High-density lipoproteins (HDL) are present in stenotic aortic valves and may interfere with the mechanisms of valvular calcification

ObjectiveTo determine whether differences exist in valvular high density lipoprotein (HDL) content between non-stenotic and stenotic aortic valves, and whether HDL could retard valvular calcification locally.MethodsStenotic aortic valves were obtained from valve replacement surgery and non-stenotic control valves from cardiac transplantations or at autopsy. The valvular localization and concentration of apolipoproteinA-I (apoA-I) were analyzed by immunohistochemistry and ELISA. The effects of HDL on the secretion of calcifying mediators and proinflammatory cytokines by cultured aortic valve myofibroblasts were assessed by ELISA and real-time PCR.ResultsThe concentration of apoA-I was higher in control than in stenotic valves (p < 0.05). ApoA-I surrounded the calcific deposits in stenotic valves, co-localizing with apoB, apoE, and osteoprotegerin (OPG). Incubation of cultured valve myofibroblasts with HDL increased their secretion of OPG (p < 0.001). Furthermore, incubation of myofibroblasts with HDL led to decreased mRNA expression of tumor necrosis factor alpha (TNF-α) (p < 0.05).ConclusionsThe amount of valvular HDL is reduced in aortic valve stenosis. HDL both induces the secretion of OPG and reduces the expression of TNF-α in vitro. Since OPG is known to inhibit and TNF-α to promote aortic valve calcification, HDL may have an anti-calcifying effect in human aortic valves.     

Interleukin-1 beta promotes matrix metalloproteinase expression and cell proliferation in calcific aortic valve stenosis

Calcific aortic valve stenosis (AS), the main heart valve disease in the elderly, is characterized by extensive remodeling of the extracellular matrix. Matrix metalloproteinases (MMPs) are upregulated in calcific AS and might modulate matrix remodeling. The regulatory mechanisms are unclear. As recent studies have suggested that calcific AS might result from an inflammatory process involving leukocyte invasion and activation, the present study aimed to elucidate the role of the pro-inflammatory cytokine interleukin (IL)-1β on MMP expression and cell proliferation in human aortic valves. Immunohistochemistry for leukocytes, IL-1β and MMP-1 was performed on aortic valves with (n=6) and without (n=6) calcification obtained at valve replacement or autopsy. Stenotic valves showed marked leukocyte infiltration and associated expression of IL-1β and MMP-1. In control valves only scattered leukocytes, low staining for MMP-1 and no staining for IL-1β were present. Double-label immunostaining localized IL-1β expression mainly to leukocytes and MMP-1 expression to myofibroblasts. Stimulation of cultured human aortic valve myofibroblasts with IL-1β lead to a time-dependently increased expression of MMP-1 and MMP-2 by Western blotting and zymography, whereas MMP-9 remained unchanged. Cell proliferation was increased by IL-1β as determined by bromodesoxyuridine incorporation. Thus, IL-1β may regulate remodeling of the extracellular matrix in calcific AS.     

Differential profile of the OPG/RANKL/RANK-system in degenerative aortic native and bioprosthetic valves

BACKGROUND AND AIM OF THE STUDY: Although degenerative calcific aortic valve stenosis is the most common valvular disease among the elderly, neither the etiology underlying the condition nor degeneration of the bioprostheses is yet fully understood. The study aim was to assess the expression profile of those OPG/RANKL/RANK-system determinants known to act as key regulators of bone metabolism and the immune system in calcific aortic valve stenosis and porcine aortic bioprostheses. METHODS: Valve probes from a total of 69 patients (41 with end-stage aortic stenosis, 11 with mild-to-moderate aortic sclerosis, 17 with degenerative porcine aortic bioprostheses) were explanted either during surgery or at autopsy. The presence and localization of OPG, RANKL, RANK and NF-kappaB were analyzed by immunostaining and morphometry. RESULTS: The majority of stenotic and sclerotic valves exhibited cell-bound signals of OPG, RANKL, RANK and NF-kappaB, while bioprostheses showed only sparse signaling. As key findings, the percentage of cells labeled by OPG, RANK and NF-kappaB was increased in sclerotic valves compared with stenotic valves (each p < 0.001), whereas the frequency of RANKL was higher in stenotic compared to sclerotic valves (p < 0.001). As a consequence, the OPG/RANKL ratio was decreased in stenotic (0.83) compared to sclerotic valves (20.2). CONCLUSION: The differential expression profile of specific members of the OPG/RANKL/RANK axis suggests an involvement of their determinants in native valve calcification, but not in the degeneration of porcine bioprostheses. Thus, these mediators of bone homeostasis may represent new targets for a more specified prevention and/or therapy of native aortic stenosis.     

RANKL Expression Is Increased in Circulating Mononuclear Cells of Patients with Calcific Aortic Stenosis

We aimed to investigate whether the expression of the OPG/RANK/RANKL triad in peripheral blood mononuclear cells (PBMC) and circulating levels of markers of ectopic mineralization (OPG, FGF-23, PPi) are modified in patients with calcific aortic valve disease (CAVD). We found that patients affected by CAVD (n?=?50) had significantly higher circulating levels of OPG as compared to control individuals (p?=?0.003). No differences between the two groups were found in FGF-23 and PPi levels. RANKL expression was higher in the PBMC from CAVD patients (p?=?0.018) and was directly correlated with the amount of valve calcification (p?=?0.032). In vitro studies showed that treatment of valve interstitial cells (VIC) with RANKL plus phosphate was followed by increase in matrix mineralization (p?=?0.001). In conclusion, RANKL expression is increased in PBMC of patients with CAVD, is directly correlated with the degree of valve calcification, and promotes pro-calcific differentiation of VIC.     

Expression of bone sialoprotein and bone morphogenetic protein-2 in calcific aortic stenosis

BACKGROUND AND AIM OF THE STUDY: Calcific aortic stenosis, the major heart valve disease encountered in the elderly, leads to massive calcium deposition in the valve leaflets that morphologically resembles bone formation. Recent studies have demonstrated the expression of various bone-associated proteins in stenotic valves, suggesting that valvular calcification may be an actively regulated process. Bone sialoprotein (BSP), a non-collagenous bone matrix protein, and bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor cytokine superfamily, are known to participate in the regulation of bone development and maturation. Their pathogenetic role in calcific aortic stenosis is unknown. METHODS: Using an immunoperoxidase technique and antibodies against BSP and BMP-2, the expression of BSP and BMP-2 was examined in 16 human aortic valves with calcific aortic stenosis obtained at valve replacement, and in seven normal autopsy controls without signs of aortic stenosis. RESULTS: By semiquantitative scoring, stenotic valves showed a significantly increased staining of BSP in cells and extracellular matrix as compared to control valves (2.7 +/- 0.1 versus 0.6 +/- 0.2 score units, p <0.001). Marked BMP-2 expression was detected in stenotic valves, mostly in cell-rich areas associated with focal calcium deposits, but no specific staining for BMP-2 was detected in control valves (1.5 +/- 0.2 versus 0.0 +/- 0.0 score units, p <0.001). CONCLUSION: These results demonstrate for the first time that BSP and BMP-2 are differentially expressed in normal aortic valves and in aortic stenosis, thereby supporting the concept that valvular calcification might be based on an actively regulated process involving BSP and BMP-2.     

Identification and characterization of calcifying valve cells from human and canine aortic valves.

BACKGROUND AND AIM OF THE STUDY: Cardiac valve calcification is the predominant pathology in patients needing valve replacement. The aim of this study was to determine if aortic valve cells calcify spontaneously and, if so, to characterize the nodular complex and response to growth factors. METHODS: Aortic valves were obtained from humans undergoing surgical valve replacement, and from female dogs. The valvular endothelium was removed and explants cultured in medium. RESULTS: A population of valvular interstitial cells spontaneously formed distinct calcified nodules containing hydroxyapatite within two to three weeks in canine and within six weeks in human aortic valves. The nodules contained an inner ring of dead cells surrounded by an outer ring of living cells. Cells associated with nodules had osteoblast-like characteristics and stained positively for extracellular bone matrix proteins. Incubating canine cells with potential calcifying stimuli tested the stimulus for calcification. The rate of nodule formation was increased with transforming growth factor beta-1 (+25 nodules), 25-hydroxycholesterol (+9 nodules) and bone morphogenetic protein 2 (+4 nodules) as compared with vehicle control (+3 nodules) over 25 days. CONCLUSIONS: We identified a population of valvular interstitial cells with osteoblast-like characteristics that spontaneously form calcific nodules in cell culture. In addition, the rate of calcific nodule formation was increased with transforming growth factor beta-1 and 25-hydroxycholesterol. Further study of these 'calcifying valve cells' may yield a new in vitro model for testing therapy aimed at preventing calcific valve stenosis.     

Lower serum calcium levels are associated with greater calcium hydroxyapatite deposition in native aortic valves of male patients with severe calcific aortic stenosis

BACKGROUND AND AIM OF THE STUDY: The study aim was to evaluate the relationship between serum calcium levels and the degree of calcification found in stenotic aortic valves. METHODS: Using atomic absorption spectroscopy, the hydroxyapatite content of 228 excised human stenotic aortic valves was determined and expressed as a percentage of valve mass. Left heart catheterization preceded valve replacement. In addition, serum levels of calcium and creatinine were determined before native calcific aortic valve excision. RESULTS: Valves from male patients contained more hydroxyapatite than those of female patients (26 +/- 9 versus 22 +/- 9 mass%; p < 0.001). Patients presenting with lower serum calcium levels showed a slight trend towards higher levels of valve calcification (r = -0.15, p = 0.026), but this association appeared only within the subgroup of male patients. Male patients with lowest serum calcium levels displayed greatest valvular hydroxyapatite deposition (1st calcium tertiary: 29.5 +/- 8.9 mass% versus 2nd calcium tertiary 26.4 +/- 7.8 mass% versus 3rd calcium tertiary 21.4 +/- 8.9 mass%; n = 122; p = 0.001; r = -0.25; p = 0.006). This association was even more distinct in male patients with normal serum creatinine levels. Furthermore, serum calcium was inversely and significantly associated with serum C-reactive protein in male patients (r = - 0.34; p < 0.001). CONCLUSION: Serum calcium levels appear to be inversely related to valve calcification in patients with severe calcific aortic stenosis (AS). This finding indicates the importance of systemic calcium metabolism in calcific AS, independent of manifest disorders of calcium metabolism or renal function. Interestingly, this association was evident only in male patients, suggesting a gender-dependent pathogenesis.     

强直性脊柱炎外周血破骨细胞前体细胞活性的研究

目的 研究强直性脊柱炎(AS)患者外周血破骨细胞前体细胞(OCP)的数量及其与血清核因子(NF)-κB受体活化因子配体(RANKL)和骨保护素(OPG)浓度以及与病情活动性的相关性.方法 采用RANKL和巨噬细胞集落刺激因子(M-CSF)体外诱导8例As患者和5名健康对照的外周血培养破骨细胞(OC).应用组织化学染色法对OC中抗酒石酸酸性磷酸酶(TRAP)染色.计数染色阳性胞核≥3个的细胞.骨吸收实验考察OC的功能.运用酶联免疫吸附试验(ELISA)法检测23例As患者和17名健康对照血清RANKL和OPG水平.对AS疾病活动性进行评估包括Bath强直性脊柱炎疾病活动指数(BASDAI)、红细胞沉降率(ESR)、C反应蛋白(CRP).对AS患者OCP数量与血清RANKUOPG比值及与病情活动性进行相关分析.统计分析采用t检验t'检验、Spearman相关分析.结果 ①As组外周血生成OC数量显著高于健康对照组(10.9±3.4与6.2±1.3,P<0.05);②AS组血清RANKL浓度、OPG浓度、RANKL/OPG比值显著高于健康对照组[(5.4±3.8)pg/ml与(1.6±0.8)pg/ml,(157±49)pg,ml与(105±20)pg/ml,0.037±0.026与0.016±0.008,P均<0.01)];③外周血生成OC数量与RANKL、RANKL/OPG比值呈正相关(r=0.692, P=0.009;r=0.813,P=0.001);④AS患者血清OPG浓度与BASDAI呈负相关(r=-0.444,P=0.044),血清RANKL浓度与BASDAI呈正相关(r=0.543,P=0.011),RANKL/OPG比值与BASI)AI呈正相关(r=0.672,R=0.001).结论 ①As患者外周血OCP数量显著增高,与关节骨质破坏程度密切相关可能是造成关节骨质破坏的机制;②AS患者OC活性增高的机制可能是炎症引起RANKL产量增多,RANKUOPG比值升高所致.     

强直性脊柱炎继发骨质疏松及相关因素分析

目的 测定强直性脊柱炎(AS)患者骨密度(BMD)、血清骨保护素(OPG)、可溶性核因子κB受体活化因子配体(sRANKL)等骨代谢指标及外周血T细胞表面RANKL表达情况,研究RANKI/RANK/OPG系统在AS骨代谢中的作用.方法 双能X线吸收法(DEXA)测定AS患者BMD;酶联免疫吸附试验(ELISA)法检测血清OPG、sRANKL、抗酒石酸酸性磷酸酶异构体5b(TRACP-5b)、骨特异性碱性磷酸酶(BALP)水平;分析BMD、上述骨代谢指标及临床指标间相关性;流式细胞术(FC)检测外周血CD4+/RANKL+及CD8+/RANKL+细胞表达率;分析它们与红细胞沉降率(ESR)、C反应蛋白(CRP)相关性.计量资料采用成组设计的t检验,计数资料采用x2检验,相关性采用直线相关分析.结果 ①AS患者骨量减少、骨质疏松(OP)发生率分别为47%、37%.②AS组血清sRANKL、TRACP-5b水平及sRANKL/OPG比值均高于对照组(P<0.05);2组血清OPG、BALP水平差异无统计学意义.③AS组血清sRANKL水平与OPG呈正相关,两者均与TRACP-5b呈正相关(P<0.01.④AS组外周血CD4+/RANKL+细胞表达率高于对照组(P<0.05).结论 AS存在较高的骨量丢失率,其骨代谢特点以骨吸收增强为主,RANKL/RANK/OPG系统在其中起着重要作用,该系统失衡可能是AS骨量丢失机制之一;CD4+T细胞可能通过上调RANKL表达参与AS破骨细胞分化成熟及骨吸收机制.     

Genetics of Calcific Aortic Stenosis – Time to Move Forward

Knowledge of the genetic determinants of aortic stenosis (AS) could help elucidate the underlying mechanisms leading to clinical valvular heart disease and could potentially aid in the development of new therapies to prevent or retard valvular heart disease. However, despite the large burden of disease due to AS, no large-scale genetic studies of AS have been reported. Research on the genetics of valvular heart disease remains at least several years behind that of myocardial infarction, coronary artery disease and several other cardiovascular diseases. To date, with few important exceptions, human genetic research in calcific aortic valve disease has been quite limited. This review will provide a summary of the genetic literature on human calcific AS and provide future directions for genetic research in aortic valve disease.     

高糖诱导大鼠心脏瓣膜间质细胞钙化的初步研究

目的初步探讨高糖诱导大鼠主动脉瓣膜间质细胞钙化的现象。方法采用细胞培养,免疫荧光(IF)鉴定,von-kossa钙化测定,western blot,real-time PCR等方法对高糖诱导大鼠主动脉瓣间质细胞钙化的现象进行初步研究。结果在高糖刺激下,随着培养时间的延长,主动脉瓣膜间质细胞中碱性磷酸酶(ALP)的表达增加,骨形态生成蛋白2(BMP2)、BMP4呈现上升趋势,钙化结节形成的数目增多(P<0.05),0 d组结节数目为(0.30±0.05),7 d组为(1.10±0.12),而14 d组结节数目为(2.70±0.30),明显增多。而在低糖条件下,培养相同时间,没有观察到钙化结节的形成。结论高糖能够诱导主动脉瓣膜间质细胞发生钙化,可能与骨化相关因子ALP、BMP2,BMP4的表达增加有关。     

The Global Burden of Aortic Stenosis

Degenerative, calcific valvular aortic stenosis (AS), caused by an active process of atherosclerosis, calcification and ossification, is the most common cause of AS in industrialized nations. The prevalence of calcific AS is age-dependent, and thus is expected to increase due to demographic aging of the global population. It is well recognized that severe AS carries a poor prognosis if left untreated. Despite this recognition, many patients are inappropriately denied surgery because of perceived risk. This article will examine the etiology, prevalence, and current trends in the treatment of degenerative AS focusing on indications for surgical aortic valve replacement.     

Risk factors for progression of calcific aortic stenosis and potential therapeutic targets

Current thought regarding the progression of calcific aortic stenosis (AS) is presented. After summarizing contemporary ideas about AS pathogenesis, the present article examines the factors that may affect disease progression. Data indicate that this process may be accelerated by aortic valve structure, degree of valvular calcification, chronic renal insufficiency and cardiovascular risk factors such as diabetes and dyslipidemia. Finally, the present review discusses potential therapeutic targets to slow AS progression.     

Serum gamma-glutamyltransferase activity is increased in patients with calcific aortic valve stenosis

BACKGROUND AND AIM OF THE STUDY: A growing body of data indicates an independent association between serum gamma-glutamyltransferase (GGT) activity, a marker of increased oxidative stress, and cardiovascular diseases. The process of calcific aortic valve disease has been shown to present characteristics of atherosclerosis. The study aim was to evaluate the possible role of serum GGT in patients with calcific aortic valve disease. METHODS: The results of patients' echocardiography studies from 2005 for the presence of calcific aortic valve disease in the forms of aortic stenosis (AS) and aortic valve calcification (AVC) without significant valve stenosis, were retrospectively evaluated. Age-and gender-matched patients with normal aortic valve morphology were selected at random as a control group. A total of 383 patients was enrolled into the study (126 with AS, 133 with AVC, 124 controls). Serum GGT activity, along with other liver enzyme analyses and laboratory results, were determined and compared among the groups. RESULTS: Age, gender and clinical and laboratory results were similar among the three groups. Median serum GGT levels in the AS, AVC and control groups were 23.0 U/1 (mean 31.5 +/- 24.9 U/1), 22.0 U/1 (mean 27.6 +/- 18.6 U/) and 18.0 U/l (mean 22.4 +/- 16.4 U/l), respectively. Compared to controls, AS patients had significantly higher serum GGT and C-reactive protein levels, while the differences between AVC patients and controls for these parameters were not significant. CONCLUSION: The study results suggest that serum GGT activity is increased in patients with calcific AS. These increases seem to occur in advanced rather than milder forms of calcific aortic valve disease.     

Aortic valvular interstitial cells apoptosis and calcification are mediated by TNF-related apoptosis-inducing ligand

Background/Objectives

Calcific aortic valvular disease (CAVD) is an actively regulated process characterized by the activation of specific osteogenic signaling pathways and apoptosis. We evaluated the involvement in CAVD of the TNF-related apoptosis-inducing ligand (TRAIL), an apoptotic molecule which induces apoptosis by interacting with the death receptor (DR)-4 and DR5, and whose activity is modulated by the decoy receptor (DcR)-1 and DcR2.

Methods

Sections of calcific and normal aortic valves, obtained at surgery time, were subjected to immunohistochemistry and confocal microscopy for TRAIL immunostaining. Valvular interstitial cells (VICs) isolated from calcific (C-VICs) and normal (N-VICs) aortic valves were investigated for the gene and protein expression of TRAIL receptors. Cell viability was assayed by MTT. Von Kossa staining was performed to verify C-VIC ability to produce mineralized nodules. TRAIL serum levels were detected by ELISA.

Results

Higher levels of TRAIL were detected in calcific aortic valves and in sera from the same patients respect to controls. C-VICs express significantly higher mRNA and protein levels of DR4, DR5, DcR1, DcR2 and Runx2 compared to N-VICs. C-VICs and N-VICs, cultured in osteogenic medium, express significantly higher mRNA levels of DR4, Runx2 and Osteocalcin compared to baseline. C-VICs and N-VICs were sensitive to TRAIL-apoptotic effect at baseline and after osteogenic differentiation, as demonstrated by MTT assay and caspase-3 activation. TRAIL enhanced mineralized matrix nodule synthesis by C-VICs cultured in osteogenic medium.

Conclusions

TRAIL is characteristically present within calcific aortic valves, and mediates the calcification of aortic valve interstitial cells in culture through mechanism involving apoptosis.     

老年人钙化主动脉瓣中基质金属蛋白酶和细胞凋亡的表达及意义

目的研究基质金属蛋白酶2(MMP-2)及基质金属蛋白酶组织抑制剂2(TIMP-2)和细胞凋亡在主动脉瓣钙化病理过程中的作用。方法选择行主动脉瓣置换术、且主动脉瓣钙化的患者15例作为钙化组,同期选择相同手术、且主动脉瓣正常的患者10例作为非钙化组。术中取主动脉瓣组织,免疫组织化学染色检测MMP-2和TIMP-2表达、TUNEL检测细胞凋亡。结果与非钙化组比较,钙化组主动脉瓣MMP 2、TIMP-2为中、重度着色,阳性细胞显著增多,差异有统计学意义(P<0.01)。钙化组主动脉瓣细胞凋亡指数较非钙化组明显增多(0.71±9.07 vs0.21±6.83,P<0.05)。结论老年钙化主动脉瓣MMP 2和TIMP 2表达异常增高,细胞凋亡数明显增多。     

Increased expression of profibrotic neutral endopeptidase and bradykinin type 1 receptors in stenotic aortic valves.

AIMS: In aortic stenosis (AS), adverse remodelling of the valves may depend on altered local regulation of pro- and antifibrotic systems. We have recently shown that angiotensin-converting enzyme (ACE), which generates profibrotic angiotensin II and inactivates antifibrotic bradykinin (BK), is upregulated in stenotic aortic valves. Here, we analyse the expression of neutral endopeptidase (NEP), another profibrotic and BK-degrading enzyme, and of BK receptors in aortic valves in AS. METHODS AND RESULTS: Stenotic aortic valves (n = 86) were obtained at valve replacement surgery and control valves (n = 13) at cardiac transplantation. Expression levels of NEP and BK type 1 and 2 receptors (BK-1R and BK-2R) in aortic valves and in isolated valvular myofibroblasts were analysed by real-time PCR and immunohistochemistry, and NEP activity was quantified by autoradiography. NEP, BK-1R, and BK-2R mRNA levels were higher in stenotic than in non-stenotic valves (P < 0.05 for each) and the respective proteins localized to valvular endothelial cells and myofibroblasts. In stenotic valves, the proteolytic activity of NEP was significantly increased (4.5-fold, P < 0.001), and tumour necrosis factor-alpha induced the expression of NEP in cultured myofibroblasts. Finally, treatment of cultured myofibroblasts with an NEP inhibitor (phosphoramidon) downregulated the expression of profibrotic transforming growth factor-beta1, whereas addition of BK decreased the expression of collagens I and III which was reversed by a BK-2R antagonist. CONCLUSION: NEP activity is increased in stenotic aortic valves in parallel with increased expression of BK-receptors. The upregulation of NEP and BK-1R have the potential to promote valvular fibrosis and remodelling while the increase in BK-2R may represent a compensatory antifibrotic response. These findings add novel pathogenic insight and raise potential new therapeutic targets in AS.     

Type I and type III collagen synthesis and composition in the valve matrix in aortic valve stenosis

Changes in the collagenous matrix may contribute to the pathogenesis and progression of human aortic valve stenosis (AS). To evaluate the significance of collagen I and III in the pathogenesis of AS, we studied their synthesis in diseased valves. Type I and type III collagen mRNA expression and the immunohistochemical localization of the collagen antigens were studied from 36 AS and 2 normal aortic valves. The concentrations of propeptides and telopeptide structure of type I (PINP, PICP, and ICTP) and those of III collagens (PIIINP and IIINTP) were measured by radioimmunoassays in soluble tissue extracts and trypsin-solubilized calcified and non-calcified matrices of 11 AS and 24 healthy aortic valves of different ages. The synthesis of type I collagen, localized in the myofibroblasts adjacent to calcified nodules, was two- to three-fold in the AS samples compared to the controls. The proportion of collagen in the total protein fraction was 90% in the healthy valves, 50% in the non-calcified matrix, and 10% in the calcified matrix of AS valves. In the calcified valves, the ICTP content was six-fold compared to the age-matched controls and two-fold compared to the young control group. In the controls, the amount of ICTP in type I collagen decreased with age (r=-0.908, p<0.001) and was replaced by other cross-linked C-telopeptide structure. The concentration of type III collagen decreased during aging (r=-0.753, p<0.001). The decrease in total collagen content, despite the increase in type I collagen synthesis indicates an increase in collagen turnover in AS. The calcification of the aortic valves is accompanied by increased amount of ICTP in type I collagen.     

Relation of oral anticoagulation to cardiac valvular and coronary calcium assessed by multislice spiral computed tomography

Vitamin K antagonists, known as oral anticoagulants, affect the synthesis and function of the matrix Gla protein, which is a potent inhibitor of tissue calcification. We performed multislice spiral computed tomography in 86 patients (53 men, mean age 71 +/- 8 years) with calcific aortic valve disease to quantitate the amount of calcification in the aortic valve and coronary arteries. Patients with long-term oral anticoagulation therapy (mean duration 88 +/- 113 months; n = 23) were compared with those without anticoagulation (n = 63). No differences were found in the demographic, clinical, or echocardiographic characteristics between the 2 study groups. Patients on oral anticoagulant therapy had increased coronary calcium (coronary Agatston score 1,561 +/- 1,141 vs 738 +/- 978, respectively; p = 0.024) and valvular calcium (valvular Agatston score 2,410 +/- 1,759 vs 1,070 +/- 1,085, respectively; p = 0.002) compared with patients without anticoagulation treatment. The results of our study have demonstrated that oral anticoagulation may be associated with increased valvular and coronary calcium in patients with aortic valve disease, presumably due to decreased activation of the matrix Gla protein.     

NOTCH1 regulates matrix gla protein and calcification gene networks in human valve endothelium

Valvular and vascular calcification are common causes of cardiovascular morbidity and mortality. Developing effective treatments requires understanding the molecular underpinnings of these processes. Shear stress is thought to play a role in inhibiting calcification. Furthermore, NOTCH1 regulates vascular and valvular endothelium, and human mutations in NOTCH1 can cause calcific aortic valve disease. Here, we determined the genome-wide impact of altering shear stress and NOTCH signaling on human aortic valve endothelium. mRNA-sequencing of primary human aortic valve endothelial cells (HAVECs) with or without knockdown of NOTCH1, in the presence or absence of shear stress, revealed NOTCH1-dependency of the atherosclerosis-related gene connexin 40 (GJA5), and numerous repressors of endochondral ossification. Among these, matrix gla protein (MGP) is highly expressed in aortic valve and vasculature, and inhibits soft tissue calcification by sequestering bone morphogenetic proteins (BMPs). Altering NOTCH1 levels affected MGP mRNA and protein in HAVECs. Furthermore, shear stress activated NOTCH signaling and MGP in a NOTCH1-dependent manner. NOTCH1 positively regulated endothelial MGP in vivo through specific binding motifs upstream of MGP. Our studies suggest that shear stress activates NOTCH1 in primary human aortic valve endothelial cells leading to downregulation of osteoblast-like gene networks that play a role in tissue calcification.