Mir‐29b promotes human aortic valve interstitial cell calcification via inhibiting TGF‐β3 through activation of wnt3/β‐catenin/Smad3 signaling

Herein, we hypothesized that pro‐osteogenic MicroRNAs (miRs) could play functional roles in the calcification of the aortic valve and aimed to explore the functional role of miR‐29b in the osteoblastic differentiation of human aortic valve interstitial cells (hAVICs) and the underlying molecular mechanism. Osteoblastic differentiation of hAVICs isolated from human calcific aortic valve leaflets obtained intraoperatively was induced with an osteogenic medium. Alizarin red S staining was used to evaluate calcium deposition. The protein levels of osteogenic markers and other proteins were evaluated using western blotting and/or immunofluorescence while qRT‐PCR was applied for miR and mRNA determination. Bioinformatics and luciferase reporter assay were used to identify the possible interaction between miR‐29b and TGF‐β3. Calcium deposition and the number of calcification nodules were pointedly and progressively increased in hAVICs during osteogenic differentiation. The levels of osteogenic and calcification markers were equally increased, thus confirming the mineralization of hAVICs. The expression of miR‐29b was significantly increased during osteoblastic differentiation. Furthermore, the osteoblastic differentiation of hAVICs was significantly inhibited by the miR‐29b inhibition. TGF‐β3 was markedly downregulated while Smad3, Runx2, wnt3, and β‐catenin were significantly upregulated during osteogenic induction at both the mRNA and protein levels. These effects were systematically induced by miR‐29b overexpression while the inhibition of miR‐29b showed the inverse trends. Moreover, TGF‐β3 was a direct target of miR‐29b. Inhibition of miR‐29b hinders valvular calcification through the upregulation of the TGF‐β3 via inhibition of wnt/β‐catenin and RUNX2/Smad3 signaling pathways.     

BMP7基因沉默抑制钙盐诱导猪主动脉瓣膜间质细胞成骨分化

目的:探究小干扰RNA(small interference RNA,siRNA)介导的骨形态发生蛋白7(bone morphogenetic protein7,BMP7)基因沉默对钙盐诱导猪主动脉瓣膜间质细胞成骨分化的影响及机制,为钙化性主动脉瓣膜病(calcific aortic valve disease,CAVD)的干预及治疗提供理论依据。方法:非CAVD瓣膜组织(non-CAVD组)取自手术治疗的主动脉夹层患者,CAVD瓣膜组织(CAVD组)取自因钙化性主动脉瓣狭窄而进行主动脉瓣膜置换术的患者,采用免疫组化和Western blot法检测non-CAVD组和CAVD组中BMP7、Runt相关转录因子2(Runx2)的蛋白质表达水平。选取健康家猪处死后即刻于无菌条件下取主动脉瓣叶,采用胶原酶连续消化法分离主动脉瓣膜间质细胞,观察其形态特征,并用免疫荧光染色行表型鉴定。采用脂质体转染法将BMP7-siRNA转染猪主动脉瓣膜间质细胞,采用qPCR和Western blot法验证BMP7表达的变化;利用钙盐培养基诱导细胞成骨分化,建立体外主动脉瓣膜间质细胞钙化模型后,采用ALP染色和茜素红S染色实验分别检测细胞早期及晚期成骨分化能力;采用qPCR和Western blot法分别检测细胞成骨相关基因及蛋白质Runx2、OCN和OPN的表达情况。并用Western blot法检测BMP7下游信号通路中Smad1/5/8的磷酸化水平。结果:BMP7和Runx2蛋白在CAVD组中表达明显高于non-CAVD组。成功分离出原代猪主动脉瓣膜间质细胞,α-平滑肌肌动蛋白(α-SMA)及波形蛋白(vimentin)染色阳性,血管性血友病因子(von willebrand factor,vWF)染色阴性。转染BMP7-siRNA后猪主动脉瓣膜间质细胞中BMP7的mRNA和蛋白质水平均明显下调,早期及晚期成骨分化能力均明显降低。沉默BMP7基因的表达,可下调Runx2、OCN和OPN的基因及蛋白质表达,且磷酸化的Smad1/5/8(p-Smad1/5/8)蛋白水平明显降低。结论:BMP7基因沉默抑制钙盐诱导的主动脉瓣膜间质细胞的成骨分化能力,BMP7/Smads信号通路可能在该过程中发挥重要作用。     

LPS通过上调BMP4促进猪主动脉瓣膜间质细胞成骨样分化

该文主要探究了LPS通过上调骨形态发生蛋白4(bone morphogenetic protein 4,BMP4)促进猪主动脉瓣膜间质细胞(valve interstitial cells,VICs)成骨样分化的作用及机制,为钙化性主动脉瓣膜病(calcific aortic valve disease,CAVD)的干...     

The new insight into extracellular NAD+ degradation-the contribution of CD38 and CD73 in calcific aortic valve disease

Nicotinamide adenine dinucleotide (NAD⁺) is crucial for cell energy metabolism and many signalling processes. Recently, we proved the role of ecto-enzymes in controlling adenine nucleotide–dependent pathways during calcific aortic valve disease (CAVD). This study aimed to investigate extracellular hydrolysis of NAD⁺ and mononucleotide nicotinamide (NMN) in aortic valves and aorta fragments of CAVD patients and on the inner aortic surface of ecto-5′-nucleotidase knockout mice (CD73−/−). Human non-stenotic valves (n = 10) actively converted NAD⁺ and NMN via both CD73 and NAD⁺-glycohydrolase (CD38) according to our analysis with RP-HPLC and immunofluorescence. In stenotic valves (n = 50), due to reduced CD73 activity, NAD⁺ was degraded predominantly by CD38 and additionally by ALP and eNPP1. CAVD patients had significantly higher hydrolytic rates of NAD⁺ (0.81 ± 0.07 vs 0.56 ± 0.10) and NMN (1.12 ± 0.10 vs 0.71 ± 0.08 nmol/min/cm²) compared with controls. CD38 was also primarily engaged in human vascular NAD⁺ metabolism. Studies using specific ecto-enzyme inhibitors and CD73−/− mice confirmed that CD73 is not the only enzyme involved in NAD⁺ and NMN hydrolysis and that CD38 had a significant contribution to these pathways. Modifications of extracellular NAD⁺ and NMN metabolism in aortic valve cells may be particularly important in valve pathology and could be a potential therapeutic target.     

颗粒蛋白前体抑制钙盐诱导主动脉瓣膜间质细胞成骨分化

该文主要研究颗粒蛋白前体(progranulin,PGRN)对猪主动脉瓣膜间质细胞(valve interstitial cells,VICs)成骨分化的影响及机制,为钙化性主动脉瓣膜病(calcific aortic valve disease,CAVD)的早期干预及治疗提供理论依据。采用免疫组化检测正常组和CAVD组中Runx2、OPN的表达,Western blot检测PGRN、纤维化指标α-SMA、钙化指标(Runx2、OPN)的表达以及AKT磷酸化水平。采用胶原酶连续消化法分离VICs,并用免疫荧光染色行表型鉴定。体外实验加入人PGRN重组蛋白,采用ALP染色、茜素红S染色、qPCR和Western blot检测细胞早期及晚期成骨分化能力以及AKT的磷酸化水平;并加入AKT的激活剂SC-79进行反向验证。结果表明,与正常组织相比,CAVD瓣膜组织中PGRN明显降低,α-SMA、Runx2、OPN和p-AKT在CAVD组中表达均明显高于正常组。成功分离出原代VICs,α-SMA和vimentin阳性,vWF阴性。PGRN可使VICs的ALP活性降低、钙盐沉积明显减少;PGRN可下调纤维化/钙化指标,且AKT的磷酸化水平降低;SC-79可减弱PGRN对纤维化/钙化指标的下调作用。提示PGRN能够抑制静止的VIC向肌纤维母细胞样的活化VIC乃至成骨样VIC进行转化,AKT信号通路可能在该过程中发挥重要作用。     

End stage renal disease‐induced hypercalcemia may promote aortic valve calcification via Annexin VI enrichment of valve interstitial cell derived‐matrix vesicles

Patients with end‐stage renal disease (ESRD) have elevated circulating calcium (Ca) and phosphate (Pi), and exhibit accelerated progression of calcific aortic valve disease (CAVD). We hypothesized that matrix vesicles (MVs) initiate the calcification process in CAVD. Ca induced rat valve interstitial cells (VICs) calcification at 4.5 mM (16.4‐fold; p < 0.05) whereas Pi treatment alone had no effect. Ca (2.7 mM) and Pi (2.5 mM) synergistically induced calcium deposition (10.8‐fold; p < 0.001) in VICs. Ca treatment increased the mRNA of the osteogenic markers Msx2, Runx2, and Alpl (p < 0.01). MVs were harvested by ultracentrifugation from VICs cultured with control or calcification media (containing 2.7 mM Ca and 2.5 mM Pi) for 16 hr. Proteomics analysis revealed the marked enrichment of exosomal proteins, including CD9, CD63, LAMP‐1, and LAMP‐2 and a concomitant up‐regulation of the Annexin family of calcium‐binding proteins. Of particular note Annexin VI was shown to be enriched in calcifying VIC‐derived MVs (51.9‐fold; p < 0.05). Through bioinformatic analysis using Ingenuity Pathway Analysis (IPA), the up‐regulation of canonical signaling pathways relevant to cardiovascular function were identified in calcifying VIC‐derived MVs, including aldosterone, Rho kinase, and metal binding. Further studies using human calcified valve tissue revealed the co‐localization of Annexin VI with areas of MVs in the extracellular matrix by transmission electron microscopy (TEM). Together these findings highlight a critical role for VIC‐derived MVs in CAVD. Furthermore, we identify calcium as a key driver of aortic valve calcification, which may directly underpin the increased susceptibility of ESRD patients to accelerated development of CAVD.     

Osteogenesis inducers promote distinct biological responses in aortic and mitral valve interstitial cells

Both aortic and mitral valves calcify in pathological conditions; however, the prevalence of aortic valve calcification is high whereas mitral valve leaflet calcification is somewhat rare. Patterns of valvular calcification may differ due to valvular architecture, but little is known to that effect. In this study, we investigated the intrinsic osteogenic differentiation potential of aortic versus mitral valve interstitial cells provided minimal differentiation conditions. For the assessment of calcification at the cellular level, we used classic inducers of osteogenesis in stem cells: β-glycerophosphate (β-Gly), dexamethasone (Dex), and ascorbate (Asc). In addition to proteomic analyses, osteogenic markers and calcium precipitates were evaluated across treatments of aortic and mitral valve cells. The combination of β-Gly, Asc, and Dex induced aortic valve interstitial cells to synthesize extracellular matrix, overexpress osteoblastic markers, and deposit calcium. However, no strong evidence showed the calcification of mitral valve interstitial cells. Mitral cells mainly responded to Asc and Dex by cell activation. These findings provide a deeper understanding of the physiological properties of aortic and mitral valves and tendencies for calcific changes within each valve type, contributing to the development of future therapeutics for heart valve diseases.     

趋化因子受体CX3CR1调控人主动脉瓣膜间质细胞成骨分化的作用研究

目的:探究趋化因子受体CX3CR1调控人主动脉瓣膜间质细胞成骨分化的作用和机制,为钙化性主动脉瓣膜疾病的早期干预和治疗提供新思路。方法:取非钙化主动脉瓣(3例)和钙化主动脉瓣(5例),免疫组织化学染色检测成骨相关转录因子Runx2、骨桥蛋白OPN和骨钙蛋白OCN的表达;取3例非钙化的主动脉瓣,采用胶原酶连续消化法分离人主动脉瓣膜间质细胞,观察细胞形态及生长状态,并采用细胞免疫荧光进行表型鉴定。对成骨诱导培养的人主动脉瓣膜间质细胞分别过表达和干扰趋化因子受体CX3CR1,平行设置CM组、OM组和negative control+OM组,采用qPCR和Western blot检测Runx2、OPN和OCN的表达,Western blot检测AKT和p-AKT的表达。茜素红S染色评价晚期钙结节形成情况。结果:临床标本显示钙化的主动脉瓣较非钙化的主动脉瓣高表达CX3CR1(P 0. 05);成功分离人主动脉瓣膜间质细胞,α-SMA和Vimentin阳性,vWF阴性。与CM、OM、negative control组比较,CX3CR1+OM组Runx2、OPN和p-AKT表达上调(P 0. 05),且茜素红S染色可见明显钙结节;与CM、OM、negative siRNA control+OM组比较,si CX3CR1+OM组Runx2、OPN和p-AKT表达下调(P 0. 05),且茜素红S染色可见钙结节减少。结论:趋化因子受体CX3CR1可能通过AKT信号通路促进人主动脉瓣膜间质细胞成骨分化。     

In‐vitro analysis of early calcification in aortic valvular interstitial cells using Laser‐Induced Breakdown Spectroscopy (LIBS)

Calcific aortic valve disease (CAVD) is a major cardiovascular disorder caused by osteogenic differentiation of valvular interstitial cells (VICs) within aortic valves. Conventional methods like colorimetric assays and histology fail to detect small calcium depositions during in‐vitro VIC cultures. Laser‐induced breakdown spectroscopy (LIBS) is a robust analytical tool used for inorganic materials characterizations, but relatively new to biomedical applications. We employ LIBS, for the first time, for quantitative in‐vitro detection of calcium depositions in VICs at various osteogenic differentiation stages. VICs isolated from porcine aortic valves were cultured in osteogenic media over various days. Colorimetric calcium assays based on arsenazo dye and Von Kossa staining measured the calcium depositions within VICs. Simultaneously, LIBS signatures for Ca I (422.67 nm) atomic emission lines were collected for estimating calcium depositions in lyophilized VIC samples. Our results indicate excellent linear correlation between the calcium assay and our LIBS measurements. Furthermore, unlike the assay results, the LIBS results could resolve calcium signals from cell samples with as early as 2 days of osteogenic culture. Quantitatively, the LIBS measurements establish the limit of detection for calcium content in VICs to be ～0.17±0.04 μg which indicates a 5‐fold improvement over calcium assay. Picture : Quantitative LIBS enables in‐vitro analysis for early stage detection of calcium deposition within aortic valvular interstitial cells (VICs).

     

Aortic valve disease in diabetes: Molecular mechanisms and novel therapies

Valve disease and particularly calcific aortic valve disease (CAVD) and diabetes (DM) are progressive diseases constituting a global health burden for all aging societies (Progress in Cardiovascular Diseases. 2014;56(6):565: Circulation Research. 2021;128(9):1344). Compared to non-diabetic individuals (The Lancet. 2008;371(9626):1800: The American Journal of Cardiology. 1983;51(3):403: Journal of the American College of Cardiology. 2017;69(12):1523), the diabetic patients have a significantly greater propensity for cardiovascular disorders and faster degeneration of implanted bioprosthetic aortic valves. Previously, using an original experimental model, the diabetic-hyperlipemic hamsters, we have shown that the earliest alterations induced by these conditions occur at the level of the aortic valves and, with time these changes lead to calcifications and CAVD. However, there are no pharmacological treatments available to reverse or retard the progression of aortic valve disease in diabetes, despite the significant advances in the field. Therefore, it is critical to uncover the mechanisms of valve disease progression, find biomarkers for diagnosis and new targets for therapies. This review aims at presenting an update on the basic research in CAVD in the context of diabetes. We provide an insight into the accumulated data including our results on diabetes-induced progressive cell and molecular alterations in the aortic valve, new potential biomarkers to assess the evolution and therapy of the disease, advancement in targeted nanotherapies, tissue engineering and the potential use of circulating endothelial progenitor cells in CAVD.     

Augmented Osteogenic Responses in Human Aortic Valve Cells Exposed to oxLDL and TLR4 Agonist: A Mechanistic Role of Notch1 and NF-κB Interaction

Aortic valve calcification causes the progression of calcific aortic valve disease (CAVD). Stimulation of aortic valve interstitial cells (AVICs) with lipopolysaccharide (LPS) up-regulates the expression of osteogenic mediators, and NF-κB plays a central role in mediating AVIC osteogenic responses to Toll-like receptor 4 (TLR4) stimulation. Diseased aortic valves exhibit greater levels of oxidized low-density lipoprotein (oxLDL). This study tested the hypothesis that oxLDL augments the osteogenic responses in human AVICs through modulation of NF-κB and Notch1 activation. AVICs isolated from normal human aortic valves were treated with LPS (0.1 µg/ml), oxLDL (20 µg/ml) or LPS plus oxLDL for 48 h. OxLDL alone increased cellular bone morphogenetic protein-2 (BMP-2) levels while it had no effect on alkaline phosphatase (ALP) levels. Cells exposed to LPS plus oxLDL produced higher levels of BMP-2 and ALP than cells exposed to LPS alone. Further, LPS plus oxLDL induced greater NF-κB activation, and inhibition of NF-κB markedly reduced the expression of BMP-2 and ALP in cells treated with LPS plus oxLDL. OxLDL also induced Notch1 activation and resulted in augmented Notch1 activation when it was combined with LPS. Inhibition of Notch1 cleavage attenuated NF-κB activation induced by LPS plus oxLDL, and inhibition of NF-κB suppressed the expression of BMP-2 and ALP induced by the synergistic effect of Jagged1 and LPS. These findings demonstrate that oxLDL up-regulates BMP-2 expression in human AVICs and synergizes with LPS to elicit augmented AVIC osteogenic responses. OxLDL exerts its effect through modulation of the Notch1-NF-κB signaling cascade. Thus, oxLDL may play a role in the mechanism underlying CAVD progression.     

The elastic properties of valve interstitial cells undergoing pathological differentiation

Increasing evidence indicates that the progression of calcific aortic valve disease (CAVD) is influenced by the mechanical forces experienced by valvular interstitial cells (VICs) embedded within the valve matrix. The ability of VICs to sense and respond to tissue-level mechanical stimuli depends in part on cellular-level biomechanical properties, which may change with disease. In this study, we used micropipette aspiration to measure the instantaneous elastic modulus of normal VICs and of VICs induced to undergo pathological differentiation in vitro to osteoblast or myofibroblast lineages on compliant and stiff collagen gels, respectively. We found that VIC elastic modulus increased after subculturing on stiff tissue culture-treated polystyrene and with pathological differentiation on the collagen gels. Fibroblast, osteoblast, and myofibroblast VICs had distinct cellular-level elastic properties that were not fully explained by substrate stiffness, but were correlated with α-smooth muscle actin expression levels. C-type natriuretic peptide, a peptide expressed in aortic valves in vivo, prevented VIC stiffening in vitro, consistent with its ability to inhibit α-smooth muscle actin expression and VIC pathological differentiation. These data demonstrate that VIC phenotypic plasticity and mechanical adaptability are linked and regulated both biomechanically and biochemically, with the potential to influence the progression of CAVD.     

A calcified polymeric valve for valve-in-valve applications

The prevalence of aortic valve stenosis (AS) is increasing in the aging society. More recently, novel treatments and devices for AS, especially transcatheter aortic valve replacement (TAVR) have significantly changed the therapeutic approach to this disease. Research and development related to TAVR require testing these devices in the calcified heart valves that closely mimic a native calcific valve. However, no animal model of AS has yet been available. Alternatively, animals with normal aortic valve that are currently used for TAVR experiments do not closely replicate the aortic valve pathology required for proper testing of these devices. To solve this limitation, for the first time, we developed a novel polymeric valve whose leaflets possess calcium hydroxyapatite inclusions immersed in them. This study reports the characteristics and feasibility of these valves. Two types of the polymeric valve, i.e., moderate and severe calcified AS models were developed and tested by deploying a transcatheter valve in those and measuring the related hemodynamics. The valves were tested in a heart flow simulator, and were studied using echocardiography. Our results showed high echogenicity of the polymeric valve, that was correlated to the severity of the calcification. Aortic valve area of the polymeric valves was measured, and the severity of stenosis was defined according to the clinical guidelines. Accordingly, we showed that these novel polymeric valves closely mimic AS, and can be a desired cost-saving solution for testing the performance of the transcatheter aortic valve systems in vitro.     

MiR‐199b‐5p inhibits osteogenic differentiation in ligamentum flavum cells by targeting JAG1 and modulating the Notch signalling pathway

Ossification of the ligamentum flavum (OLF) is a pathology almost only reported in East Asian countries. The leading cause of OLF is thoracic spinal canal stenosis and myelopathy. In this study, the role of miR‐199b‐5p and jagged 1 (JAG1) in primary ligamentum flavum cell osteogenesis was examined. MiR‐199b‐5p was found to be down‐regulated during osteogenic differentiation in ligamentum flavum cells, while miR‐199b‐5p overexpression inhibited osteogenic differentiation. In addition, JAG1 was found to be up‐regulated during osteogenic differentiation in ligamentum flavum cells, while JAG1 knockdown via RNA interference caused an inhibition of Notch signalling and osteogenic differentiation. Moreover, target prediction analysis and dual luciferase reporter assays supported the notion that JAG1 was a direct target of miR‐199b‐5p, with miR‐199b‐5p found to down‐regulate both JAG1 and Notch. Further, JAG1 knockdown was demonstrated to block the effect of miR‐199b‐5p inhibition. These findings imply that miR‐199b‐5p performs an inhibitory role in osteogenic differentiation in ligamentum flavum cells by potentially targeting JAG1 and influencing the Notch signalling pathway.     

Oxidative-mechanical stress signals stem cell niche mediated Lrp5 osteogenesis in eNOS(-/-) null mice

Calcific aortic valve disease (CAVD) is the most common indication for valve surgery in the USA. This study hypothesizes that CAVD develops secondary to Wnt3a/Lrp5 activation via oxidative‐mechanical stress in eNOS null mice. eNOS^?/? mice were tested with experimental diets including a control (n = 20), cholesterol (n = 20), cholesterol + Atorvastatin (n = 20). After 23 weeks the mice were tested for the development of aortic stenosis by Echo, Histology, MicroCT, and RTPCR for bone markers. In vitro studies measured Wnt3a secretion from aortic valve endothelial cells and confirmed oxidative stress via eNOS activity. Anion exchange chromatography was performed to isolate the mitogenic protein. Myofibroblast cells were tested to induce bone formation. Cholesterol treated eNOS mice develop severe stenosis with an increase in Wnt3a, Lrp5, Runx2 (threefold increase (P < 0.0001) in the bicuspid versus tricuspid aortic valves. Secretion of Wnt3a from aortic valve endothelium in the presence of abnormal oxidative stress was correlated with diminished eNOS enzymatic activity and tissue nitrite levels. Initial characterization of the architecture for a stem cell nice was determined by protein isolation using anion‐exchange chromatography and cell proliferation via thymidine incorporation. Osteoblastogenesis in the myofibroblast cell occurred via Lrp5 receptor upregulation in the presence of osteogenic media. Targeting the Wnt3a/Lrp5 pathway in valve calcification and activation of osteogenesis is via an oxidative‐mechanical stress in CAVD. These findings provide a foundation for treating this disease process by targeting the cross talk mechanism in a resident stem cell niche. J. Cell. Biochem. 113: 1623–1634, 2012. © 2011 Wiley Periodicals, Inc.