Aortic Valve Stenosis Causes Accumulation of Extracellular Hemoglobin and Systemic Endothelial Dysfunction.

BACKGROUND: Whether aortic valve stenosis (AS) can adversely affect systemic endothelial function independently of standard modifiable cardiovascular risk factors is unknown. METHODS: We therefore investigated endothelial and cardiac function in an experimental model of AS mice devoid of standard modifiable cardiovascular risk factors and human cohorts with AS scheduled for transcatheter aortic valve replacement. Endothelial function was determined by flow-mediated dilation using ultrasound. Extracellular hemoglobin (eHb) concentrations and nitric oxide (NO) consumption were determined in blood plasma of mice and humans by ELISA and chemiluminescence. This was complemented by measurements of aortic blood flow using 4-dimensional flow acquisition by magnetic resonance imaging and computational fluid dynamics simulations. The effects of plasma and red blood cell (RBC) suspensions on vascular function were determined in transfer experiments in a murine vasorelaxation bioassay system. RESULTS: In mice, the induction of AS caused systemic endothelial dysfunction. In the presence of normal systolic left ventricular function and mild hypertrophy, the increase in the transvalvular gradient was associated with elevated eryptosis, increased eHb, and increased plasma NO consumption; eHb sequestration by haptoglobin restored endothelial function. Because the aortic valve orifice area in patients with AS decreased, postvalvular mechanical stress in the central ascending aorta increased. This was associated with elevated eHb, circulating RBC-derived microvesicles, eryptotic cells, lower haptoglobin levels without clinically relevant anemia, and consecutive endothelial dysfunction. Transfer experiments demonstrated that reduction of eHb by treatment with haptoglobin or elimination of fluid dynamic stress by transcatheter aortic valve replacement restored endothelial function. In patients with AS and subclinical RBC fragmentation, the remaining circulating RBCs before and after transcatheter aortic valve replacement exhibited intact membrane function, deformability, and resistance to osmotic and hypoxic stress. CONCLUSIONS: AS increases postvalvular swirling blood flow in the central ascending aorta, triggering RBC fragmentation with the accumulation of hemoglobin in the plasma. This increases NO consumption in blood, thereby limiting vascular NO bioavailability. Thus, AS itself promotes systemic endothelial dysfunction independent of other established risk factors. Transcatheter aortic valve replacement is capable of limiting NO scavenging and rescuing endothelial function by realigning postvalvular blood flow to near physiological patterns. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifiers: NCT05603520 and NCT01805739.

AVCAPIR: A Novel Procalcific PIWI-Interacting RNA in Calcific Aortic Valve Disease.

BACKGROUND: Calcification of the aortic valve leads to increased leaflet stiffness and consequently results in the development of calcific aortic valve disease (CAVD). However, the underlying molecular and cellular mechanisms of calcification remain unclear. Here, we identified a novel aortic valve calcification-associated PIWI-interacting RNA (piRNA; AVCAPIR) that increases valvular calcification and promotes CAVD progression. METHODS: Using piRNA sequencing, we identified piRNAs contributing to the pathogenesis of CAVD that we termed AVCAPIRs. High-cholesterol diet-fed ApoE-/- mice with AVCAPIR knockout were used to examine the role of AVCAPIR in aortic valve calcification (AVC). Gain- and loss-of-function assays were conducted to determine the role of AVCAPIR in the induced osteogenic differentiation of human valvular interstitial cells. To dissect the mechanisms underlying AVCAPIR-elicited procalcific effects, we performed various analyses, including an RNA pulldown assay followed by liquid chromatography-tandem mass spectrometry, methylated RNA immunoprecipitation sequencing, and RNA sequencing. RNA pulldown and RNA immunoprecipitation assays were used to study piRNA interactions with proteins. RESULTS: We found that AVCAPIR was significantly upregulated during AVC and exhibited potential diagnostic value for CAVD. AVCAPIR deletion markedly ameliorated AVC in high-cholesterol diet-fed ApoE-/- mice, as shown by reduced thickness and calcium deposition in the aortic valve leaflets, improved echocardiographic parameters (decreased peak transvalvular jet velocity and mean transvalvular pressure gradient, as well as increased aortic valve area), and diminished levels of osteogenic markers (Runx2 and Osterix) in aortic valves. These results were confirmed in osteogenic medium-induced human valvular interstitial cells. Using unbiased protein-RNA screening and molecular validation, we found that AVCAPIR directly interacts with FTO (fat mass and obesity-associated protein), subsequently blocking its N6-methyladenosine demethylase activity. Further transcriptomic and N6-methyladenosine modification epitranscriptomic screening followed by molecular validation confirmed that AVCAPIR hindered FTO-mediated demethylation of CD36 mRNA transcripts, thus enhancing CD36 mRNA stability through the N6-methyladenosine reader IGF2BP1 (insulin-like growth factor 2 mRNA binding protein 1). In turn, the AVCAPIR-dependent increase in CD36 stabilizes its binding partner PCSK9 (proprotein convertase subtilisin/kexin type 9), a procalcific gene, at the protein level, which accelerates the progression of AVC. CONCLUSIONS: We identified a novel piRNA that induced AVC through an RNA epigenetic mechanism and provide novel insights into piRNA-directed theranostics in CAVD.

Multimodal Analytical Tools to Enhance Mechanistic Understanding of Aortic Valve Calcification.

This review focuses on technologies at the core of calcific aortic valve disease (CAVD) and drug target research advancement, including transcriptomics, proteomics, and molecular imaging. We examine how bulk RNA sequencing and single-cell RNA sequencing have engendered organismal genomes and transcriptomes, promoting the analysis of tissue gene expression profiles and cell subpopulations, respectively. We bring into focus how the field is also largely influenced by increasingly accessible proteome profiling techniques. In unison, global transcriptional and protein expression analyses allow for increased understanding of cellular behavior and pathogenic pathways under pathologic stimuli including stress, inflammation, low-density lipoprotein accumulation, increased calcium and phosphate levels, and vascular injury. We also look at how direct investigation of protein signatures paves the way for identification of targetable pathways for pharmacologic intervention. Here, we note that imaging techniques, once a clinical diagnostic tool for late-stage CAVD, have since been refined to address a clinical need to identify microcalcifications using positron emission tomography/computed tomography and even detect in vivo cellular events indicative of early stage CAVD and map the expression of identified proteins in animal models. Together, these techniques generate a holistic approach to CAVD investigation, with the potential to identify additional novel regulatory pathways.

Sex-dependent expression of galectin-1, a cardioprotective ß-galactoside-binding lectin, in human calcific aortic stenosis.

We aimed to analyze sex-related differences in galectin-1 (Gal-1), a ß-galactoside-binding lectin, in aortic stenosis (AS) and its association with the inflammatory and fibrocalcific progression of AS. Gal-1 was determined in serum and aortic valves (AVs) from control and AS donors by western blot and immunohistochemistry. Differences were validated by ELISA and qPCR in AS samples. In vitro experiments were conducted in primary cultured valve interstitial cells (VICs). Serum Gal-1 was not different neither between control and AS nor between men and women. There was no association between circulating and valvular Gal-1 levels. The expression of Gal-1 in stenotic AVs was higher in men than women, even after adjusting for confounding factors, and was associated with inflammation, oxidative stress, extracellular matrix remodeling, fibrosis, and osteogenesis. Gal-1 (LGALS1) mRNA was enhanced within fibrocalcific areas of stenotic AVs, especially in men. Secretion of Gal-1 was up-regulated over a time course of 2, 4, and 8 days in men's calcifying VICs, only peaking at day 4 in women's VICs. In vitro, Gal-1 was associated with similar mechanisms to those in our clinical cohort. ß-estradiol significantly up-regulated the activity of an LGALS1 promoter vector and the secretion of Gal-1, only in women's VICs. Supplementation with rGal-1 prevented the effects elicited by calcific challenge including the metabolic shift to glycolysis. In conclusion, Gal-1 is up-regulated in stenotic AVs and VICs from men in association with inflammation, oxidative stress, matrix remodeling, and osteogenesis. Estrogens can regulate Gal-1 expression with potential implications in post-menopause women. Exogenous rGal-1 can diminish calcific phenotypes in both women and men.

Macrophage Migration Inhibitory Factor Promotes Thromboinflammation and Predicts Fast Progression of Aortic Stenosis.

BACKGROUND: Aortic stenosis (AS) is driven by progressive inflammatory and fibrocalcific processes regulated by circulating inflammatory and valve resident endothelial and interstitial cells. The impact of platelets, platelet-derived mediators, and platelet-monocyte interactions on the acceleration of local valvular inflammation and mineralization is presently unknown. METHODS: We prospectively enrolled 475 consecutive patients with severe symptomatic AS undergoing aortic valve replacement. Clinical workup included repetitive echocardiography, analysis of platelets, monocytes, chemokine profiling, aortic valve tissue samples for immunohistochemistry, and gene expression analysis. RESULTS: The patients were classified as fast-progressive AS by the median ∆Vmax of 0.45 m/s per year determined by echocardiography. Immunohistological aortic valve analysis revealed enhanced cellularity in fast-progressive AS (slow- versus fast-progressive AS; median [interquartile range], 247 [142.3-504] versus 717.5 [360.5-1234]; P<0.001) with less calcification (calcification area, mm2: 33.74 [27.82-41.86] versus 20.54 [13.52-33.41]; P<0.001). MIF (macrophage migration inhibitory factor)-associated gene expression was significantly enhanced in fast-progressive AS accompanied by significantly elevated MIF plasma levels (mean±SEM; 6877±379.1 versus 9959±749.1; P<0.001), increased platelet activation, and decreased intracellular MIF expression indicating enhanced MIF release upon platelet activation (CD62P, %: median [interquartile range], 16.8 [11.58-23.8] versus 20.55 [12.48-32.28], P=0.005; MIF, %: 4.85 [1.48-9.75] versus 2.3 [0.78-5.9], P<0.001). Regression analysis confirmed that MIF-associated biomarkers are strongly associated with an accelerated course of AS. CONCLUSIONS: Our findings suggest a key role for platelet-derived MIF and its interplay with circulating and valve resident monocytes/macrophages in local and systemic thromboinflammation during accelerated AS. MIF-based biomarkers predict an accelerated course of AS and represent a novel pharmacological target to attenuate progression of AS.

Genome-wide chromatin contacts of super-enhancer-associated lncRNA identify LINC01013 as a regulator of fibrosis in the aortic valve.

Calcific aortic valve disease (CAVD) is characterized by a fibrocalcific process. The regulatory mechanisms that drive the fibrotic response in the aortic valve (AV) are poorly understood. Long noncoding RNAs derived from super-enhancers (lncRNA-SE) control gene expression and cell fate. Herein, multidimensional profiling including chromatin immunoprecipitation and sequencing, transposase-accessible chromatin sequencing, genome-wide 3D chromatin contacts of enhancer-promoter identified LINC01013 as an overexpressed lncRNA-SE during CAVD. LINC01013 is within a loop anchor, which has contact with the promoter of CCN2 (CTGF) located at ~180 kb upstream. Investigation showed that LINC01013 acts as a decoy factor for the negative transcription elongation factor E (NELF-E), whereby it controls the expression of CCN2. LINC01013-CCN2 is part of a transforming growth factor beta 1 (TGFB1) network and exerts a control over fibrogenesis. These findings illustrate a novel mechanism whereby a dysregulated lncRNA-SE controls, through a looping process, the expression of CCN2 and fibrogenesis of the AV.

Interleukin 38 alleviates aortic valve calcification by inhibition of NLRP3.

Calcific aortic valve disease (CAVD) is common in people over the age of 65. Progressive valvular calcification is a characteristic of CAVD and due to chronic inflammation in aortic valve interstitial cells (AVICs) resulting in CAVD progression. IL-38 is a naturally occurring anti-inflammatory cytokine; here, we report lower levels of endogenous IL-38 in AVICs isolated from patients' CAVD valves compared to AVICs from non-CAVD valves. Recombinant IL-38 suppressed spontaneous inflammatory activity and calcium deposition in cultured AVICs. In mice, knockdown of IL-38 enhanced the production of inflammatory mediators in murine AVICs exposed to the proinflammatory stimulant matrilin-2. We also observed that in cultured AVICs matrilin-2 stimulation activated the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome with procaspase-1 cleavage into active caspase-1. The addition of IL-38 to matrilin-2-treated AVICs suppressed caspase-1 activation and reduced the expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, runt-related transcription factor 2, and alkaline phosphatase. Aged IL-38-deficient mice fed a high-fat diet exhibited aortic valve lesions compared to aged wild-type mice fed the same diet. The interleukin-1 receptor 9 (IL-1R9) is the putative receptor mediating the anti-inflammatory properties of IL-38; we observed that IL-1R9-deficient mice exhibited spontaneous aortic valve thickening and greater calcium deposition in AVICs compared to wild-type mice. These data demonstrate that IL-38 suppresses spontaneous and stimulated osteogenic activity in aortic valve via inhibition of the NLRP3 inflammasome and caspase-1. The findings of this study suggest that IL-38 has therapeutic potential for prevention of CAVD progression.

Network modeling predicts personalized gene expression and drug responses in valve myofibroblasts cultured with patient sera.

Aortic valve stenosis (AVS) patients experience pathogenic valve leaflet stiffening due to excessive extracellular matrix (ECM) remodeling. Numerous microenvironmental cues influence pathogenic expression of ECM remodeling genes in tissue-resident valvular myofibroblasts, and the regulation of complex myofibroblast signaling networks depends on patient-specific extracellular factors. Here, we combined a manually curated myofibroblast signaling network with a data-driven transcription factor network to predict patient-specific myofibroblast gene expression signatures and drug responses. Using transcriptomic data from myofibroblasts cultured with AVS patient sera, we produced a large-scale, logic-gated differential equation model in which 11 biochemical and biomechanical signals were transduced via a network of 334 signaling and transcription reactions to accurately predict the expression of 27 fibrosis-related genes. Correlations were found between personalized model-predicted gene expression and AVS patient echocardiography data, suggesting links between fibrosis-related signaling and patient-specific AVS severity. Further, global network perturbation analyses revealed signaling molecules with the most influence over network-wide activity, including endothelin 1 (ET1), interleukin 6 (IL6), and transforming growth factor ß (TGFß), along with downstream mediators c-Jun N-terminal kinase (JNK), signal transducer and activator of transcription (STAT), and reactive oxygen species (ROS). Lastly, we performed virtual drug screening to identify patient-specific drug responses, which were experimentally validated via fibrotic gene expression measurements in valvular interstitial cells cultured with AVS patient sera and treated with or without bosentan-a clinically approved ET1 receptor inhibitor. In sum, our work advances the ability of computational approaches to provide a mechanistic basis for clinical decisions including patient stratification and personalized drug screening.

Lumican promotes calcific aortic valve disease through H3 histone lactylation.

BACKGROUND AND AIMS: Valve interstitial cells (VICs) undergo a transition to intermediate state cells before ultimately transforming into the osteogenic cell population, which is a pivotal cellular process in calcific aortic valve disease (CAVD). Herein, this study successfully delineated the stages of VIC osteogenic transformation and elucidated a novel key regulatory role of lumican (LUM) in this process. METHODS: Single-cell RNA-sequencing (scRNA-seq) from nine human aortic valves was used to characterize the pathological switch process and identify key regulatory factors. The in vitro, ex vivo, in vivo, and double knockout mice were constructed to further unravel the calcification-promoting effect of LUM. Moreover, the multi-omic approaches were employed to analyse the molecular mechanism of LUM in CAVD. RESULTS: ScRNA-seq successfully delineated the process of VIC pathological transformation and highlighted the significance of LUM as a novel molecule in this process. The pro-calcification role of LUM is confirmed on the in vitro, ex vivo, in vivo level, and ApoE-/-//LUM-/- double knockout mice. The LUM induces osteogenesis in VICs via activation of inflammatory pathways and augmentation of cellular glycolysis, resulting in the accumulation of lactate. Subsequent investigation has unveiled a novel LUM driving histone modification, lactylation, which plays a role in facilitating valve calcification. More importantly, this study has identified two specific sites of histone lactylation, namely, H3K14la and H3K9la, which have been found to facilitate the process of calcification. The confirmation of these modification sites' association with the expression of calcific genes Runx2 and BMP2 has been achieved through ChIP-PCR analysis. CONCLUSIONS: The study presents novel findings, being the first to establish the involvement of lumican in mediating H3 histone lactylation, thus facilitating the development of aortic valve calcification. Consequently, lumican would be a promising therapeutic target for intervention in the treatment of CAVD.

ATF6 protects against protein misfolding during cardiac hypertrophy.

Cardiomyocytes activate the unfolded protein response (UPR) transcription factor ATF6 during pressure overload-induced hypertrophic growth. The UPR is thought to increase ER protein folding capacity and maintain proteostasis. ATF6 deficiency during pressure overload leads to heart failure, suggesting that ATF6 protects against myocardial dysfunction by preventing protein misfolding. However, conclusive evidence that ATF6 prevents toxic protein misfolding during cardiac hypertrophy is still pending. Here, we found that activation of the UPR, including ATF6, is a common response to pathological cardiac hypertrophy in mice. ATF6 KO mice failed to induce sufficient levels of UPR target genes in response to chronic isoproterenol infusion or transverse aortic constriction (TAC), resulting in impaired cardiac growth. To investigate the effects of ATF6 on protein folding, the accumulation of poly-ubiquitinated proteins as well as soluble amyloid oligomers were directly quantified in hypertrophied hearts of WT and ATF6 KO mice. Whereas only low levels of protein misfolding was observed in WT hearts after TAC, ATF6 KO mice accumulated increased quantities of misfolded protein, which was associated with impaired myocardial function. Collectively, the data suggest that ATF6 plays a critical adaptive role during cardiac hypertrophy by protecting against protein misfolding.

PALMD haploinsufficiency aggravates extracellular matrix remodeling in vascular smooth muscle cells and promotes calcification.

Reduced PALMD expression is strongly associated with the development of calcified aortic valve stenosis; however, the role of PALMD in vascular calcification remains unknown. Calcified arteries were collected from mice to detect PALMD expression. Heterozygous Palmd knockout (Palmd+/-) mice were established to explore the role of PALMD in subtotal nephrectomy-induced vascular calcification. RNA sequencing was applied to detect molecular changes in aortas from Palmd+/- mice. Primary Palmd+/- vascular smooth muscle cells (VSMCs) or PALMD-silenced VSMCs by short interfering RNA were used to analyze PALMD function in phenotypic changes and calcification. PALMD haploinsufficiency aggravated subtotal nephrectomy-induced vascular calcification. RNA sequencing analysis showed that loss of PALMD disturbed the synthesis and degradation of the extracellular matrix (ECM) in aortas, including collagens and matrix metalloproteinases (Col6a6, Mmp2, Mmp9, etc.). In vitro experiments revealed that PALMD-deficient VSMCs were more susceptible to high phosphate-induced calcification. Downregulation of SMAD6 expression and increased levels of p-SMAD2 were detected in Palmd+/- VSMCs, suggesting that transforming growth factor-ß signaling may be involved in PALMD haploinsufficiency-induced vascular calcification. Our data revealed that PALMD haploinsufficiency causes ECM dysregulation in VSMCs and aggravates vascular calcification. Our findings suggest that reduced PALMD expression is also linked to vascular calcification, and PALMD may be a potential therapeutic target for this disease. NEW & NOTEWORTHY We found that PALMD haploinsufficiency causes extracellular matrix dysregulation, reduced PALMD expression links to vascular calcification, and PALMD mutations may lead to the risk of both calcific aortic valve stenosis and vascular calcification.

Myocardial proteome changes in aortic stenosis rats subjected to long-term aerobic exercise.

The effects of exercise training (ET) on the heart of aortic stenosis (AS) rats are controversial and the mechanisms involved in alterations induced by ET have been poorly clarified. In this study, we analyzed the myocardial proteome to identify proteins modulated by moderate-intensity aerobic ET in rats with chronic supravalvular AS. Wistar rats were divided into four groups: sedentary control (C-Sed), exercised control (C-Ex), sedentary aortic stenosis (AS-Sed), and exercised AS (AS-Ex). ET consisted of five treadmill running sessions per week for 16 weeks. Statistical analysis was performed by ANOVA or Kruskal-Wallis and Goodman tests. Results were discussed at a significance level of 5%. At the end of the experiment, AS-Ex rats had higher functional capacity, lower blood lactate concentration, and better cardiac structural and left ventricular (LV) functional parameters than the AS-Sed. Myocardial proteome analysis showed that AS-Sed had higher relative protein abundance related to the glycolytic pathway, oxidative stress, and inflammation, and lower relative protein abundance related to beta-oxidation than C-Sed. AS-Ex had higher abundance of one protein related to mitochondrial biogenesis and lower relative protein abundance associated with oxidative stress and inflammation than AS-Sed. Proteomic data were validated for proteins related to lipid and glycolytic metabolism. Chronic pressure overload changes the abundance of myocardial proteins that are mainly involved in lipid and glycolytic energy metabolism in rats. Moderate-intensity aerobic training attenuates changes in proteins related to oxidative stress and inflammation and increases the COX4I1 protein, related to mitochondrial biogenesis. Protein changes are combined with improved functional capacity, cardiac remodeling, and LV function in AS rats.

Th1 cells reduce the osteoblast-like phenotype in valvular interstitial cells by inhibiting NLRP3 inflammasome activation in macrophages.

BACKGROUND AND AIMS: Inflammation is initiates the propagation phase of aortic valve calcification. The activation of NLRP3 signaling in macrophages plays a crucial role in the progression of calcific aortic valve stenosis (CAVS). IFN-γ regulates NLRP3 activity in macrophages. This study aimed to explore the mechanism of IFN-γ regulation and its impact on CAVS progression and valve interstitial cell transdifferentiation. METHODS AND RESULTS: The number of Th1 cells and the expression of IFN-γ and STAT1 in the aortic valve, spleen and peripheral blood increased significantly as CAVS progressed. To explore the mechanisms underlying the roles of Th1 cells and IFN-γ, we treated CAVS mice with IFN-γ-AAV9 or an anti-IFN-γ neutralizing antibody. While IFN-γ promoted aortic valve calcification and dysfunction, it significantly decreased NLRP3 signaling in splenic macrophages and Ly6C+ monocytes. In vitro coculture showed that Th1 cells inhibited NLPR3 activation in ox-LDL-treated macrophages through the IFN-γR1/IFN-γR2-STAT1 pathway. Compared with untreated medium, conditioned medium from Th1-treated bone marrow-derived macrophages reduced the osteogenic calcification of valvular interstitial cells. CONCLUSION: Inhibition of the NLRP3 inflammasome by Th1 cells protects against valvular interstitial cell calcification as a negative feedback mechanism of adaptive immunity toward innate immunity. This study provides a precision medicine strategy for CAVS based on the targeting of anti-inflammatory mechanisms.

Advanced glycation end product-modified low-density lipoprotein promotes pro-osteogenic reprogramming via RAGE/NF-κB pathway and exaggerates aortic valve calcification in hamsters.

BACKGROUND: Advanced glycation end product-modified low-density lipoprotein (AGE-LDL) is related to inflammation and the development of atherosclerosis. Additionally, it has been demonstrated that receptor for advanced glycation end products (RAGE) has a role in the condition known as calcific aortic valve disease (CAVD). Here, we hypothesized that the AGE-LDL/RAGE axis could also be involved in the pathophysiological mechanism of CAVD. METHODS: Human aortic valve interstitial cells (HAVICs) were stimulated with AGE-LDL following pre-treatment with or without interleukin 37 (IL-37). Low-density lipoprotein receptor deletion (Ldlr-/-) hamsters were randomly allocated to chow diet (CD) group and high carbohydrate and high fat diet (HCHFD) group. RESULTS: AGE-LDL levels were significantly elevated in patients with CAVD and in a hamster model of aortic valve calcification. Our in vitro data further demonstrated that AGE-LDL augmented the expression of intercellular cell adhesion molecule-1 (ICAM-1), interleukin-6 (IL-6) and alkaline phosphatase (ALP) in a dose-dependent manner through NF-κB activation, which was attenuated by nuclear factor kappa-B (NF-κB) inhibitor Bay11-7082. The expression of RAGE was augmented in calcified aortic valves, and knockdown of RAGE in HAVICs attenuated the AGE-LDL-induced inflammatory and osteogenic responses as well as NF-κB activation. IL-37 suppressed inflammatory and osteogenic responses and NF-κB activation in HAVICs. The vivo experiment also demonstrate that supplementation with IL-37 inhibited valvular inflammatory response and thereby suppressed valvular osteogenic activities. CONCLUSIONS: AGE-LDL promoted inflammatory responses and osteogenic differentiation through RAGE/NF-κB pathway in vitro and aortic valve lesions in vivo. IL-37 suppressed the AGE-LDL-induced inflammatory and osteogenic responses in vitro and attenuated aortic valve lesions in a hamster model of CAVD.

Fibrosis and expression of extracellular matrix proteins in human interventricular septum in aortic valve stenosis and regurgitation.

Valvular heart disease leads to ventricular pressure and/or volume overload. Pressure overload leads to fibrosis, which might regress with its resolution, but the limits and details of this reverse remodeling are not known. To gain more insight into the extent and nature of cardiac fibrosis in valve disease, we analyzed needle biopsies taken from the interventricular septum of patients undergoing surgery for valve replacement focusing on the expression and distribution of major extracellular matrix protein involved in this process. Proteomic analysis performed using mass spectrometry revealed an excellent correlation between the expression of collagen type I and III, but there was little correlation with the immunohistochemical staining performed on sister sections, which included antibodies against collagen I, III, fibronectin, sarcomeric actin, and histochemistry for wheat germ agglutinin. Surprisingly, the immunofluorescence intensity did not correlate significantly with the gold standard for fibrosis quantification, which was performed using Picrosirius Red (PSR) staining, unless multiplexed on the same tissue section. There was also little correlation between the immunohistochemical markers and pressure gradient severity. It appears that at least in humans, the immunohistochemical pattern of fibrosis is not clearly correlated with standard Picrosirius Red staining on sister sections or quantitative proteomic data, possibly due to tissue heterogeneity at microscale, comorbidities, or other patient-specific factors. For precise correlation of different types of staining, multiplexing on the same section is the best approach.

Myocardial transcriptomic analysis of diabetic patients with aortic stenosis: key role for mitochondrial calcium signaling.

BACKGROUND: Type 2 diabetes (T2D) is a frequent comorbidity encountered in patients with severe aortic stenosis (AS), leading to an adverse left ventricular (LV) remodeling and dysfunction. Metabolic alterations have been suggested as contributors of the deleterious effect of T2D on LV remodeling and function in patients with severe AS, but so far, the underlying mechanisms remain unclear. Mitochondria play a central role in the regulation of cardiac energy metabolism. OBJECTIVES: We aimed to explore the mitochondrial alterations associated with the deleterious effect of T2D on LV remodeling and function in patients with AS, preserved ejection fraction, and no additional heart disease. METHODS: We combined an in-depth clinical, biological and echocardiography phenotype of patients with severe AS, with (n = 34) or without (n = 50) T2D, referred for a valve replacement, with transcriptomic and histological analyses of an intra-operative myocardial LV biopsy. RESULTS: T2D patients had similar AS severity but displayed worse cardiac remodeling, systolic and diastolic function than non-diabetics. RNAseq analysis identified 1029 significantly differentially expressed genes. Functional enrichment analysis revealed several T2D-specific upregulated pathways despite comorbidity adjustment, gathering regulation of inflammation, extracellular matrix organization, endothelial function/angiogenesis, and adaptation to cardiac hypertrophy. Downregulated gene sets independently associated with T2D were related to mitochondrial respiratory chain organization/function and mitochondrial organization. Generation of causal networks suggested a reduced Ca2+ signaling up to the mitochondria, with the measured gene remodeling of the mitochondrial Ca2+ uniporter in favor of enhanced uptake. Histological analyses supported a greater cardiomyocyte hypertrophy and a decreased proximity between the mitochondrial VDAC porin and the reticular IP3-receptor in T2D. CONCLUSIONS: Our data support a crucial role for mitochondrial Ca2+ signaling in T2D-induced cardiac dysfunction in severe AS patients, from a structural reticulum-mitochondria Ca2+ uncoupling to a mitochondrial gene remodeling. Thus, our findings open a new therapeutic avenue to be tested in animal models and further human cardiac biopsies in order to propose new treatments for T2D patients suffering from AS. TRIAL REGISTRATION: URL: https://www. CLINICALTRIALS: gov ; Unique Identifier: NCT01862237.

Role of CD34 in calcification of human aortic valve interstitial cells from patients with aortic valve stenosis.

Various osteogenic factors are involved in ectopic human aortic valve calcification; however, the key cell species involved in calcification remains unclear. In a previous study, we reported that mesenchymal stem (CD73, 90, 105) and endothelial (VEGFR2) cell markers are positive in almost all human aortic valve interstitial cells (HAVICs) obtained from a patient with calcified aortic valve stenosis (CAVS). Further, CD34-negative HAVICs are highly sensitive to calcification stimulations. Here, we aimed to pathophysiologically clarify the role of CD34 in HAVICs obtained from individual patients with severe CAVS. A DNA microarray between CD34-positive and CD34-negative HAVICs, separated by fluorescence-activated cell sorting, indicated that tenascin X (TNX) mRNA expression significantly decreased in CD34-negative cells. Furthermore, the inflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1ß significantly downregulated CD34 expression in HAVICs. TGF-ß, a key cytokine of endothelial-mesenchymal transition, did not affect HAVIC calcification. CD34 overexpression strongly inhibited TNF-α- and IL-1ß-induced calcification and maintained TNX mRNA expression. These results suggest one possibility that CD34 is an inhibitory regulator of valve calcification. Furthermore, TNF-α- and IL-1ß-induced CD34 downregulation in HAVICs contributes to HAVIC calcification by downregulating TNX protein expression.

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.

Oxidized phospholipids are proinflammatory and proatherogenic in hypercholesterolaemic mice.

Oxidized phospholipids (OxPL) are ubiquitous, are formed in many inflammatory tissues, including atherosclerotic lesions, and frequently mediate proinflammatory changes 1 . Because OxPL are mostly the products of non-enzymatic lipid peroxidation, mechanisms to specifically neutralize them are unavailable and their roles in vivo are largely unknown. We previously cloned the IgM natural antibody E06, which binds to the phosphocholine headgroup of OxPL, and blocks the uptake of oxidized low-density lipoprotein (OxLDL) by macrophages and inhibits the proinflammatory properties of OxPL2-4. Here, to determine the role of OxPL in vivo in the context of atherogenesis, we generated transgenic mice in the Ldlr-/- background that expressed a single-chain variable fragment of E06 (E06-scFv) using the Apoe promoter. E06-scFv was secreted into the plasma from the liver and macrophages, and achieved sufficient plasma levels to inhibit in vivo macrophage uptake of OxLDL and to prevent OxPL-induced inflammatory signalling. Compared to Ldlr-/- mice, Ldlr -/- E06-scFv mice had 57-28% less atherosclerosis after 4, 7 and even 12 months of 1% high-cholesterol diet. Echocardiographic and histologic evaluation of the aortic valves demonstrated that E06-scFv ameliorated the development of aortic valve gradients and decreased aortic valve calcification. Both cholesterol accumulation and in vivo uptake of OxLDL were decreased in peritoneal macrophages, and both peritoneal and aortic macrophages had a decreased inflammatory phenotype. Serum amyloid A was decreased by 32%, indicating decreased systemic inflammation, and hepatic steatosis and inflammation were also decreased. Finally, the E06-scFv prolonged life as measured over 15 months. Because the E06-scFv lacks the functional effects of an intact antibody other than the ability to bind OxPL and inhibit OxLDL uptake in macrophages, these data support a major proatherogenic role of OxLDL and demonstrate that OxPL are proinflammatory and proatherogenic, which E06 counteracts in vivo. These studies suggest that therapies inactivating OxPL may be beneficial for reducing generalized inflammation, including the progression of atherosclerosis, aortic stenosis and hepatic steatosis.

Hyperglycemia-simulating environment attenuated experimentally induced calcification in cultured human aortic valve interstitial cells.

Objectives: The role of diabetes mellitus as a risk factor for the development of calcific aortic valve disease has not been fully clarified. Aortic valve interstitial cells (VICs) have been suggested to be crucial for calcification of the valve. Induced calcification in cultured VICs is a good in vitro model for aortic valve calcification. The purpose of this study was to investigate whether increased glucose levels increase experimentally induced calcification in cultured human VICs. Design: VICs were isolated from explanted calcified aortic valves after valve replacement. Osteogenic medium induced calcification of cultured VICs at different glucose levels (5, 15, and 25 mM). Calcium deposits were visualized using Alizarin Red staining and measured spectrophotometrically. Results: The higher the glucose concentration, the lower the level of calcification. High glucose (25 mM) reduced calcification by 52% compared with calcification at a physiological (5 mM) glucose concentration (correlation and regression analysis: r = -0.55, p = .025 with increased concentration of glucose). Conclusions: In vitro hyperglycemia-like conditions attenuated calcification in VICs. High glucose levels may trigger a series of events that secondarily stimulate calcification of VICs in vivo.