Modulation of inflammatory M1-macrophages phenotype by valvular interstitial cells.

BACKGROUND: Aortic valve stenosis involves inflammation, excess deposition of a collagen-rich extracellular matrix, and calcification. Recent studies have shown that M1 or inflammatory macrophages derived from infiltrating monocytes promote calcification of valvular interstitial cells, the most prevalent cell type of the aortic valve. We hypothesized that valvular interstitial cells could modulate inflammatory macrophages phenotype. METHODS: We first assessed macrophage phenotype in human aortic valve stenosis and control aortic valves from donors. Then, we examined profibrotic and inflammatory-related gene expression in valves and valvular interstitial cells. Finally, we investigated whether valvular interstitial cells can modify the phenotype of inflammatory macrophages. RESULTS: Circulating monocytes and plasma transforming growth factor beta-1 levels of patients with aortic valve stenosis were significantly higher compared with patients without aortic valve stenosis. Histologic analysis of thickened spongiosa of the aortic valve from patients with aortic valve stenosis showed a high macrophage infiltration but a low matrix metalloproteinase-9 expression compared with control aortic valves. On the other hand, valvular interstitial cell culture of aortic valve stenosis exhibited a profibrotic phenotype with a high expression of transforming growth factor beta-1 and transforming growth factor beta-1/transforming growth factor beta-3 ratio but a decreased expression of the peroxisome proliferator-activated receptor gamma nuclear receptor. Valvular interstitial cell-conditioned media of aortic valve stenosis led to a decrease in enzymatic activity of matrix metalloproteinase-9 and an increase in production of collagen in inflammatory macrophages compared with valvular interstitial cell-conditioned media from control aortic valve donors. CONCLUSIONS: These findings indicate that profibrotic valvular interstitial cells promote the imbalance of extracellular matrix remodeling by reducing matrix metalloproteinase-9 production on inflammatory macrophages that lead to excessive collagen deposition observed in aortic valve stenosis. Further investigation is needed to clarify the role of transforming growth factor beta-1/proliferator-activated receptor gamma nuclear receptor/matrix metalloproteinase-9 in aortic valve stenosis.

Circulating Monocyte Subsets and Transcatheter Aortic Valve Replacement.

Transcatheter aortic valve replacement (TAVR), as an alternative to open heart surgery, has revolutionized the treatment of severe aortic valve stenosis (AVS), the most common valvular disorder in the elderly. AVS is now considered a form of atherosclerosis and, like the latter, partly of inflammatory origin. Patients with high-grade AVS have a highly disturbed blood flow associated with high levels of shear stress. The immediate reopening of the valve during TAVR leads to a sudden restoration of a normal blood flow hemodynamic. Despite its good prognosis for patients, TAVR remains associated with bleeding or thrombotic postprocedural complications, involving mechanisms that are still poorly understood. Many studies report the close link between blood coagulation and inflammation, termed thromboinflammation, including monocytes as a major actor. The TAVR procedure represents a unique opportunity to study the influence of shear stress on human monocytes, key mediators of inflammation and hemostasis processes. The purpose of this study was to conduct a review of the literature to provide a comprehensive overview of the impact of TAVR on monocyte phenotype and subset repartition and the association of these parameters with the clinical outcomes of patients with severe AVS who underwent TAVR.

Human Aortic Valve Interstitial Cells Display Proangiogenic Properties During Calcific Aortic Valve Disease.

OBJECTIVE: The study's aim was to analyze the capacity of human valve interstitial cells (VICs) to participate in aortic valve angiogenesis. Approach and Results: VICs were isolated from human aortic valves obtained after surgery for calcific aortic valve disease and from normal aortic valves unsuitable for grafting (control VICs). We examined VIC in vitro and in vivo potential to differentiate in endothelial and perivascular lineages. VIC paracrine effect was also examined on human endothelial colony-forming cells. A pathological VIC (VICp) mesenchymal-like phenotype was confirmed by CD90+/CD73+/CD44+ expression and multipotent-like differentiation ability. When VICp were cocultured with endothelial colony-forming cells, they formed microvessels by differentiating into perivascular cells both in vivo and in vitro. VICp and control VIC conditioned media were compared using serial ELISA regarding quantification of endothelial and angiogenic factors. Higher expression of VEGF (vascular endothelial growth factor)-A was observed at the protein level in VICp-conditioned media and confirmed at the mRNA level in VICp compared with control VIC. Conditioned media from VICp induced in vitro a significant increase in endothelial colony-forming cell proliferation, migration, and sprouting compared with conditioned media from control VIC. These effects were inhibited by blocking VEGF-A with blocking antibody or siRNA approach, confirming VICp involvement in angiogenesis by a VEGF-A dependent mechanism. CONCLUSIONS: We provide here the first proof of an angiogenic potential of human VICs isolated from patients with calcific aortic valve disease. These results point to a novel function of VICp in valve vascularization during calcific aortic valve disease, with a perivascular differentiation ability and a VEGF-A paracrine effect. Targeting perivascular differentiation and VEGF-A to slow calcific aortic valve disease progression warrants further investigation.

Age, Sex, and Valve Phenotype Differences in Fibro-Calcific Remodeling of Calcified Aortic Valve.

Background In calcific aortic valve disease on tricuspid aortic valves (TAVs), men have higher aortic valve calcification and less fibrosis than women. However, little is known in bicuspid aortic valves (BAV). We thus aimed to investigate the impact of age, sex, and valve phenotype (TAVs versus BAVs) on fibro-calcific remodeling in calcific aortic valve disease. Methods and Results We included 2 cohorts: 411 patients who underwent multidetector computed tomography (37% women) for aortic valve calcification density assessment and 138 explanted aortic valves (histological cohort; 50% women). The cohorts were divided in younger (<60 years old) or older patients with BAV (≥60 years old), and TAV patients. In each group, women and men were matched. Women presented less aortic valve calcification density than men in each group of the multidetector computed tomography cohort (all P≤0.01). Moreover, in women, younger patients with BAV had the lowest aortic valve calcification density (both P=0.02). In multivariate analysis, aortic valve calcification density correlated with age (ß estimate±standard error: 6.5±1.8; P=0.0004) and male sex (109.2±18.4; P<0.0001), and there was a trend with TAVs (41.5±23.0; P=0.07). Women presented a higher collagen content than men (77.8±10.8 versus 69.9±12.9%; P<0.001) in the entire cohort. In women, younger patients with BAV had denser connective tissue than TAV and older patients with BAV (both P≤0.05), while no difference was observed between men. Conclusions In calcific aortic valve disease, women had less calcification and more fibrotic remodeling than men, regardless of the phenotype of the valve or age of the patient. Moreover, younger women with BAVs had less valve calcification. Thus, mineralization/fibrosis of the aortic valve is likely to have sex/age-specific mechanisms and be influenced by the valve morphology.

Real-Time Monitoring of von Willebrand Factor in the Catheterization Laboratory: The Seatbelt of Mini-Invasive Transcatheter Aortic Valve Replacement?

Significant paravalvular regurgitation (PVR) remains a relatively frequent (4% to 9%) and deleterious complication of transcatheter aortic valve replacement (TAVR), even with the latest generation of bioprosthesis. Although mini-invasive TAVR without general anesthesia or transesophageal echocardiography (TEE) is progressively becoming the predominant approach, identification and grading of PVR in the catheterization laboratory remain an important and challenging clinical issue. The authors discuss how a recently reported blood biomarker reflecting the von Willebrand factor activity, that is, the closure time with adenosine diphosphate, can be successfully applied during the TAVR procedure to detect and monitor PVR in real time, with an excellent negative predictive value. This point-of-care testing performed directly in the catheterization laboratory may improve the diagnosis of PVR and rationalize the decision of whether or not to perform corrective measures. They further discuss how such a test could be a substitute for the multimodal approach combining TEE, hemodynamics, and cine-angiography, and help to secure the transition to the mini-invasive approach and facilitate the expanding indications of less invasive procedures to lower-risk patients without jeopardizing procedural and clinical outcomes.

DNA methylation of a PLPP3 MIR transposon-based enhancer promotes an osteogenic programme in calcific aortic valve disease.

Aims: Calcific aortic valve disease (CAVD) is characterized by the osteogenic transition of valve interstitial cells (VICs). In CAVD, lysophosphatidic acid (LysoPA), a lipid mediator with potent osteogenic activity, is produced in the aortic valve (AV) and is degraded by membrane-associated phospholipid phosphatases (PLPPs). We thus hypothesized that a dysregulation of PLPPs could participate to the osteogenic reprograming of VICs during CAVD. Methods and results: The expression of PLPPs was examined in human control and mineralized AVs and comprehensive analyses were performed to document the gene regulation and impact of PLPPs on the osteogenic transition of VICs. We found that PLPP3 gene and enzymatic activity were downregulated in mineralized AVs. Multidimensional gene profiling in 21 human AVs showed that expression of PLPP3 was inversely correlated with the level of 5-methylcytosine (5meC) located in an intronic mammalian interspersed repeat (MIR) element. Bisulphite pyrosequencing in a larger series of 67 AVs confirmed that 5meC in intron 1 was increased by 2.2-fold in CAVD compared with control AVs. In isolated cells, epigenome editing with clustered regularly interspersed short palindromic repeats-Cas9 system containing a deficient Cas9 fused with DNA methyltransferase (dCas9-DNMT) was used to increase 5meC in the intronic enhancer and showed that it reduced significantly the expression of PLPP3. Knockdown experiments showed that lower expression of PLPP3 in VICs promotes an osteogenic programme. Conclusions: DNA methylation of a MIR-based enhancer downregulates the expression of PLPP3 and promotes the mineralization of the AV.

A transcriptome-wide association study identifies PALMD as a susceptibility gene for calcific aortic valve stenosis.

Calcific aortic valve stenosis (CAVS) is a common and life-threatening heart disease and the current treatment options cannot stop or delay its progression. A GWAS on 1009 cases and 1017 ethnically matched controls was combined with a large-scale eQTL mapping study of human aortic valve tissues (n = 233) to identify susceptibility genes for CAVS. Replication was performed in the UK Biobank, including 1391 cases and 352,195 controls. A transcriptome-wide association study (TWAS) reveals PALMD (palmdelphin) as significantly associated with CAVS. The CAVS risk alleles and increasing disease severity are both associated with decreased mRNA expression levels of PALMD in valve tissues. The top variant identified shows a similar effect and strong association with CAVS (P = 1.53 × 10-10) in UK Biobank. The identification of PALMD as a susceptibility gene for CAVS provides insights into the genetic nature of this disease, opens avenues to investigate its etiology and to develop much-needed therapeutic options.

Human Alternative Macrophages Populate Calcified Areas of Atherosclerotic Lesions and Display Impaired RANKL-Induced Osteoclastic Bone Resorption Activity.

RATIONALE: Vascular calcification is a process similar to bone formation leading to an inappropriate deposition of calcium phosphate minerals in advanced atherosclerotic plaques. Monocyte-derived macrophages, located in atherosclerotic lesions and presenting heterogeneous phenotypes, from classical proinflammatory M1 to alternative anti-inflammatory M2 macrophages, could potentially display osteoclast-like functions. OBJECTIVE: To characterize the phenotype of macrophages located in areas surrounding the calcium deposits in human atherosclerotic plaques. METHODS AND RESULTS: Macrophages near calcium deposits display an alternative phenotype being both CD68 and mannose receptor-positive, expressing carbonic anhydrase type II, but relatively low levels of cathepsin K. In vitro interleukin-4-polarization of human primary monocytes into macrophages results in lower expression and activity of cathepsin K compared with resting unpolarized macrophages. Moreover, interleukin-4 polarization lowers expression levels of the osteoclast transcriptional activator nuclear factor of activated T cells type c-1, associated with increased gene promoter levels of the transcriptional repression mark H3K27me3 (histone 3 lysine 27 trimethylation). Despite higher expression of the receptor activator of nuclear factor κB receptor, receptor activator of nuclear factor κB ligand/macrophage colony-stimulating factor induction of nuclear factor of activated T cells type c-1 and cathepsin K expression is defective in these macrophages because of reduced Erk/c-fos-mediated downstream signaling resulting in impaired bone resorption capacity. CONCLUSIONS: These results indicate that macrophages surrounding calcium deposits in human atherosclerotic plaques are phenotypically defective being unable to resorb calcification.

Leptin induces osteoblast differentiation of human valvular interstitial cells via the Akt and ERK pathways.

AIMS: Calcific aortic valve disease (CAVD) affects 2-6% of the population over 65 years, and age, gender, smoking, overweight, dyslipidemia, diabetes contribute to the development of this disease. CAVD results, in part, from the osteoblast differentiation of human valvular interstitial cells (VICs). This study aims to elucidate the effects of leptin on osteoblast phenotype of VICs and the signalling pathways involved. METHODS: Patients who underwent aortic valve replacement for CAVD (n = 43) were included in this study. Patients with coronary artery disease (CAD) without CAVD (n = 129) were used as controls. RESULTS: Patients with CAVD had higher serum leptin concentrations than CAD patients (p = 0.002). Leptin was found in calcific aortic valves, with higher concentrations in calcified versus non-calcified zones (p = 0.01). Chronic leptin stimulation of human VICs enhanced alkaline phosphatase (ALP) activity and ALP, BMP-2 and RUNX2 expression and decreased osteopontin expression. Moreover, inhibiting Akt or ERK during leptin stimulation lowered the expression of osteoblast markers in VIC. CONCLUSIONS: Taken together, these findings indicate that leptin plays a critical role in CAVD development by promoting osteoblast differentiation of human aortic VICs in an Akt- and ERK-dependent manner. This study highlights the role of leptin in CAVD development, and further studies are needed to determine whether reducing circulating leptin levels or blocking leptin actions on VICs is efficient to slow CAVD progression.

Meta-Analysis of Abdominal Aortic Aneurysm in Patients With Coronary Artery Disease.

The high coronary artery disease (CAD) prevalence in patients with abdominal aortic aneurysm (AAA) is well known. However, the inverse relation has been little explored. We present, based on a systematic review and meta-analysis of the published evidence, a critical appraisal of the issue of AAA prevalence and also AAA predictive risk factors in patients with CAD, comparing it with AAA prevalence in subjects without CAD. A total of 22 studies involving 13,388 patients with CAD met the inclusion criteria. Overall, AAA prevalence in patients with CAD was 8.4% (95% confidence interval [CI] 6.9 to 10.3), significantly higher than in subjects without CAD (odds ratio [OR] 2.42, 95% CI 2.08 to 2.81). Pooled analysis revealed that smoking, arterial hypertension, and concomitant carotid artery stenosis were significantly associated with AAA in patients with CAD (OR 1.72, 95% CI 1.14 to 2.61; OR 1.57, 95% CI 1.06 to 2.35; OR 2.14, 95% CI 1.20 to 3.79, respectively). In patients with CAD, AAA prevalence tended to be higher with concomitant peripheral artery disease (OR 2.66, 95% CI 0.82 to 8.61, p = 0.08). In conclusion, AAA prevalence was significantly higher in patients with CAD versus subjects without CAD.