Pathogenesis Of Aortic Valve Stenosis Research Articles

Aortic valve disease augments vesicular microRNA-145-5p to regulate the calcification of valvular interstitial cells via cellular crosstalk

Abstract Rationale: Aortic valve stenosis (AVS) is a major contributor to cardiovascular death in the elderly population worldwide. MicroRNAs (miRNAs) are highly dysregulated in patients with AVS undergoing surgical aortic valve replacement (SAVR). However, miRNA-dependent mechanisms regulating inflammation and calcification or miRNA-mediated cell-cell crossstalk during the pathogenesis of AVS are still poorly understood. Here, we explored the role of extracellular vesicles (EV)-associated miR-145-5p, which we showed to be highly upregulated upon valvular calcification in AVS in mice and humans. Methods Human TaqMan miRNA arrays identified dysregulated miRNAs in aortic valve tissue explants from AVS patients compared to non-calcified valvular tissue explants of patients undergoing SAVR. Echocardiographic parameters were measured in association with the quantification of dysregulated miRNAs in a murine AVS model. In vitro calcification experiments were performed to explore the effects of EV-miR-145-5p on calcification and crosstalk in valvular cells. To dissect molecular miRNA signatures and their effect on signaling pathways, integrated OMICS analyses were performed. RNA sequencing (RNA-seq), high-throughput transcription factor (TF) and proteome arrays showed that a number of genes, miRNAs, TFs, and proteins are crucial for calcification and apoptosis, which are involved in the pathogenesis of AVS. Results Among several miRNAs dysregulated in valve explants of AVS patients, miR-145-5p was the most highly gender-independently dysregulated miRNA (AUC, 0.780, p-value, 0.01). MiRNA arrays utilizing patient-derived- and murine aortic-stenosis samples demonstrated that the expression of miR-145-5p is significantly upregulated and correlates positively with cardiac function based on echocardiography. In vitro experiments confirmed that miR-145-5p is encapsulated into EVs and shuttled into valvular interstitial cells. Based on the integrated OMICs results, miR-145-5p interrelates with markers of inflammation, calcification, and apoptosis. In vitro calcification experiments demonstrated that miR-145-5p regulates the ALPL gene, a hallmark of calcification in vascular and valvular cells. EV-mediated shuttling of miR-145-5p suppressed the expression of ZEB2, a negative regulator of the ALPL gene, by binding to its 3' untranslated region to inhibit its translation, thereby diminishing the calcification of target valvular interstitial cells. Conclusion Elevated levels of pro-calcific and pro-apoptotic EV-associated miR-145-5p contribute to the progression of AVS via the ZEB2-ALPL axis, which could potentially be therapeutically targeted to minimize the burden of AVS.

Aortic valve stenosis augments long noncoding RNA H19 to govern angiogenic responses and phenotypes of valvular endothelial cell through endothelial to mesenchymal transition

Abstract Background Long noncoding RNAs (lncRNAs) have emerged as important regulators of cardiovascular disease, including aortic valve stenosis (AVS). Although several recent studies have reported that lncRNAs are dysregulated and might play a crucial role in (Endothelial to mesenchymal transition) EndMT, no specific role of lncRNA was reported in AVS pathogenesis. Here, we sought to investigate the role of H19 in EndMT during the pathogenesis of AVS. Methods and results High-throughput lncRNA sequencing of human aortic valve tissue explanted from patients with AVS due to severe stenosis during surgical aortic valve replacement (SAVR) demonstrated that certain lncRNAs [H19, AGAP2-AS1 (PUNISHER), GAS5, MALAT1] are significantly dysregulated compared to patients without AVS. To validate the RNA-seq data, 60 patients with AVS (n=30) or without AVS (n=30) were prospectively investigated. Among these, H19 (p=0.002), AGAP2-AS1 (p=0.002), and GAS5 (0.02) were significantly upregulated in patients with AVS, compared to non-AVS patients. Aortic valvular endothelial cells (VECs) were isolated from explanted valve tissue from AVS patients or obtained commercially. In vitro EndMT-promoting stimuli (TNFα or TGFß+IL1ß) were used for VEC stimulation for 5 days. RT-qPCR was used to quantify the expression of EndMT markers (EC markers, vWF, and NOS3, and mesenchymal markers, αSMA, SM22, Vimentin, and SNAI2). Loss of H19 led to dysregulation of the EC gene network and protein expression patterns that were analyzed by qRT-PCR, immunoblotting, and immunofluorescence, respectively. Further experiments show that silencing of H19 affects migration and proliferation, and promotes EndMT in EC. Functional assays suggested that H19 is a critical regulator of angiogenic properties. The molecular signature of angiogenesis was investigated using a RT-qPCR-based angiogenesis array to identify the regulation of angiogenesis with 84-angiogenesis-related genes. Mechanistically, the angiogenesis array revealed that H19 regulates Jagged Canonical Notch Ligand 1 (JAG1) (4.43-fold vs. control EC), an important regulator of NOTCH1 signaling, and pro-angiogenic factor in EC. Accordingly, EndMT marker expressions were dysregulated upon H19 depletion. A high-throughput luciferase reporter array demonstrated STAT3 activation upon H19 knockdown. Depletion of H19 increased the phosphorylation of STAT3 at TYR705 and pharmacological inhibition of STAT3 activation abolished the effects of H19 silencing on EndMT marker expression as well as on angiogenesis. Overexpression of H19 in VEC abrogated the H19-mediated promotion of JAG1- and VEGFA gene- and protein expression. Our data suggest that H19 might play a role in EC angiogenic gene expression and EndMT. The data reveal a pivotal role of H19 in controlling EndMT in AVS via regulation of the JAG1-NOTCH axis, which could potentially be therapeutically targeted to halt EndMT during AVS.

Interleukin 6 in asymptomatic patients with nonsevere aortic valve stenosis: a post-hoc study of the SEAS trial

Abstract Background Inflammation plays a pivotal role in the pathogenesis of aortic valve stenosis (AS) through cytokine-mediated valve calcification. Studies of explanted valves found associations of an elevated pro-inflammatory cytokine, Interleukin 6 (IL-6), with increased postoperative mortality. However, limited knowledge exists regarding the prognostic value of elevated circulating IL-6 in asymptomatic patients with nonsevere AS. Purpose To investigate the hypothesis that elevated plasma IL-6 concentration ≥7 pg/mL (above the upper limit of normal 95th percentile) correlates with more severe AS and a higher risk of adverse outcomes in asymptomatic patients with nonsevere AS. Methods We analyzed data of 1574 asymptomatic patients who had mild-to-moderate AS, preserved systolic and kidney function, no overt cardiovascular diseases, and an event-free follow-up one year after enrollment in the randomized Simvastatin and Ezetimibe in Aortic Stenosis (SEAS) trial. We ascertained the correlation of year-0 and year-1 IL-6 levels with echocardiographic AS severity, defined as moderate-severe AS in the presence of ≥2 measurements at year-0: aortic valve area <1 cm², mean pressure gradient ≥40 mmHg, maximal transaortic velocity ≥4 m/s, and velocity ratio <0.25. Multivariable regression examined correlations between IL-6 at year-0 and clinical variables. Cox proportional hazard models and competing risk analyses examined associations of elevated IL-6 at year-1 and 4-year risk of the primary composite endpoint of major cardiovascular events (MACE) comprising the first event of cardiovascular death, aortic valve replacement, hospitalization with heart failure, myocardial infarction, percutaneous coronary intervention, coronary artery bypass graft, or ischemic stroke. Results At year-0, among 1574 patients with a mean (SD) age of 67.5 (9.7) years, 629 (40.0%) were women, 263 (16.7%) had moderate-severe AS, and 194 (12.3%) had elevated IL-6. The median relative IL-6 change from year-0 to year-1 was 1.0 (IQR, 0.8-1.4) across AS-severity groups (P=0.12) (Figure 1). In adjusted models, IL-6 at year-0 did not correlate with AS-severity (P=0.30). In mild AS, an elevated IL-6 at year-1 was associated with an increased 4-year MACE risk (HR 1.40; 95% CI, 1.02-1.92; p=0.04) in adjusted Cox regression analyses (Figure 2A). In sensitivity analyses, the annual IL-6 increase >50% combined with normal IL-6 was associated with an increased 4-year MACE-risk of HR 1.48 (95% CI, 1.10-2.00; P=0.009) in patients with mild AS and 1.82 (95% CI, 0.68-1.74; P=0.73) in patients with moderate-severe AS (all P for interaction>0.25) (Figure 2C-D). Conclusion In asymptomatic patients with nonsevere AS, circulating IL-6 remains stable within the reference range in 7 out of 8 patients during a 1-year follow-up, regardless of AS severity. An elevated IL-6 was associated with a 1.4-fold increased 4-year MACE-risk in patients with mild AS, suggesting its possible role as an early risk marker.Figure 1Figure 2

Aortic valve calcification is promoted by clonal hematopoiesis associated with DNMT3A and TET2 gene driver mutations

Abstract Background Clonal hematopoiesis (CH) due to DNMT3A or TET2-driver mutations is associated with coronary heart disease and worse prognosis among patients with aortic valve stenosis (AVS) and inflammation. However, it is unknown what role CH plays in the pathogenesis of AVS. AVS is the most common age-related heart valve disease, with no medical therapy to halt progression. Methods and Results Single-cell RNA-sequencing of immune cells of CAVD patients (n=13) with and without the most prevalent CH-mutations (DNMT3A and TET2), with mean CH-driver gene variant allele frequency was 6.5% for DNMT3A and 17.9% for TET2. Monocytes of patients harboring DNMT3A and TET2 mutations shared 836 upregulated genes, many associated with glycolytic, pro-inflammatory activation (CXCL10, IL38) and paracrine pro-calcific factors (oncostatin M (OSM), S100A9), suggesting that CH might be causally involved in AVS. Indeed, silencing of TET2 or DNMT3A in macrophages promotes M1-like polarization and increases release of pro-calcific mediators, particularly S100A9 and OSM. To assess whether CH-gene silenced macrophages may stimulate mesenchymal cells to secrete calcium, secreted factors from TET2 or DNMT3A-silenced macrophages were applied mesenchymal cells. Indeed, CH-gene silenced secretomes induced osteoblastic differentiation of mesenchymal cells, evidenced by increased Alizarin red calcium staining and elevated levels of ALP and RUNX2. This CH-mediated enhancement in osteoblastic conversion could be ablated by silencing of OSM. Finally, valves from atheroprone Ldlr-/- mice receiving TET2-/- bone marrow transplants demonstrated increased total calcified area (1.49-fold) along with increased numbers of calcification deposits (2.33-fold) as evidenced by von Kossa staining, along with enhanced OSM and S100A9 levels. Conclusion This is the first study to demonstrate that somatic CH-driver mutations in monocytes may accelerate AVS. Moreover, calcification was ablated via silencing of OSM in CH-gene silenced macrophages. These data suggest Oncostatin M may be a therapeutically promising target in halting the progression of AVS in patients harboring CH mutations.

Long noncoding RNA H19 is elevated in aortic valve stenosis and governs antigenic response and valvular endothelial cell phenotypes through endothelial to mesenchymal transition

Abstract Background Long noncoding RNAs (lncRNAs) have emerged as important regulators of cardiovascular disease, including aortic valve stenosis (AVS). Endothelial to mesenchymal transition (EndMT) is a transdifferentiation process that is associated with the development of AVS in the elderly population. Although several recent studies have reported that lncRNAs are dysregulated and might play a crucial role in EndMT, no specific role of lncRNA was reported in AVS pathogenesis. Here, we sought to investigate the role of H19 in EndMT during the pathogenesis of AVS. Methods and results High-throughput lncRNA sequencing of human aortic valve tissue explanted from patients with AVS due to severe stenosis during surgical aortic valve replacement (SAVR) demonstrated that certain lncRNAs [H19, AGAP2-AS1 (PUNISHER), GAS5, MALAT1] are significantly dysregulated compared to patients without AVS. To validate the RNA-seq data, 60 patients with AVS (n=30) or without AVS (n=30) were prospectively investigated. Among these, H19 (p=0.002), AGAP2-AS1 (p=0.002), and GAS5 (0.02) were significantly upregulated in patients with AVS, compared to non-AVS patients. Aortic valvular endothelial cells (VECs) were isolated from explanted valve tissue from AVS patients or obtained commercially. In vitro EndMT-promoting stimuli (TNFα or TGFß+IL1ß) were used for VEC stimulation for 5 days. RT-qPCR was used to quantify the expression of EndMT markers (EC markers, vWF, and NOS3, and mesenchymal markers, αSMA, SM22, Vimentin, and SNAI2). Loss of H19 led to dysregulation of the EC gene network and protein expression patterns that were analyzed by qRT-PCR, immunoblotting, and immunofluorescence, respectively. Further experiments show that silencing of H19 affects migration and proliferation, and promotes EndMT in EC. Functional assays suggested that H19 is a critical regulator of angiogenic properties. The molecular signature of angiogenesis was investigated using a RT-qPCR-based angiogenesis array to identify the regulation of angiogenesis with 84-angiogenesis-related genes. Mechanistically, the angiogenesis array revealed that H19 regulates Jagged Canonical Notch Ligand 1 (JAG1) (4.43-fold vs. control EC), an important regulator of NOTCH1 signaling, and pro-angiogenic factor in EC. Accordingly, EndMT marker expressions were dysregulated upon H19 depletion. A high-throughput luciferase reporter array demonstrated STAT3 activation upon H19 knockdown. Depletion of H19 increased the phosphorylation of STAT3 at TYR705 and pharmacological inhibition of STAT3 activation abolished the effects of H19 silencing on EndMT marker expression as well as on angiogenesis. Overexpression of H19 in VEC abrogated the H19-mediated promotion of JAG1- and VEGFA gene- and protein expression.

Abstract 165: Large Extracellular Vesicles-derived MicroRNA-150-5p Is Upregulated In Patients With Aortic Valve Stenosis And Regulates Valvular Endothelial Cell Function

Aortic valve stenosis (AVS) is the most common type of valvular disease. Recent evidence indicates that extracellular vesicles (EVs) might play an important role in cardiovascular disease, particularly AVS. However, the contribution of EV-encapsulated microRNAs (miRNAs) in valvular endothelial cells (VECs) and AVS has been poorly investigated. Herein, we identified the differentially expressed miRNAs in large EV-derived aortic valve tissue from patients with AVS and explored the novel role of miR-150-5p in endothelial to mesenchymal transition (EndMT). Our miRNA sequencing revealed several EV-miRNAs (miRNA-150-5p, miR-486-5p, and miR-486-3p) were significantly dysregulated in large EVs from aortic valve tissue in patients with AVS. In silico analysis indicated that miRNA-150-5p is mainly enriched in Wnt, TGF-β, and PI3k/AKT signaling pathways, known to be altered in the pathogenesis of AVS. To elucidate the role of miR-150-5p in AVS, we validated the upregulation of miR-150-5p in AVS patients by RT-qPCR. Importantly, the overexpression of miR-150-5p in VECs treated with TNF-α reduced EndMT, which was quantized by significant expression of EndMT markers (vWF, ACTA1, VIM, and SLUG). To investigate the role of miRNA-150-5p in VEC function, cell migration and tube formation were performed. The data revealed that miR-150-5p regulates VEC function via increased tube formation and reduced cell migration. To further investigate VEC function under the laminar flow, which regulates EC function, we exposed VEC under the flow and found that the expression of miR-150-5p is increased suggesting a protective role in VECs. To study the role of miR-150-5p in intercellular communication, we overexpressed miR-150-5p in donor VECs and incubated them with recipient VECs prior to the labeling of EVs with PKH67. Fluorescent microscopy showed an increased expression of miR-150-5p in recipient VECs. Further, we induced EndMT in VECs after co-incubation with EVs (miR-150-5p overexpression vs. control). Our qPCR-analysis revealed that EV-miR-150-5p rescued the expression of EndMT markers. Our data showed that large EV-associated miR-150-5p was significantly upregulated in AVS and may play a crucial role in cellular crosstalk via regulating function of VEC via EndMT.

LXR-beta signalling is a key mediator in the pathogenesis of aortic valve stenosis and its prevention by saringosterol

Abstract Background Cholesterol metabolism contributes as a risk factor for aortic valve stenosis (AS), but pharmacological approaches remained unsatisfying. The liver-X-receptor (LXR) is a key regulator in cholesterol metabolism, though its clinical use is limited due to unwanted side effects. The seaweed-derived oxysterol saringosterol is an agonist of the LXRβ, promising a more favourable tolerability. Purpose This study aimed to better understand the pathophysiology of aortic valve stenosis and to assess the potential of saringosterol as a targeted pharmacotherapy. Methods Tissue samples from aortic valves were collected from patients with AS or aortic valve regurgitation (AR). Transcriptomics were performed and gene ontology (GO) analysis was used to determine pathways and genes that are relevant to AS, and then validated using qPCR. In vivo, mice received a wire-induced aortic valve stenosis and were either fed a diet supplemented with saringosterol or control diet. Haemodynamic characteristics were assessed using echocardiography. Additionally, hepatic concentrations of saringosterol, expression of LXRβ regulated genes as well as aortic valve thickness and composition were assessed. In vitro, human aortic valve interstitial cells (VIC) were cultured in a procalcifing medium and stimulated with saringosterol to investigate the underlying molecular mechanisms. Results Transcriptomic analysis of AS samples revealed the regulation of several GO-terms related to cholesterol- or lipid metabolism. Many of the genes identified were regulated by LXRβ, suggesting its pathophysiological relevance in AS. We validated this assumption by performing qPCR from aortic valves for the most prominent downstream targets of LXRβ, ABCA1 and ABCG1, with both being differentially regulated. In vivo, treatment with saringosterol for six weeks resulted in a significant accumulation of saringosterol in liver tissue as well as induction of LXRβ-regulated genes. Furthermore, treatment with saringosterol strikingly reduced the development of AS after wire injury as assessed by echocardiographic and histological measurements. In vitro, the differentiation of VIC into osteoblastic and myofibroblastic phenotypes was abolished by saringosterol, which reduced the expression of the procalcifying mediators RUNX-2 and ACTA-2 in a dose-dependent manner. Conclusion We identified LXRβ-signalling as a key regulator in the pathophysiology of AS. Transcriptomic analyses revealed that cholesterol metabolism was altered in human AS, and many of the genes involved were linked to the LXRβ. In a murine model, we demonstrated that oral application of saringosterol induced LXRβ-activity and mitigated the development of AS. In vitro experiments demonstrated that saringosterol prevents adverse cell differentiation of VIC, which provides a mechanistic explanation. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): BONFOR Universität Bonn

Lipoprotein(a) and aortic valve stenosis: work in progress.

Schematic illustration of the observational and genetic evidence that LDL-cholesterol (LDL-C) and lipoprotein(a) [Lp(a)] might be involved in the pathogenesis of aortic valve stenosis and/or aortic valve calcification. Recent trials with statins had no effect on the aortic valve outcomes. Questions concerning trials to be planned with Lp(a)-lowering drugs are listed. OxPL, oxidized phospholipids.

The Role of Wnt/β-Catenin Pathway Mediators in Aortic Valve Stenosis.

Aortic valve stenosis (AVS) is a prevailing and life-threatening cardiovascular disease in adults over 75 years of age. However, the molecular mechanisms governing the pathogenesis of AVS are yet to be fully unraveled. With accumulating evidence that Wnt signaling plays a key role in the development of AVS, the involvement of Wnt molecules has become an integral study target in AVS pathogenesis. Thus, we hypothesized that the Wnt/β-catenin pathway mediators, SFRP2, DVL2, GSK3β and β-catenin are dysregulated in patients with AVS. Using immunohistochemistry, Real-Time qPCR and Western blotting, we investigated the presence of SFRP2, GSK-3β, DVL2, and β-catenin in normal and stenotic human aortic valves. Markedly higher mRNA and protein expression of GSK-3β, DVL2, β-catenin and SFRP2 were found in stenotic aortic valves. This was further corroborated by observation of their abundant immunostaining, which displayed strong immunoreactivity in diseased aortic valves. Proteomic analyses of selective GSK3b inhibition in calcifying human aortic valve interstitial cells (HAVICs) revealed enrichment of proteins involved organophosphate metabolism, while reducing the activation of pathogenic biomolecular processes. Lastly, use of the potent calcification inhibitor, Fetuin A, in calcifying HAVICs significantly reduced the expression of Wnt signaling genes Wnt3a, Wnt5a, Wnt5b, and Wnt11. The current findings of altered expression of canonical Wnt signaling in AVS suggest a possible role for regulatory Wnts in AVS. Hence, future studies focused on targeting these molecules are warranted to underline their role in the pathogenesis of the disease.

Impact of several proinflammatory and cell degradation factors in patients with aortic valve stenosis.

Aortic valve (AoV) stenosis is the third most common cardiovascular disease. The pathogenesis of AoV stenosis is associated with an inflammatory process where MMPs serve important roles. The aim of the present study was to determine the association between matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and inflammatory factors, and AoV stenosis at various degrees of severity compared with the control. A total of 18 patients with mild, 19 with moderate and 15 with severe AoV stenosis were included in the present stud, and 50 individuals were enrolled in the control group. The severity of stenosis was determined by echocardiography. The expression levels of chemerin, fibroblast growth factor 21, MMP-1, −3, and −9, and TIMP-1 and −3 were analyzed by ELISA. Data were analyzed using GraphPad Prism7 software. The expression levels of MMP-1 was increased in patients with stenosis compared with the control group (P=0.0043). Distribution of the trimodal MMP-1 values was obtained in the stenosis group and monomodal in the control group. A total of 80% of patients in the stenosis group presented significantly increased expression levels of MMP-1 compared with the control group (P=0.0002). Expression of MMP-1 was significantly higher in all stenosis groups compared with the control. The highest expression level of MMP-1 appeared in patients with moderate stenosis (P<0.0001). There was no significant difference in the expression of MMP-3, MMP-9 and TIMP-1 in the aortic stenosis group, compared with the control group. A positive correlation between MMP-1 and MMP-9 expression levels was identified (r=0.37; P=0.017). The increase of MMP-1 was correlated with the increase of MMP-9, but not with the level of MMP-3. The expression levels of chemerin was significantly elevated in patients with stenosis compared with healthy patients. The highest expression levels of chemerin were determined in patients with mild (P=0.0001) and moderate (P=0.0007) stenosis and decreased with the grade of severity compared with the control group. The expression of FGF-21 was significantly different between the control and mild (P=0.013), moderate (P=0.015) and severe stenosis (P=0.003) groups. The expression levels of FGF-21 increased with the increase in severity grade, reaching the maximum for severe stenosis. The results of the present study indicated that the inflammatory process is predominantly occurring at the early, mild stage of stenosis and the most prominent extracellular matrix remodeling occurs in moderate stenosis (demonstrated by MMP-1 levels). In patients with severe stenosis, the levels of MMP-1 and chemerin (which are lower than in a case of mild or moderate stenosis) could indicate the development of calcinosis and the reduction in activity or inactivation of the inflammatory process.

Genome-Wide DNA Methylation Analysis and Epigenetic Variations Associated with Congenital Aortic Valve Stenosis (AVS).

Congenital heart defect (CHD) is the most common cause of death from congenital anomaly. Among several candidate epigenetic mechanisms, DNA methylation may play an important role in the etiology of CHDs. We conducted a genome-wide DNA methylation analysis using an Illumina Infinium 450k human methylation assay in a cohort of 24 newborns who had aortic valve stenosis (AVS), with gestational-age matched controls. The study identified significantly-altered CpG methylation at 59 sites in 52 genes in AVS subjects as compared to controls (either hypermethylated or demethylated). Gene Ontology analysis identified biological processes and functions for these genes including positive regulation of receptor-mediated endocytosis. Consistent with prior clinical data, the molecular function categories as determined using DAVID identified low-density lipoprotein receptor binding, lipoprotein receptor binding and identical protein binding to be over-represented in the AVS group. A significant epigenetic change in the APOA5 and PCSK9 genes known to be involved in AVS was also observed. A large number CpG methylation sites individually demonstrated good to excellent diagnostic accuracy for the prediction of AVS status, thus raising possibility of molecular screening markers for this disorder. Using epigenetic analysis we were able to identify genes significantly involved in the pathogenesis of AVS.

Increased Smad2/3 phosphorylation in circulating leukocytes and platelet-leukocyte aggregates in a mouse model of aortic valve stenosis: Evidence of systemic activation of platelet-derived TGF-β1 and correlation with cardiac dysfunction

BackgroundTransforming growth factor-β1 (TGF-β1) has been implicated in the pathogenesis of aortic valve stenosis (AS). There is, however, little direct evidence for a role of active TGF-β1 in AS due to the sensitivity of current assays. We searched for evidence of plasma TGF-β1 activation by assaying Smad2/3 phosphorylation in circulating leukocytes and platelet-leukocyte aggregates (PLAs) in a mouse model of AS (Reversa). MethodsEchocardiography was used to measure AS and cardiac function. Intracellular phospho-flow cytometry in combination with optical fluorescence microscopy was used to detect PLAs and p-Smad2/3 levels. ResultsReversa mice on a western diet developed AS, had significantly increased numbers of PLAs and more intense staining for p-Smad2/3 in both PLAs and single leukocytes (all p<0.05). p-Smad2/3 staining was more intense in PLAs than in single leukocytes in both diet groups (p<0.05) and correlated with plasma total TGF-β1 levels (r=0.38, p=0.05 for PLAs and r=0.37, p=0.06 for single leukocytes) and reductions in ejection fraction (r=−0.42, p=0.03 for PLAs and r=−0.37, p=0.06 for single leukocytes). Conclusionsp-Smad2/3 staining is more intense in leukocytes of hypercholesterolemic mice that developed AS, suggesting increased circulating active TGF-β1 levels. Leukocyte p-Smad2/3 may be a valuable surrogate indicator of circulating active TGF-β1.

Targeting Advanced Glycation End Products in Cardiac Surgery: The Unexplored Alternative.

Dear Editor, In the previous issue of “Research in Cardiovascular Medicine”, our group presented a review of pharmacologic approaches against advanced glycation end products (AGEs) in diabetic cardiovascular disease (1). Over the last years, clinicians' viewpoint about diabetes moved from “glycaemia” to “glycaemia, glycated haemoglobin and glycation pathway”, and the recognized pivotal role of AGEs in genesis and progression of cardiovascular disease led to the development of new anti-AGEs compounds, with alagebrium, statins and thiazolidinediones being the most promising. These pharmacological studies were always performed in cardiology settings, but evidence-based medicine states that diabetic patients are more likely to have long-term benefits with surgical than percutaneous procedures (2). So, what is the state-of-the-art about AGEs awareness and anti-AGEs treatment in cardiac surgery? AGEs levels and its pathways are closely related to post-operative outcomes and length of hospital stay. In fact, higher pre-operative plasma soluble receptor for AGEs (sRAGE) values are associated with prolonged duration of care (3), and higher carboxy-methyl-lysine (CML) values correlate with prolonged ventilation time after cardiac surgery (4). Also, sRAGE are a sensitive and early marker of post-operative lung distress, and this represents the most attractive field of investigation in both paediatric and adult procedures (5-7). sRAGE level was also reported to be lower in patients scheduled for surgical aortic valve replacement compared to healthy subjects, and its role in the pathogenesis of aortic valve stenosis is being currently investigated (8). As for diastolic dysfunction, plasma CML values are associated with mitral E/A ratio in patients undergoing coronary artery bypass graft surgery (9). The prognostic implication of AGEs appears also in orthotopic heart transplantation, as AGEs deposition in diabetic patients was related to acute rejection episodes (10). However, despite its potential interest, there is no unanimous consensus in the literature regarding the optimal technique for AGEs measurements. Plasma essays, skin autofluorescence and immunochemical methods have been differently reported throughout the literature and this complicates the possibility of a clear comparisons among the clinical studies (11). In the next years, the standardization of AGEs measurement would improve the actual understanding of the mechanisms underlying AGEs biological pathways and the effects of anti-AGEs treatments. As far as pharmacologic approaches against AGEs are concerned, despite the flourishing production of literature in the field of cardiology on this topic, there are no current or terminated clinical trials investigating anti-AGEs role in cardiac surgery. This may be because newer drugs, such as alagebrium and aminoguanidine, are associated with side effects that seem to outnumber the potential benefits in AGEs modification. On the other hand, statins and thiazolidinediones are well-known drugs exerting significant anti-AGEs effect, but targeted clinical trials are still lacking. Thus the under-use of anti-AGEs compounds may be due to the under-comprehension of their potential benefits, which can be evaluated only during a long-term time span. In fact, notwithstanding the general lack of clinical trial on anti-AGEs treatments, the available studies only focus on short-term follow up which might elucidate side effects of anti-AGEs compounds, but cannot reveal their actual long-term benefits in the progression of diabetic cardiovascular disease. Alexis Carrel, French surgeon, Nobel laureate in 1912, wrote that “A few observation and much reasoning lead to error; many observations and a little reasoning to truth”. Studies on anti-AGEs compounds showed benefits in arterial compliance, ventricular stiffening and slowed progression of diabetic vascular disease. Spurred by the wave of optimism regarding the potential of anti-AGEs compounds, it seems to be right moment to introduce anti-AGEs drugs also into cardiac surgery study protocols, using newer safe compounds such as alagebrium and assessing the modification in the glycation pathway caused by known compounds such as statins and thiazolidinediones. As mentioned above, some investigators introduced interesting associations between AGEs and cardiac outcomes in cardiac surgery, but more structured approach are needed in this context. Also, considering the role of AGEs as marker of disease progression in the complex scenario of diabetes, measurement of AGEs levels should become part of the routine evaluation of the diabetic patient, not only to monitor blood glucose effects, but also to predict potential cardiac complications and provide prompt treatment. We hope that our considerations might shed light on this shaded topic in cardiac surgery and foster the scientific debate on this argument.

Increased risk of aortic valve stenosis in patients with psoriasis: a nationwide cohort study.

Psoriasis is a chronic inflammatory disease associated with increased risk of cardiovascular disease including atherosclerosis. The pathogenesis of aortic valve stenosis (AS) also includes an inflammatory component. We therefore investigated the risk of AS in patients with psoriasis compared with the general population in a nationwide cohort. The study comprised the entire Danish population aged ≥18 years followed from 1 January 1997 until diagnosis of AS, 31 December 2011, or death. Information on comorbidity, concomitant medication, and socioeconomic status was identified by individual-level linkage of administrative registers. Incidence rates for AS were calculated and incidence rate ratios (IRRs) adjusted for age, gender, calendar year, comorbidity, medications, and socioeconomic status, were estimated in Poisson regression models. A total of 5 107 624 subjects were eligible for analysis. During the study period, we identified 58 747 patients with mild psoriasis and 11 918 patients with severe psoriasis. The overall incidence rates for AS were 8.09, 16.07, and 20.08 per 10 000 person-years for the reference population (48 539 cases [mean follow-up 12.3 years]), mild psoriasis (509 cases [mean follow-up 6.2 years]), and severe psoriasis (99 cases [mean follow-up 5.4 years]), respectively. Correspondingly, the fully adjusted IRRs for AS were markedly increased in patients with psoriasis with IRR 1.22 (95% confidence interval [CI] 1.12-1.33) and IRR 1.61 (CI 1.32-1.96) for subjects with mild and severe disease, respectively. In a nationwide cohort, psoriasis was associated with a disease severity-dependent increased risk of AS. The mechanisms underlying this novel finding require further study.

Abstract 18627: Monocyte Subsets in Patients With Severe Aortic Valve Stenosis Undergoing TAVI

Introduction: Aortic valve stenosis (AVS) shares profound similarities with atherosclerotic plaque progression, including the presence of macrophages in the valve tissue. Monocytes are the precursor of macrophages and various studies have shown associations of individual monocyte subsets with cardiovascular risk. Therefore, we assessed the hypotheses that monocyte subsets differ in the presence of severe AVS compared to an AVS-free setting and change by intervention of the AVS. Methods: Classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) CD86 positive monocyte subsets and monocyte activation (median fluorescent intensity of CD11b) were determined by flow cytometry in peripheral blood from patients with severe AVS (n=32) and matched AVS-free controls (n=28). A follow-up evaluation of monocyte subsets and activation was performed in AVS patients undergoing transcutaneous valve implantation (TAVI) 3 months after the procedure (n=9). Results: There were no significant differences in the absolute monocyte counts between AVS patients and controls. In contrast, AVS patients exhibited a shift towards CD16+ monocytes subsets with significant higher proportion of the intermediate monocyte subset compared to controls (median [IQR] 7.0% [5.5-8.7%] versus 5.8% [4.7-6.6%]; p=0.015). Classical and non-classical monocyte subsets and monocyte activation did not differ significantly between the groups. There was no significant change in the distribution of monocyte subsets or in monocyte activation 3 months after TAVI compared to baseline. Conclusions: We found preliminary evidence for a significant difference in the distribution of monocyte subsets in patients with severe AVS compared to AVS-free controls indicating an involvement of monocyte subsets in the pathogenesis of AVS. Monocyte subsets and activation did not change after treatment of AVS by TAVI.

Interleukin-6 receptor Asp358Ala gene polymorphism is associated with plasma C-reactive protein levels and severity of aortic valve stenosis

Interleukin-6 receptor (IL-6R) gene Asp358Ala (A>C, rs2228145) polymorphism has been associated with lower circulating inflammation biomarkers in coronary artery disease (CAD), such as C-reactive protein (CRP) or fibrinogen, but its role in the pathogenesis of aortic valve stenosis (AS) remains unknown. Since the pathogenesis of AS and atherosclerosis shares several similarities, we tested the hypothesis that IL-6R Asp358Ala polymorphism is associated with the severity of AS. A total of 284 AS patients aged 64.3±11.1 years were studied, in whom IL-6R polymorphism was determined by TaqMan genotyping. The genotype distribution was as follows: AA-43.7% (n=124); AC-37.0% (n=105); and CC-19.4% (n=55). For every copy of C allele inherited, mean concentration of CRP was reduced by 22% (95% CI 13.8-30.4; p<0.0001). Carriers of the C allele compared to the AA homozygotes were also characterized by lower mean and maximal transvalvular gradients [44.8 (30.5-60.0) vs. 52.7 (40.5-69.0) mmHg, p=0.0005; and 78.1±26.5 vs. 87.3±27.6 mmHg; p=0.008, respectively], and larger aortic valve area [0.8 (0.6-1.0) vs. 0.7 (0.5-0.9) cm2, p=0.005]. Our study is the first to show that the presence of the IL-6R 358Ala allele in AS patients is associated with attenuated systemic inflammatory state and less severe AS.

Interleukin-6 receptor Asp358Ala gene polymorphism is associated with plasma C-reactive protein levels and severity of aortic valve stenosis

BACKGROUND Interleukin-6 receptor (IL-6R) gene Asp358Ala (A>C, rs2228145) polymorphism has been associated with lower circulating inflammation biomarkers in coronary artery disease (CAD), such as C-reactive protein (CRP) or fibrinogen, but its role in the pathogenesis of aortic valve stenosis (AS) remains unknown. Since the pathogenesis of AS and atherosclerosis shares several similarities, we tested the hypothesis that IL-6R Asp358Ala polymorphism is associated with the severity of AS. METHODS A total of 284 AS patients aged 64.3±11.1 years were studied, in whom IL-6R polymorphism was determined by TaqMan genotyping. RESULTS The genotype distribution was as follows: AA-43.7% (n=124); AC-37.0% (n=105); and CC-19.4% (n=55). For every copy of C allele inherited, mean concentration of CRP was reduced by 22% (95% CI 13.8-30.4; p<0.0001). Carriers of the C allele compared to the AA homozygotes were also characterized by lower mean and maximal transvalvular gradients [44.8 (30.5-60.0) vs. 52.7 (40.5-69.0) mmHg, p=0.0005; and 78.1±26.5 vs. 87.3±27.6 mmHg; p=0.008, respectively], and larger aortic valve area [0.8 (0.6-1.0) vs. 0.7 (0.5-0.9) cm2, p=0.005]. CONCLUSIONS Our study is the first to show that the presence of the IL-6R 358Ala allele in AS patients is associated with attenuated systemic inflammatory state and less severe AS.

Pathogenesis of aortic valve stenosis: a spectroscopic study

Pathogenesis of aortic valve stenosis: a spectroscopic study

Impaired fibrinolysis is associated with the severity of aortic stenosis in humans

A role of fibrinolysis in the pathogenesis of aortic valve stenosis (AS) is unknown, although fibrinolytic proteins have been detected in aortic stenotic valves. To investigate whether impaired fibrinolysis could be associated with AS. We studied 74 patients with AS (43male, 31female, aged 62.7±10.7years) without documented atherosclerotic valvular disease scheduled for isolated valve replacement and 68 controls. The plasma fibrin clot lysis time (CLT) in the presence of tissue factor (TF) and tissue plasminogen activator (tPA), along with plasma plasminogen activator inhibitor-1 (PAI-1) were determined. Valvular expression of fibrin and PAI-1 together with macrophages and mast cells (MC) was evaluated by immunostaining. Patients with AS compared with controls were characterized by a prolonged CLT (median, 110 [54-153] vs. 92.5 [58-115]min, P=0.0007) and increased plasma PAI-1 (78.6 [35.5-149] vs. 38.5 [18-61]ngmL(-1) , P<0.0001). CLT was correlated with maximal (r=0.43, P=0.0002) and mean (r=0.38, P=0.001) transvalvular pressure gradients, and aortic valve area (r=-0.59, P<0.0001). In AS patients, the CLT was positively correlated with the valve leaflet thickness (r=0.67, P=0.003), the degree of valve calcification (r=0.65, P<0.00001), valvular fibrin (r=0.54, P=0.007) and PAI-1 expression (r=0.48, P=0.007). Double-immunostaining revealed colocalization of valvular PAI-1 with MC (87±17% cells) and macrophages (48±11% cells) within stenotic valves. Hypofibrinolysis might be a marker of severe AS and be implicated in AS progression.

Lipoprotein-associated phospholipase A2 is elevated in patients with severe aortic valve stenosis without clinically overt atherosclerosis

Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an inflammatory mediator involved in atherosclerosis. Since aortic valve stenosis (AVS) is regarded as an atherosclerosis-like inflammatory disease, we sought to investigate whether AVS is associated with elevated Lp-PLA2. Plasma Lp-PLA2 levels were determined in 48 consecutive patients with severe AVS without atherosclerotic vascular disease and compared with the values obtained in 48 controls matched for age, sex and cardiovascular risk factors. Lp-PLA2 was higher in AVS than in controls (242.3±50.4 vs. 151.9±28.1 ng/mL, p<0.0001). Lp-PLA2 correlated inversely with aortic valve area (AVA) (r=-0.53; p=0.0001) and positively with mean pressure gradient (PG) (r=0.32; p=0.029). In multivariable analysis C-reactive protein (CRP) (OR=1.42; 95% CI 0.95-2.1; p=0.09) and AVA (OR=0.003; 95% CI 0.00004-0.23; p<0.01) were independently associated with Lp-PLA2 above a mean of 242 ng/mL. After adjustment for CRP, AVA was the only independent predictor of Lp-PLA2 in AVS patients (p<0.001). This study is the first to show that AVS is characterized by increased plasma Lp-PLA2 levels associated with the severity of AVS, which suggests active involvement of Lp-PLA2 in the pathogenesis of AVS.