Search Results (43)

Male sex and aging are risk factors for fibro-calcific aortic valve disease (FCAVD), indicating an understudied influence of sex hormones. Valvular interstitial cells (VICs) from female and male donors were isolated and exposed to pro-calcifying medium (PM), and the expression of matrix gla protein (MGP), fibronectin (FN1) and bone morphogenic protein 2 (BMP2) was analyzed. The effect of sex hormones on hydroxyapatite (HA) deposition by VICs was also analyzed. Exposure to PM increased MGP gene expression in male (n = 5, +5.8-fold, p = 0.031), and female VICs (n = 6, +4.9-fold, p = 0.004). In female VICs a +3.5-fold MGP increase accompanied the transition from the fibrotic to the calcific phase (p = 0.022 vs. males) while in male VICs the increase was delayed to the calcific phase. Female VICs upregulated FN1 (+1.8-fold, p = 0.003), while male VICs upregulated BMP2 (+3.7-fold, p = 0.05). 5α-dihydrotestosterone increased HA deposition +6.3-fold in male and +5.2-fold in female VICs (p ≤ 0.001 and p < 0.04, respectively). It further decreased BMP2 (p < 0.001) in male VICs and increased MGP in female VICs (p = 0.087). Female VICs decreased HA deposition when exposed to progesterone (−2.4-fold, p = 0.037 vs. PM) and estrogen (−2.0-fold, p = 0.072). In summary, VICs show donor-sex-specific gene expression which is modifiable by 5α-dihydrotestosterone. This needs to be considered when designing in vitro regulatory studies. Full article

Adipose tissue is present in aortic valves (AVs). Valve interstitial cells (VICs) could differentiate into adipogenic lineages. We here characterize whether the presence of adipose tissue in the AV influences inflammation and extracellular matrix (ECM) composition in patients with aortic regurgitation (AR). A total of 144 AVs were analyzed by histological and molecular techniques. We performed discovery studies using Olink Proteomics^® technology in 40 AVs (N = 16 without and N = 24 with adipose tissue). In vitro, human white adipocytes (HWAs) or VICs were cultured with adipogenic media and co-cultured with control VICs. Of Avs, 67% presented white-like adipocytes within the spongiosa. Discovery studies revealed increased levels of inflammatory and ECM molecules in AVs containing adipocytes. Interestingly, the presence of adipocytes was associated with greater AV thickness, higher inflammation, and ECM remodeling, which was characterized by increased proinflammatory molecules, collagen, fibronectin, proteoglycans, and metalloproteinases. AV thickness positively correlated with markers of adipose tissue, inflammation, and ECM. In vitro, adipocyte-like VICs expressed higher levels of adipocyte markers, increased cytokines, fibronectin, decorin, and MMP-13. Analyses of supernatants from co-cultured control VICs with HWA or adipocyte-like VICs showed higher expression of inflammatory mediators, collagen type I, proteoglycans, and metalloproteinases. AVs presenting adipocytes were thicker and exhibited changes characterized by increased inflammation accompanied by aberrant expression of collagen, proteoglycans, and metalloproteinases. VICs could differentiate into adipogenic pathway, affect neighbor VICs, and contribute to inflammation, collagen and proteoglycan accumulation, as well as to metalloproteinases secretion. In summary, the presence of adipose tissue in AV could modify its composition, favoring inflammation and remodeling with an impact on AV thickness. Full article

Background: Calcific aortic valve disease (CAVD) is a highly prevalent disease, especially in the elderly population, but there are no effective drug therapies other than aortic valve repair or replacement. CAVD develops preferentially on the fibrosa side, while the ventricularis side remains relatively spared through unknown mechanisms. We hypothesized that the fibrosa is prone to the disease due to side-dependent differences in transcriptomic patterns and cell phenotypes. Methods: To test this hypothesis, we performed single-cell RNA sequencing using a new method to collect endothelial-enriched samples independently from the fibrosa and ventricularis sides of freshly obtained human aortic valve leaflets from five donors, ranging from non-diseased to fibrocalcific stages. Results: From the 82,356 aortic valve cells analyzed, we found 27 cell clusters, including seven valvular endothelial cell (VEC), nine valvular interstitial cell (VIC), and seven immune, three transitional, and one stromal cell population. We identified several side-dependent VEC subtypes with unique gene expression patterns. Homeostatic VIC clusters were abundant in non-diseased tissues, while VICs enriched with fibrocalcific genes and pathways were more prevalent in diseased leaflets. Furthermore, homeostatic macrophage (MΦ) clusters decreased while inflammatory MΦ and T-cell clusters increased with disease progression. A foamy MΦ cluster was increased in the fibrosa of mildly diseased tissues. Some side-dependent VEC clusters represented non-diseased, protective phenotypes, while others were CAVD-associated and were characterized by genes enriched in pathways of inflammation, endothelial–mesenchymal transition, apoptosis, proliferation, and fibrosis. Interestingly, we found several activator protein-1 (AP-1)-related transcription factors (FOSB, FOS, JUN, JUNB) and EGR1 to be upregulated in the fibrosa and diseased aortic valve leaflets. Conclusions: Our results showed that VECs are highly heterogeneous in a side- and CAVD-dependent manner. Unique VEC clusters and their differentially regulated genes and pathways found in the fibrosa of diseased tissues may represent novel pathogenic mechanisms and potential therapeutic targets. Full article

Heart disease is a leading cause of mortality, with calcific aortic valve disease (CAVD) being the most prevalent subset. Being able to predict this disease in its early stages is important for monitoring patients before they need aortic valve replacement surgery. Thus, this study explored hydrodynamic, mechanical, and hemodynamic differences in healthy and very mildly calcified porcine small intestinal submucosa (PSIS) bioscaffold valves to determine any notable parameters between groups that could, possibly, be used for disease tracking purposes. Three valve groups were tested: raw PSIS as a control and two calcified groups that were seeded with human valvular interstitial and endothelial cells (VICs/VECs) and cultivated in calcifying media. These two calcified groups were cultured in either static or bioreactor-induced oscillatory flow conditions. Hydrodynamic assessments showed metrics were below thresholds associated for even mild calcification. Young’s modulus, however, was significantly higher in calcified valves when compared to raw PSIS, indicating the morphological changes to the tissue structure. Fluid–structure interaction (FSI) simulations agreed well with hydrodynamic results and, most notably, showed a significant increase in time-averaged wall shear stress (TAWSS) between raw and calcified groups. We conclude that tracking hemodynamics may be a viable biomarker for early-stage CAVD tracking. Full article

Lysyl oxidase (LOX)-mediated extracellular matrix crosslinking modulates calcification in atherosclerosis and aortic valve disease; however, this enzyme also induces oxidative stress. We addressed the contribution of LOX-dependent oxidative stress to cardiovascular calcification. LOX is upregulated in human-calcified atherosclerotic lesions and atheromas from atherosclerosis-challenged LOX transgenic mice (TgLOX^VSMC) and colocalized with a marker of oxidative stress (8-oxo-deoxyguanosine) in vascular smooth muscle cells (VSMCs). Similarly, in calcific aortic valves, high LOX expression was detected in valvular interstitial cells (VICs) positive for 8-oxo-deoxyguanosine, while LOX and LOXL2 expression correlated with osteogenic markers (SPP1 and RUNX2) and NOX2. In human VICs, mito-TEMPO and TEMPOL attenuated the increase in superoxide anion levels and the mineralization induced by osteogenic media (OM). Likewise, in OM-exposed VICs, β-aminopropionitrile (a LOX inhibitor) ameliorated both oxidative stress and calcification. Gain- and loss-of-function approaches in VICs demonstrated that while LOX silencing negatively modulates oxidative stress and calcification induced by OM, lentiviral LOX overexpression exacerbated oxidative stress and VIC calcification, effects that were prevented by mito-TEMPO, TEMPOL, and β-aminopropionitrile. Our data indicate that LOX-induced oxidative stress participates in the procalcifying effects of LOX activity in ectopic cardiovascular calcification, and highlight the multifaceted role played by LOX isoenzymes in cardiovascular diseases. Full article

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

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

Amyloid deposition within stenotic aortic valves (AVs) also appears frequent in the absence of cardiac amyloidosis, but its clinical and pathophysiological relevance has not been investigated. We will elucidate the rate of isolated AV amyloid deposition and its potential clinical and pathophysiological significance in aortic stenosis (AS). In 130 patients without systemic and/or cardiac amyloidosis, we collected the explanted AVs during cardiac surgery: 57 patients with calcific AS and 73 patients with AV insufficiency (41 with AV sclerosis and 32 without, who were used as controls). Amyloid deposition was found in 21 AS valves (37%), 4 sclerotic AVs (10%), and none of the controls. Patients with and without isolated AV amyloid deposition had similar clinical and echocardiographic characteristics and survival rates. Isolated AV amyloid deposition was associated with higher degrees of AV fibrosis (p = 0.0082) and calcification (p < 0.0001). Immunohistochemistry analysis suggested serum amyloid A1 (SAA1), in addition to transthyretin (TTR), as the protein possibly involved in AV amyloid deposition. Circulating SAA1 levels were within the normal range in all groups, and no difference was observed in AS patients with and without AV amyloid deposition. In vitro, AV interstitial cells (VICs) were stimulated with interleukin (IL)-1β which induced increased SAA1-mRNA both in the control VICs (+6.4 ± 0.5, p = 0.02) and the AS VICs (+7.6 ± 0.5, p = 0.008). In conclusion, isolated AV amyloid deposition is frequent in the context of AS, but it does not appear to have potential clinical relevance. Conversely, amyloid deposition within AV leaflets, probably promoted by local inflammation, could play a role in AS pathophysiology. Full article

Ischemia–reperfusion injury (IRI) in the myocardium has been thoroughly researched, especially in acute coronary syndrome and heart transplantation. However, our understanding of IRI implications on cardiac valves is still developing. This knowledge gap becomes even more pronounced given the advent of partial heart transplantation, a procedure designed to implant isolated human heart valves in young patients. This study aims to investigate the effects of IRI on aortic valvular endothelial cells (VECs), valvular interstitial cells (VICs), and whole leaflet cultures (no separation of VECs and VICs). We employed two conditions: hypoxic cold storage reperfusion (HCSR) and normothermia (NT). Key markers, secreted protein acidic and cysteine rich (SPARC) (osteonectin), and inducible nitric oxide synthase (iNOS2) were evaluated. In the isolated cells under HCSR, VICs manifested a significant 15-fold elevation in SPARC expression compared to NT (p = 0.0016). Conversely, whole leaflet cultures exhibited a 1-fold increment in SPARC expression in NT over HCSR (p = 0.0011). iNOS2 expression in VECs presented a marginal rise in HCSR, whereas, in whole leaflet settings, there was a 1-fold ascent in NT compared to HCSR (p = 0.0003). Minor escalations in the adhesion molecules intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), E-selection, and P-selection were detected in HCSR for whole leaflet cultures, albeit without statistical significance. Additionally, under HCSR, VICs released a markedly higher quantity of IL-6 and IL-8, with respective p-values of 0.0033 and <0.0001. Interestingly, the IL-6 levels in VECs remained consistent across both HCSR and NT conditions. These insights lay the groundwork for understanding graft IRI following partial heart transplantation and hint at the interdependent dynamic of VECs and VICs in valvular tissue. Full article

Thoracic radiation therapy may result in accelerated atherosclerosis and in late aortic valve stenosis (AS). In this study, we assessed the feasibility of inducing radiation-induced AS using a targeted aortic valve irradiation (10 or 20 Grays) in two groups of C57Bl6/J (WT) and ApoE^−/− mice compared to a control (no irradiation). Peak aortic jet velocity was evaluated by echocardiography to characterize AS. T2*-weighted magnetic resonance imaging after injection of MPIO-αVCAM-1 was used to examine aortic inflammation resulting from irradiation. A T2* signal void on valve leaflets and aortic sinus was considered positive. Valve remodeling and mineralization were assessed using von Kossa staining. Finally, the impact of radiation on cell viability and cycle from aortic human valvular interstitial cells (hVICs) was also assessed. The targeted aortic valve irradiation in ApoE^−/− mice resulted in an AS characterized by an increase in peak aortic jet velocity associated with valve leaflet and aortic sinus remodeling, including mineralization process, at the 3-month follow-up. There was a linear correlation between histological findings and peak aortic jet velocity (r = 0.57, p < 0.01). In addition, irradiation was associated with aortic root inflammation, evidenced by molecular MR imaging (p < 0.01). No significant effect of radiation exposure was detected on WT animals. Radiation exposure did not affect hVICs viability and cell cycle. We conclude that targeted radiation exposure of the aortic valve in mice results in ApoE^−/−, but not in WT, mice in an aortic valve remodeling mimicking the human lesions. This preclinical model could be a useful tool for future assessment of therapeutic interventions. Full article

One of the contributors to atherogenesis is enzymatically modified LDL (eLDL). eLDL was detected in all stages of aortic valve sclerosis and was demonstrated to trigger the activation of p38 mitogen-activated protein kinase (p38 MAPK), which has been identified as a pro-inflammatory protein in atherosclerosis. In this study, we investigated the influence of eLDL on IL-6 and IL-33 induction, and also the impact of eLDL on calcification in aortic valve stenosis (AS). eLDL upregulated phosphate-induced calcification in valvular interstitial cells (VICs)/myofibroblasts isolated from diseased aortic valves, as demonstrated by alizarin red staining. Functional studies demonstrated activation of p38 MAPK as well as an altered gene expression of osteogenic genes known to be involved in vascular calcification. In parallel with the activation of p38 MAPK, eLDL also induced upregulation of the cytokines IL-6 and IL-33. The results suggest a pro-calcifying role of eLDL in AS via induction of IL-6 and IL-33. Full article

To investigate the pathogenic mechanisms of calcified aortic valve disease (CAVD), it is necessary to develop a new three-dimensional model that contains valvular interstitial cells (VIC) and valvular endothelial cells (VEC). For this purpose, ovine aortic valves were processed to isolate VIC and VEC that were dissolved in an alginate/gelatin hydrogel. A 3D-bioprinter (3D-Bioplotter^® Developer Series, EnvisionTec, Gladbeck, Germany) was used to print cell-laden tissue constructs containing VIC and VEC which were cultured for up to 21 days. The 3D-architecture, the composition of the culture medium, and the hydrogels were modified, and cell viability was assessed. The composition of the culture medium directly affected the cell viability of the multicellular tissue constructs. Co-culture of VIC and VEC with a mixture of 70% valvular interstitial cell and 30% valvular endothelial cell medium components reached the cell viability best tested with about 60% more living cells compared to pure valvular interstitial cell medium (p = 0.02). The tissue constructs retained comparable cell viability after 21 days (p = 0.90) with different 3D-architectures, including a “sandwich” and a “tube” design. Good long-term cell viability was confirmed even for thick multilayer multicellular tissue constructs. The 3D-bioprinting of multicellular tissue constructs with VEC and VIC is a successful new technique to design tissue constructs that mimic the structure of the native aortic valve for research applications of aortic valve pathologies. Full article

Aortic stenosis (AS) is associated with hypofibrinolysis, but its mechanism is poorly understood. We investigated whether LDL cholesterol affects plasminogen activator inhibitor 1 (PAI-1) expression, which may contribute to hypofibrinolysis in AS. Stenotic valves were obtained from 75 severe AS patients during valve replacement to assess lipids accumulation, together with PAI-1 and nuclear factor-κB (NF-κB) expression. Five control valves from autopsy healthy individuals served as controls. The expression of PAI-1 in valve interstitial cells (VICs) after LDL stimulation was assessed at protein and mRNA levels. PAI-1 activity inhibitor (TM5275) and NF-κB inhibitor (BAY 11-7082) were used to suppress PAI-1 activity or NF-κB pathway. Clot lysis time (CLT) was performed to assess fibrinolytic capacity in VICs cultures. Solely AS valves showed PAI-1 expression, the amount of which was correlated with lipid accumulation and AS severity and co-expressed with NF-κB. In vitro VICs showed abundant PAI-1 expression. LDL stimulation increased PAI-1 levels in VICs supernatants and prolonged CLT. PAI-1 activity inhibition shortened CLT, while NF-κB inhibition decreased PAI-1 and SERPINE1 expression in VICs, its level in supernatants and shortened CLT. In severe AS, valvular PAI-1 overexpression driven by lipids accumulation contributes to hypofibrinolysis and AS severity. Full article

Objectives: Calcific aortic valve disease (CAVD) is most common in the aging population and is without effective medical treatments. Brain and muscle ARNT-like 1 (BMAL1) is related to calcification. It has unique tissue-specific characteristics and plays different roles in different tissues’ calcification processes. The purpose of the present study is to explore the role of BMAL1 in CAVD. Methods: The protein levels of BMAL1 in normal and calcified human aortic valves and valvular interstitial cells (VICs) isolated from normal and calcified human aortic valves were checked. HVICs were cultured in osteogenic medium as an in vitro model, and BMAL1 expression and location were detected. TGF-β and RhoA/ROCK inhibitors and RhoA-siRNA were applied to detect the mechanism underlying the source of BMAL1 during HVICs’ osteogenic differentiation. ChIP was applied to check whether BMAL1 could directly interact with the runx2 primer CPG region, and the expression of key proteins involved in the TNF signaling pathway and NF-κ B pathway was tested after silencing BMAL1. Results: In this study, we found that BMAL1 expression was elevated in calcified human aortic valves and VICs isolated from calcified human aortic valves. Osteogenic medium could promote BMAL1 expression in HVICs and the knockdown of BMAL1 induced the inhibition of HVICs’ osteogenic differentiation. Furthermore, the osteogenic medium promoting BMAL1 expression could be blocked by TGF-β and RhoA/ROCK inhibitors and RhoA-siRNA. Meanwhile, BMAL1 could not bind with the runx2 primer CPG region directly, but knockdown of BMAL1 led to decreased levels of P-AKT, P-IκBα, P-p65 and P-JNK. Conclusions: Osteogenic medium could promote BMAL1 expression in HVICs through the TGF-β/RhoA/ROCK pathway. BMAL1 could not act as a transcription factor, but functioned through the NF-κ B/AKT/MAPK pathway to regulate the osteogenic differentiation of HVICs. Full article

Purinergic signaling is associated with a vast spectrum of physiological processes, including cardiovascular system function and, in particular, its pathological calcifications, such as aortic valve stenosis. Aortic valve stenosis (AS) is a degenerative disease for which there is no cure other than surgical replacement of the affected valve. Purinergic signaling is known to be involved in the pathologic osteogenic differentiation of valve interstitial cells (VIC) into osteoblast-like cells, which underlies the pathogenesis of AS. ATP, its metabolites and related nucleotides also act as signaling molecules in normal osteogenic differentiation, which is observed in pro-osteoblasts and leads to bone tissue development. We show that stenotic and non-stenotic valve interstitial cells significantly differ from each other, especially under osteogenic stimuli. In osteogenic conditions, the expression of the ecto-nucleotidases ENTPD1 and ENPP1, as well as ADORA2b, is increased in AS VICs compared to normal VICs. In addition, AS VICs after osteogenic stimulation look more similar to osteoblasts than non-stenotic VICs in terms of purinergic signaling, which suggests the stronger osteogenic differentiation potential of AS VICs. Thus, purinergic signaling is impaired in stenotic aortic valves and might be used as a potential target in the search for an anti-calcification therapy. Full article