-like particle containing apolipoprotein B (apo B) that is covalently bound to apolipoprotein(a). Its level is predominantly genetically determined, and it is estimated that 20% to 25% of the population have Lp(a) levels that are associated with increased cardiovascular risk. Elevated Lp(a) is related to increased vascular inflammation, calcification, atherogenesis and thrombosis, and is considered an independent and potentially causal risk factor for atherosclerotic cardiovascular diseases and calcified aortic valve stenosis. Recent data demonstrate that Lp(a) testing has the potential to reclassify patients' risk and improve cardiovascular risk prediction, and therefore could inform clinical decision-making regarding risk management. Statins and ezetimibe are ineffective in lowering Lp(a) levels, whereas proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors have a modest effect on Lp(a) reduction. Nevertheless, RNA interference-based therapies with potent Lp(a)-lowering effects are in advanced stages of development, and clinical trials are underway to confirm their benefit in reducing cardiovascular events. This scientific consensus document was developed by a committee that consisted of representatives from the Israeli Society for the Research, Prevention and Treatment of Atherosclerosis, and the Israeli Society for Clinical Laboratory Sciences, in order to create uniformity in Lp(a) measurement methods, indications for testing and reporting of the results, aiming to improve the diagnosis and management of elevated Lp(a) in clinical practice.

Indoxyl Sulfate-Induced Valve Endothelial Cell Endothelial-to-Mesenchymal Transition and Calcification in an Integrin-Linked Kinase-Dependent Manner.

Calcific Aortic Valve Disease (CAVD) is a significant concern for cardiovascular health and is closely associated with chronic kidney disease (CKD). Aortic valve endothelial cells (VECs) play a significant role in the onset and progression of CAVD. Previous research has suggested that uremic toxins, particularly indoxyl sulfate (IS), induce vascular calcification and endothelial dysfunction, but the effect of IS on valve endothelial cells (VECs) and its contribution to CAVD is unclear. Our results show that IS reduced human VEC viability and increased pro-calcific markers RUNX2 and alkaline phosphatase (ALP) expression. Additionally, IS-exposed VECs cultured in pro-osteogenic media showed increased calcification. Mechanistically, IS induced endothelial-to-mesenchymal transition (EndMT), evidenced by the loss of endothelial markers and increased expression of mesenchymal markers. IS triggered VEC inflammation, as revealed by NF-kB activation, and decreased integrin-linked kinase (ILK) expression. ILK overexpression reversed the loss of endothelial phenotype and RUNX2, emphasizing its relevance in the pathogenesis of CAVD in CKD. Conversely, a lower dose of IS intensified some of the effects in EndMT caused by silencing ILK. These findings imply that IS affects valve endothelium directly, contributing to CAVD by inducing EndMT and calcification, with ILK acting as a crucial modulator.

Coexistence Case of Papillary Fibroelastoma and Lambl's Excrescence of the Left Heart, Which Is Occasionally Associated with Significant Stroke.

Papillary fibroelastoma (PFE) is a benign tumor that arises mostly from left-sided valves. PFE can cause stroke, and surgical resection may be needed. Lambl's excrescence (LE) is a filiform valvular lesion and is considered a possible cause of stroke. A 79-year-old man with light-headedness and left-sided hemiparesis was diagnosed with stroke. Transesophageal echocardiography (TEE) revealed a round-shaped mobile mass in the left ventricular outflow tract (LVOT), which was considered the cause of the stroke. Surgical resection was performed transaortically, and during surgery, a mass was incidentally detected on the noncoronary cusp (NCC), which was also resected followed by aortic valve replacement. Pathology confirmed that the mass in the LVOT was a PFE and that the filiform mass on the NCC was LE. We herein report a rare case of PFE in the LVOT and coexisting LE on the NCC. A careful examination via TEE helps to identify other possible causes of stroke hidden behind the obvious cause.

Association between serum beclin 1 level and cardiac valvular calcification in hemodialysis patients.

BACKGROUND: Cardiovascular calcification is a pervasive issue throughout chronic kidney disease (CKD) progression. Autophagy, a fundamental cellular process, exerts significant influence on various cardiac pathologies, including arrhythmias, atherosclerosis, heart failure, and notably, valvular, and vascular calcifications. Beclin-1, a crucial eukaryotic protein, plays a major regulatory role in autophagy as part of the phosphatidylinositol-3-kinase (PI3K) complex. Recent evidence suggests a protective role for Beclin-1-mediated autophagy in CKD vascular calcification, raising its potential as a novel therapeutic target in this context. WE AIMED TO: Investigate the association between serum Beclin 1 levels and the presence of cardiovascular valvular calcification in hemodialysis patients. RESULTS: This study evaluated a cohort of 102 hemodialysis patients, evenly divided into two groups based on echocardiographic findings. All participants underwent serum Beclin 1 measurement and transthoracic echocardiography. Patients with acute kidney injury, active malignancy, or diabetes were excluded. Our study revealed significant differences between the two groups in terms of: Serum Beclin 1 levels, all parameters of lipid profile, prevalence of ischemic heart disease, serum albumin levels and Total calcium. Echocardiography in Group 1 showed that most cases (60.78%) exhibited mild aortic valve calcification. Additionally, significant relationships were observed between Beclin 1 and: ischemic heart disease (p=0.011) Aortic valve calcification on echocardiography (p < 0.001) Interestingly, lower Beclin 1 levels were associated with more severe valve calcification. A Beclin 1 cutoff value of ≤ 35.5 ng/ml demonstrated the highest sensitivity (98%) and specificity (92%). CONCLUSION: Our findings suggest that the serum Beclin 1 level could be incorporated into a predictive model for cardiac valvular calcification in hemodialysis patients.

Autotaxin inhibition attenuates the aortic valve calcification by suppressing inflammation-driven fibro-calcific remodeling of valvular interstitial cells.

BACKGROUND: Patients with fibro-calcific aortic valve disease (FCAVD) have lipid depositions in their aortic valve that engender a proinflammatory impetus toward fibrosis and calcification and ultimately valve leaflet stenosis. Although the lipoprotein(a)-autotaxin (ATX)-lysophosphatidic acid axis has been suggested as a potential therapeutic target to prevent the development of FCAVD, supportive evidence using ATX inhibitors is lacking. We here evaluated the therapeutic potency of an ATX inhibitor to attenuate valvular calcification in the FCAVD animal models. METHODS: ATX level and activity in healthy participants and patients with FCAVD were analyzed using a bioinformatics approach using the Gene Expression Omnibus datasets, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and western blotting. To evaluate the efficacy of ATX inhibitor, interleukin-1 receptor antagonist-deficient (Il1rn-/-) mice and cholesterol-enriched diet-induced rabbits were used as the FCAVD models, and primary human valvular interstitial cells (VICs) from patients with calcification were employed. RESULTS: The global gene expression profiles of the aortic valve tissue of patients with severe FCAVD demonstrated that ATX gene expression was significantly upregulated and correlated with lipid retention (r = 0.96) or fibro-calcific remodeling-related genes (r = 0.77) in comparison to age-matched non-FCAVD controls. Orally available ATX inhibitor, BBT-877, markedly ameliorated the osteogenic differentiation and further mineralization of primary human VICs in vitro. Additionally, ATX inhibition significantly attenuated fibrosis-related factors' production, with a detectable reduction of osteogenesis-related factors, in human VICs. Mechanistically, ATX inhibitor prohibited fibrotic changes in human VICs via both canonical and non-canonical TGF-ß signaling, and subsequent induction of CTGF, a key factor in tissue fibrosis. In the in vivo FCAVD model system, ATX inhibitor exposure markedly reduced calcific lesion formation in interleukin-1 receptor antagonist-deficient mice (Il1rn-/-, P = 0.0210). This inhibition ameliorated the rate of change in the aortic valve area (P = 0.0287) and mean pressure gradient (P = 0.0249) in the FCAVD rabbit model. Moreover, transaortic maximal velocity (Vmax) was diminished with ATX inhibitor administration (mean Vmax = 1.082) compared to vehicle control (mean Vmax = 1.508, P = 0.0221). Importantly, ATX inhibitor administration suppressed the effects of a high-cholesterol diet and vitamin D2-driven fibrosis, in association with a reduction in macrophage infiltration and calcific deposition, in the aortic valves of this rabbit model. CONCLUSIONS: ATX inhibition attenuates the development of FCAVD while protecting against fibrosis and calcification in VICs, suggesting the potential of using ATX inhibitors to treat FCAVD.

Identification of hub genes in calcific aortic valve disease.

Calcific aortic valve disease (CAVD) is a heart valve disorder characterized primarily by calcification of the aortic valve, resulting in stiffness and dysfunction of the valve. CAVD is prevalent among aging populations and is linked to factors such as hypertension, dyslipidemia, tobacco use, and genetic predisposition, and can result in becoming a growing economic and health burden. Once aortic valve calcification occurs, it will inevitably progress to aortic stenosis. At present, there are no medications available that have demonstrated effectiveness in managing or delaying the progression of the disease. In this study, we mined four publicly available microarray datasets (GSE12644 GSE51472, GSE77287, GSE233819) associated with CAVD from the GEO database with the aim of identifying hub genes associated with the occurrence of CAVD and searching for possible biological targets for the early prevention and diagnosis of CAVD. This study provides preliminary evidence for therapeutic and preventive targets for CAVD and may provide a solid foundation for subsequent biological studies.

miRNA Regulation of Cell Phenotype and Parietal Remodeling in Atherosclerotic and Non-Atherosclerotic Aortic Aneurysms: Differences and Similarities.

Aortic aneurysms are a serious health concern as their rupture leads to high morbidity and mortality. Abdominal aortic aneurysms (AAAs) and thoracic aortic aneurysms (TAAs) exhibit differences and similarities in their pathophysiological and pathogenetic features. AAA is a multifactorial disease, mainly associated with atherosclerosis, characterized by a relevant inflammatory response and calcification. TAA is rarely associated with atherosclerosis and in some cases is associated with genetic mutations such as Marfan syndrome (MFS) and bicuspid aortic valve (BAV). MFS-related and non-genetic or sporadic TAA share aortic degeneration with endothelial-to-mesenchymal transition (End-Mt) and fibrosis, whereas in BAV TAA, aortic degeneration with calcification prevails. microRNA (miRNAs) contribute to the regulation of aneurysmatic aortic remodeling. miRNAs are a class of non-coding RNAs, which post-transcriptionally regulate gene expression. In this review, we report the involvement of deregulated miRNAs in the different aortic remodeling characterizing AAAs and TAAs. In AAA, miRNA deregulation appears to be involved in parietal inflammatory response, smooth muscle cell (SMC) apoptosis and aortic wall calcification. In sporadic and MFS-related TAA, miRNA deregulation promotes End-Mt, SMC myofibroblastic phenotypic switching and fibrosis with glycosaminoglycan accumulation. In BAV TAA, miRNA deregulation sustains aortic calcification. Those differences may support the development of more personalized therapeutic approaches.

Transcriptomic Analysis of Mineralized Adipose-Derived Stem Cell Tissues for Calcific Valve Disease Modelling.

Calcific aortic valve disease (CAVD) is characterized by the fibrosis and mineralization of the aortic valve, which leads to aortic stenosis and heart failure. At the cellular level, this is due to the osteoblastic-like differentiation of valve interstitial cells (VICs), resulting in the calcification of the tissue. Unfortunately, human VICs are not readily available to study CAVD pathogenesis and the implicated mechanisms in vitro; however, adipose-derived stromal/stem cells (ASCs), carrying the patient's specific genomic features, have emerged as a promising cell source to model cardiovascular diseases due to their multipotent nature, availability, and patient-specific characteristics. In this study, we describe a comprehensive transcriptomic analysis of tissue-engineered, scaffold-free, ASC-embedded mineralized tissue sheets using bulk RNA sequencing. Bioinformatic and gene set enrichment analyses revealed the up-regulation of genes associated with the organization of the extracellular matrix (ECM), suggesting that the ECM could play a vital role in the enhanced mineralization observed in these tissue-engineered ASC-embedded sheets. Upon comparison with publicly available gene expression datasets from CAVD patients, striking similarities emerged regarding cardiovascular diseases and ECM functions, suggesting a potential link between ECM gene expression and CAVDs pathogenesis. A matrisome-related sub-analysis revealed the ECM microenvironment promotes the transcriptional activation of the master gene runt-related transcription factor 2 (RUNX2), which is essential in CAVD development. Tissue-engineered ASC-embedded sheets with enhanced mineralization could be a valuable tool for research and a promising avenue for the identification of more effective aortic valve replacement therapies.

Biomechanical Remodeling of Aortic Valve Interstitial Cells During Calcified Lesion Formation In Vitro.

Healthy aortic heart valves are essential to the regulation of unidirectional blood flow. Calcific aortic valve disease (CAVD) is an actively progressive disease that involves the disorganization of valve cells and accumulation of calcium deposits on the aortic valve leaflets. CAVD involves disruption of cell environment homeostasis that prior cell culture models have found difficult to portray and model. As it is still poorly understood how tissue stiffening associates with lesion formation, here, we implement a novel 3D culture platform to characterize the relationship between mechanical stress and tissue remodeling and analyze how the application of pro-osteogenic stimulation dysregulates the native ability of valve cells to organize its matrix. Through a temporal study of macroscopic remodeling, we determine that aortic valve interstitial neo-tissues undergo varying stiffness and mechanical stress, demonstrate greater myofibroblastic gene expression, and show greater remodeling activity in the outer surface of the neo-tissue in a banding pattern when cultured in osteogenic growth medium. In human aortic valve interstitial cells cultured in osteogenic growth medium, we observed an increase in stress but significant decreases in myofibroblastic gene expression with the addition of growth factors. In summary, we are able to see the interplay of biochemical and biomechanical stimuli in valvular remodeling by using our platform to model dynamic stiffening of valve interstitial neo-tissues under different biochemical conditions.

Aortic valve and arterial calcification in patients with familial hypercholesterolemia.

Heterozygous familial hypercholesterolemia (heFH) is an autosomal dominant lipid metabolism disorder. Its prevalence is 1:250-1:300 people in the population. Patients with heFH have an up to 13-fold increased risk of premature coronary artery disease (CAD). If left untreated, men and women with heFH typically develop early CAD before the ages of 55 and 60, respectively. There is evidence that coronary artery calcification (CAC) and aortic valve calcification (AoVC) are more prevalent in FH patients than in the general population. It is documented that CAC and AoVC are predictors of increased risk of cardiovascular morbidity and mortality in heFH patients, like in the general population. However, the etiology and pathogenesis of vascular calcification in FH patients is not well understood. Risk factors for vascular calcification include age, increased levels of atherogenic lipoproteins, Lp(a), increased blood pressure, and inflammation. There are convincing data from clinical studies and animal atherosclerotic mouse models using low-density lipoprotein receptor (LDL-R) knockout mice that the vascular calcification processes in FH are associated with LDL-R mutations, probably partly due to a higher total cholesterol burden of FH subjects. Data from animal models as well as clinical studies indicate that the Wnt/beta-catenin pathway components and LDL receptor-related proteins 5 and 6 (LRP-5/6) might be involved in calcification processes in FH patients. The purpose of the review is to describe the prevalence of coronary and aortic calcification and its risk factors in FH patients. The review covers data about the role of the Wnt/beta-catenin pathway and factors modulating calcification processes.

Insights From the Histopathologic Analysis of Acquired and Genetic Thoracic Aortic Aneurysms and Dissections.

OBJECTIVE: The purpose of this study was to apply contemporary consensus criteria developed by the Society for Cardiovascular Pathology and the Association for European Cardiovascular Pathology to the evaluation of aortic pathology, with the expectation that the additional pathologic information may enhance the understanding and management of aortic diseases. METHODS: A scoring system was applied to ascending aortic specimens from 42 patients with heritable thoracic aortic disease and known genetic variations and from 86 patients from a single year, including patients with known genetic variations (n = 12) and patients with sporadic disease (n = 74). RESULTS: The various types of lesions of medial degeneration and the overall severity of medial degeneration overlapped considerably between those patients with heritable disease and those with sporadic disease; however, patients with heritable thoracic aortic disease had significantly more overall medial degeneration (P = .004) and higher levels of elastic fiber fragmentation (P = .03) and mucoid extracellular matrix accumulation (P = .04) than patients with sporadic thoracic aortic disease. Heritable thoracic aortic disease with known genetic variation was more prevalent in women than in men (27.2% vs 9.8%; P = .04), and women had more severe medial degeneration than men (P = .04). Medial degeneration scores were significantly lower for patients with bicuspid aortic valves than for patients with tricuspid aortic valves (P = .03). CONCLUSION: The study's findings indicate considerable overlap in the pattern, extent, and severity of medial degeneration between sporadic and hereditary types of thoracic aortic disease. This finding suggests that histopathologic medial degeneration represents the final common outcome of diverse pathogenetic factors and mechanisms.

Inhibition of valve mesenchymal stromal cell calcium deposition by bFGF through alternative polyadenylation regulation of the CAT gene.

OBJECTIVE: Calcific aortic valve disease (CAVD) is the leading cause of angina, heart failure, and death from aortic stenosis. However, the molecular mechanisms of its progression, especially the complex disease-related transcriptional regulatory mechanisms, remain to be further elucidated. METHODS: This study used porcine valvular interstitial cells (PVIC) as a model. We used osteogenic induced medium (OIM) to induce calcium deposition in PVICs to calcify them, followed by basic fibroblast growth factor (bFGF) treatment to inhibit calcium deposition. Transcriptome sequencing was used to study the mRNA expression profile of PVICs and its related transcriptional regulation. We used DaPars to further examine alternative polyadenylation (APA) between different treatment groups. RESULTS: We successfully induced calcium deposition of PVICs through OIM. Subsequently, mRNA-seq was used to identify differentially expressed mRNAs for three different treatments: control, OIM-induced and OIM-induced bFGF treatment. Global APA events were identified in the OIM and bFGF treatment groups by bioinformatics analysis. Finally, it was discovered and proven that catalase (CAT) is one of the potential targets of bFGF-induced APA regulation. CONCLUSION: We described a global APA change in a calcium deposition model related to CAVD. We revealed that transcriptional regulation of the CAT gene may contribute to bFGF-induced calcium deposition inhibition.

Virtual non-iodine photon-counting CT-angiography for aortic valve calcification scoring.

Photon-counting detector (PCD)-CT allows for reconstruction of virtual non-iodine (VNI) images from contrast-enhanced datasets. This study assesses the diagnostic performance of aortic valve calcification scoring (AVCS) derived from VNI datasets generated with a 1st generation clinical dual-source PCD-CT. AVCS was evaluated in 123 patients (statistical analysis only comprising patients with aortic valve calcifications [n = 56; 63.2 ± 11.6 years]), who underwent contrast enhanced electrocardiogram-gated (either prospective or retrospective or both) cardiac CT on a clinical PCD system. Patient data was reconstructed at 70 keV employing a VNI reconstruction algorithm. True non-contrast (TNC) scans at 70 keV without quantum iterative reconstruction served as reference in all individuals. Subgroup analysis was performed in 17 patients who received both, prospectively and retrospectively gated contrast enhanced scans (n = 8 with aortic valve calcifications). VNI images with prospective/retrospective gating had an overall sensitivity of 69.2%/56.0%, specificity of 100%/100%, accuracy of 85.4%/81.0%, positive predictive value of 100%/100%, and a negative predictive value of 78.2%/75.0%. VNI images with retrospective gating achieved similar results. For both gating approaches, AVCSVNI showed high correlation (r = 0.983, P < 0.001 for prospective; r = 0.986, P < 0.001 for retrospective) with AVCSTNC. Subgroup analyses demonstrated excellent intra-individual correlation between different acquisition modes (r = 0.986, P < 0.001). Thus, VNI images derived from cardiac PCD-CT allow for excellent diagnostic performance in the assessment of AVCS, suggesting potential for the omission of true non-contrast scans in the clinical workup of patients with aortic calcifications.

The association of peritoneal dialysis and hemodialysis on mitral and aortic valve calcification associated mortality: a meta-analysis.

Cardiac valve calcification (CVC), characterized by the accumulation of calcium in the heart valves, is highly prevalent among patients undergoing dialysis. This meta-analysis aimed to provide an updated summary of recent studies on the prognostic value of CVC in patients undergoing dialysis. We conducted a search of PubMed, Embase, and Web of Science to identify observational studies investigating cardiovascular or all-cause mortality associated with CVC in dialysis patients until March 2023. Hazard ratios (HRs) and the corresponding 95% confidence intervals (CIs) were calculated for the meta-analysis, and the strength and significance of the associations between CVC and mortality outcomes in dialysis patients were assessed. From 6218 initially identified studies, we included 10 critical studies with a total of 3376 dialysis patients in a further meta-analysis. Pooled analyses demonstrated a significant association between CVC and an elevated risk of all-cause and cardiovascular mortality in dialysis patients. In our study, we discovered HRs of 1.592 (95% CI 1.410-1.797) for all-cause mortality and 2.444 (95% CI 1.632-3.659) for cardiovascular mortality. Furthermore, subgroup analysis revealed elevated all-cause mortality among patients with mitral valve calcification (HR 1.572; 95% CI 1.200-2.060) compared to those with aortic valve calcification (HR 1.456; 95% CI 1.105-1.917). Similarly, patients undergoing peritoneal dialysis faced a greater risk for all-cause mortality (HR 2.094; 95% CI 1.374-3.191) than those on hemodialysis (HR 1.553; 95% CI 1.369-1.763). This highlights the possibility of CVC being an independent risk factor for dialysis patients, particularly in relation to mitral valve calcification or peritoneal dialysis.

Intracellular proteomics and extracellular vesiculomics as a metric of disease recapitulation in 3D-bioprinted aortic valve arrays.

In calcific aortic valve disease (CAVD), mechanosensitive valvular cells respond to fibrosis- and calcification-induced tissue stiffening, further driving pathophysiology. No pharmacotherapeutics are available to treat CAVD because of the paucity of (i) appropriate experimental models that recapitulate this complex environment and (ii) benchmarking novel engineered aortic valve (AV)-model performance. We established a biomaterial-based CAVD model mimicking the biomechanics of the human AV disease-prone fibrosa layer, three-dimensional (3D)-bioprinted into 96-well arrays. Liquid chromatography-tandem mass spectrometry analyses probed the cellular proteome and vesiculome to compare the 3D-bioprinted model versus traditional 2D monoculture, against human CAVD tissue. The 3D-bioprinted model highly recapitulated the CAVD cellular proteome (94% versus 70% of 2D proteins). Integration of cellular and vesicular datasets identified known and unknown proteins ubiquitous to AV calcification. This study explores how 2D versus 3D-bioengineered systems recapitulate unique aspects of human disease, positions multiomics as a technique for the evaluation of high throughput-based bioengineered model systems, and potentiates future drug discovery.

SIRT6-mediated Runx2 downregulation inhibits osteogenic differentiation of human aortic valve interstitial cells in calcific aortic valve disease.

Calcific aortic valve disease (CAVD) is a progressive cardiovascular disorder involving multiple pathogenesis. Effective pharmacological therapies are currently unavailable. Sirtuin6 (SIRT6) has been shown to protect against aortic valve calcification in CAVD. The exact regulatory mechanism of SIRT6 in osteoblastic differentiation remains to be determined, although it inhibits osteogenic differentiation of aortic valve interstitial cells. We demonstrated that SIRT6 was markedly downregulated in calcific human aortic valves. Mechanistically, SIRT6 suppressed osteogenic differentiation in human aortic valve interstitial cells (HAVICs), as confirmed by loss- and gain-of-function experiments. SIRT6 directly interacted with Runx2, decreased Runx2 acetylation levels, and facilitated Runx2 nuclear export to inhibit the osteoblastic phenotype transition of HAVICs. In addition, the AKT signaling pathway acted upstream of SIRT6. Together, these findings elucidate that SIRT6-mediated Runx2 downregulation inhibits aortic valve calcification and provide novel insights into therapeutic strategies for CAVD.

Defining the Role of the miR-145-KLF4-αSMA Axis in Mitral Valvular Interstitial Cell Activation in Myxomatous Mitral Valve Prolapse Using the Canine Model.

Mitral valve prolapse (MVP) is a common valvular disease, affecting 2-3% of the adult human population and is a degenerative condition. A total of 5-10% of the afflicted will develop severe mitral regurgitation, cardiac dysfunction, congestive heart failure, and sudden cardiac death. Naturally occurring myxomatous MVP in dogs closely resembles MVP in humans structurally, and functional consequences are similar. In both species, valvular interstitial cells (VICs) in affected valves exhibit phenotype consistent with activated myofibroblasts with increased alpha-smooth muscle actin (αSMA) expression. Using VICs collected from normal and MVP-affected valves of dogs, we analyzed the miRNA expression profile of the cells and their associated small extracellular vesicles (sEV) using RNA sequencing to understand the role of non-coding RNAs and sEV in MVP pathogenesis. miR-145 was shown to be upregulated in both the affected VICs and sEV, and overexpression of miR-145 by mimic transfection in quiescent VIC recapitulates the activated myofibroblastic phenotype. Concurrently, KLF4 expression was noted to be suppressed by miR-145, confirming the miR-145-KLF4-αSMA axis. Targeting this axis may serve as a potential therapy in controlling pathologic abnormalities found in MVP valves.