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Background: Active involvement of extracellular matrix (ECM) and its composition regulating factors may have a central role in the pathogenesis of calcific aortic valve disease (CAVD). Thrombospondins (TSPs) are highly conserved matricellular proteins regulating inflammation, angiogenesis and ECM remodeling. These processes are strongly associated with progression of aortic valve stenosis (AS). However, the expression of TSPs in CAVD is not known., Methods: We characterized the expression of TSPs 1-4 in human aortic valves by real-time quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry. Control valves (n=8), thickened and stiffened fibro(sclero)tic valves (n=8), and calcified AS valves (n=24) were compared. Furthermore, potential factors regulating TSP-2 expression was studied by western blotting and gel mobility shift assay in another set of control (n=10) and AS (n=20) valves., Results: TSP-2 mRNA levels were increased 4.9-fold (P=0.037) and 4.8-fold (P=0.001) in fibro(sclero)tic and stenotic valves, respectively, whereas the expression of other TSPs did not change significantly. All TSPs 1-4 were detected from aortic valves by immunohistochemistry. Positive TSP-2 immunostaining was seen in the valvular myofibroblasts and patchily in endothelial cells. Semiquantitative analysis of TSP-2 staining indicated increased immunoreactivity for TSP-2 in neo vessels of fibro(sclero)tic and calcified aortic valves. Finally, when compared to controls, AS was associated with significant down regulation of Akt-pathway and diminished binding activity of nuclear factor-κB (NF-κB)., Conclusions: We report for the first time that TSPs 1-4 are expressed in human aortic valves. CAVD is characterized by myofibroblastic proliferation and neovascularization associated upregulation of TSP-2 expression, as well as inactivation of Akt and NF-κB., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Background: There is increasing evidence that renin-angiotensin system (RAS) may play a major role in the actively regulated fibrocalcific process in aortic valve stenosis (AS), but the gene expression or function of (pro)renin receptor ((P)RR), prorenin and renin or angiotensin converting enzyme 2(ACE2)/angiotensin-(1-7)/Mas receptor axis in calcific aortic valve disease is not known., Methods and Results: We characterized expression of (P)RR, ACE2 and Mas receptor as well as renin, prorenin and angiotensin II type 2 (AT(2)) receptors in human aortic valves, and compared normal control valves (n = 11) with valves obtained from patients with aortic regurgitation (AR, n = 14), AR with fibrosis (n = 20) and AS (n = 61). By immunohistochemistry (P)RR positive staining was seen in the valvular endothelial cells of control and in the neovessels of stenotic valves. By RT-PCR, renin mRNA levels were 72% (P = 0.001) and prorenin mRNA levels 64% lower (P = 0.002) in stenotic aortic valves compared to control valves. ACE2, Mas receptor and AT(2)-receptor mRNA levels were 69% (P < 0.001), 58% (P = 0.008) and 75% (P = 0.001) lower, respectively, in stenotic valves. ACE2 positive staining, existing to lesser extent in stenotic aortic valves, was localized mainly to stromal area in spongiosa layer in control valves., Conclusions: (P)RR, prorenin and renin are expressed in human aortic valves. We also report for the first time expression of ACE2/angiotensin-(1-7)/-Mas receptor axis in human aortic valve cusps. The downregulation of ACE2/angiotensin-(1-7)/-Mas receptor axis as well as AT(2)-receptors may promote fibrosis, proliferation and inflammation in patients with AS., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Background and Aim of the Study: Aortic valve stenosis (AS) is an actively regulated pathobiological process that shows some hallmarks of atherosclerosis. Apelin and its receptor, APJ, are highly expressed in the heart, and the proposed effects of the apelin-APJ system are opposite to those of the angiotensin II-AT1-receptor pathway. The role of the apelin-APJ signaling pathway in calcified aortic valve disease is unknown., Methods: The study involved the characterization and comparison of expression of apelin and APJ as well as angiotensin II receptors (AT1 and AT2) in the aortic valves of patients with normal valves (n = 6), aortic regurgitation (n = 9 AR), regurgitation and fibrosis/mild sclerosis (n = 14), and AS (n = 25)., Results: By employing the reverse-transcriptase polymerase chain reaction (RT-PCR), the gene expression of apelin (3.63-fold, p = 0.001) and the APJ receptor (2.70-fold, p = 0.01) were shown to be significantly up-regulated in stenotic valves when compared to controls. In addition, APJ receptor mRNA levels were higher (2.9-fold, p = 0.010) in the AR + sclerosis group when compared to controls. Using immunohistochemistry, apelin was shown to be localized in stenotic aortic valves to the valvular endothelial layer of the aortic valve, to vascular endothelial cells in neovessels, and to fibroblasts and macrophages adjacent to vessels in the stromal area. AT2-receptor mRNA levels were 90% (p < 0.001) lower in stenotic valves. In contrast, the gene expression of AT1-receptors did not differ significantly among the groups., Conclusion: Aortic valve stenosis is characterized by an up-regulation of the apelin-APJ signaling pathway, revealing a possible novel target for drug discovery in calcified aortic valve disease by suppressing chemotaxis, angiogenesis and osteoblast activity, all of which are well-documented phenomena in the disease process.

Aims: Aortic valve stenosis (AS) is an actively regulated process like atherosclerosis, which is accompanied by changes e.g. in endothelin-related genes. However, the role of endothelin peptides in AS is unknown., Methods and Results: We characterized the expression of the endothelin system in aortic valves of patients with normal valves (n = 12), regurgitation, and fibrosis (n = 6) and AS (n = 18) by reverse-transcriptase-polymerase chain reaction and immunohistochemistry. The number of endothelin-1 (ET-1) positive cells was higher in AS than in control valves, while levels of ET-1 mRNA did not differ between groups. Endothelin receptor-A (ET(A)) mRNA levels were upregulated in stenotic valves (4.3-fold, P = 0.032) associated with a remarkable increase in number of ET(A)-immunopositive cells. ET(B)-receptor mRNA levels did not change during disease progression. Endothelin-converting enzyme-1 (ECE-1) mRNA levels were 42% lower (P = 0.007) in stenotic valves. Finally, because ET-1 and ECE-1 have binding site for activator protein-1 (AP-1), we measured AP-1 DNA binding by gel shift assays, which showed significantly lower (76%, P = 0.003) activity in AS., Conclusion: AS is characterized by distinct upregulation of ET-1 and its target receptor ET(A), promoting growth, inflammation, and fibrosis. These findings suggest therapeutic potential for ET(A)-receptor antagonists in aortic valve calcification.

Clinically, calcific aortic valve disease is a progressive continuum from obstructive fibro(sclero)tic valve thickening to aortic stenosis. Recent evidence suggests that, in addition to nonbone miscellaneous mineralization, calcified valves present distinct signs of active bone remodeling; and in this context, noncollagenous bone-associated proteins are assumed to have a critical role. The expression of 5 bone matrix proteins-bone morphogenetic protein-2 and -4, bone sialoprotein II, osteopontin, and osteoprotegerin-was examined by reverse transcriptase polymerase chain reaction (n = 31) and immunolabeling (n = 83) in the clinical continuum from healthy pliable valves to heavily calcified ones. As a known structural pathologic sign, the extent of neovascularization was also examined. We observed progressive increase in the gene expression of osteopontin (7.4-fold elevation, P < .001) and bone sialoprotein II (5.8-fold elevation, P < .05), and also 1.7-fold elevation (P < .05) in osteoprotegerin gene expression during the disease course. These findings were congruent with that of immunohistochemical analysis. Surprisingly, bone morphogenetic protein-2 and -4 showed a comparable significant decrease in messenger RNA levels in calcified valves (P < .01 and P < .05, respectively). Our results support the view that aortic valve calcification is an actively regulated process. Furthermore, the results suggest that the expression of pro- and anticalcific noncollagenous bone-associated matrix proteins is altered during the disease continuum and that this imbalance may contribute to the pathology of calcific aortic valve disease.

Background: Aortic valve calcification is an actively regulated process that displays hallmarks of atherosclerosis. Natriuretic peptides (A-, B-, and C-type natriuretic peptides [ANP, BNP, and CNP]) have been reported to have a role in the pathogenesis of vascular atherosclerosis, but their expression in aortic valves is not known. Here, we characterized and compared expression of natriuretic peptide system in aortic valves of patients with normal valves (n=4), aortic regurgitation (n=11), regurgitation and fibrosis (n=6), and aortic valve stenosis (n=21)., Methods and Results: By reverse-transcription polymerase chain reaction, all 3 natriuretic peptides were found to be expressed in aortic valves. CNP mRNA levels were 92% lower (P<0.001) in stenotic valves, whereas no significant changes in the expression of ANP and BNP genes were found compared with valves obtained from patients with aortic regurgitation. CNP was localized by immunohistochemistry with specific CNP (32-53) antibody to valvular endothelial cells and myofibroblasts. Gene expression of furin, which proteolytically cleaves proCNP into active CNP, was 54% lower in aortic valve stenosis (P=0.04). Moreover, natriuretic peptide receptor-A and natriuretic peptide receptor-B mRNA levels were 78% and 76% lower, respectively, in stenotic valves. In contrast, gene expression of corin, a proANP- and proBNP-converting enzyme, and natriuretic peptide receptor-C did not differ between groups., Conclusions: We show that natriuretic peptides, their processing enzymes, and their receptors are expressed in human aortic valves. Aortic valve stenosis is characterized by distinct downregulation of gene expression of CNP, its processing enzyme furin, and the target receptors natriuretic peptide receptor-B and natriuretic peptide receptor-A, which suggests that CNP acts as a paracrine regulator of the aortic valve calcification process.

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.

Objective: Tetracycline derivatives affect many cellular functions relevant to chronic cardiovascular pathologies, including cell proliferation, migration and matrix remodelling. Accordingly, we sought to determine whether they may modulate the pathologic characteristics known to be significantly involved in human aortic valve stenosis, such as gelatinase production, apoptosis, expression of vascular endothelial growth factor (VEGF) and tumour necrosis factor-alpha (TNF-alpha)., Methods: The effects of tetracycline derivatives (tetracycline and CMTs-3, -5, -8) on MMP-2 and -9 and their endogenous tissue inhibitor (TIMP-1 and -2) production profiles in explanted human aortic valve pieces were examined by means of gelatine zymography and reverse zymography. Chemiluminescent ELISA was performed to assess VEGF and TNF-alpha concentrations in the medium, and in order to evaluate programmed cell death, in situ labelling of the 3'-ends of the DNA fragments generated by apoptosis-associated endonucleases was performed., Results: CMT-3 and -8 lowered the MMP-9 and VEGF levels significantly in a drug-, dose-, and time-dependent manner. MMP-2 and TIMPs remained unchanged, emphasizing the specificity of CMTs to MMP-9 production on the one hand and restoring the beneficial equilibrium of MMP-9 and TIMPs on the other. Tetracycline was the only drug with a significant impact on net gelatinolytic activity, suggesting that the effect of tetracycline is more extensive concerning total MMP activity., Conclusions: Tetracycline derivatives may have therapeutic effects on the pathologic remodellation of advanced human aortic stenosis through the inhibition of MMP-9 and VEGF production.

Background: Recently, aortic valve stenosis has been demonstrated to exhibit increased expression of certain matrix metalloproteinases (MMPs), and this has relevantly raised the question about possible interdependency between these and their tissue inhibitors. We sought to assess the expression of elastolytic MMPs and their inhibitors (TIMPs) in nonrheumatic aortic stenosis., Methods: The study comprised 30 stenotic and six noncalcified human aortic valves. To measure the expression levels and the amount and molecular forms of gelatinases (MMP-2, MMP-9) and TIMPs (1, 2), in situ hybridization, gelatin zymography, and reverse zymography were carried out. Antielastin staining by a monoclonal BA-4 antibody was performed to investigate the changes of one of the main substrates of these MMPs, and to substantiate the nature of the putative MMP- synthesizing cell. The cases were also immunostained with an antibody to alpha-smooth muscle actin. Inflammatory cell characterization was managed by monoclonal mouse antibodies (UCHL-1, L26, and PGM-1)., Results: Compared with the controls, the calcific valves showed increased mRNA expression and activation of MMP-9, and this was associated with typical characteristics of valve disease. MMP-2 mRNA production was rare, but proMMP-2 protein was detected in all valves. In agreement with the interdependency between MMP-9 and its inhibitors, a suggestive imbalance came out in diseased valves., Conclusions: The disproportion between MMP-9 and its tissue inhibitors may favor a persistent MMP activation state within the calcific valve and likely contribute to the valvular remodeling process in the setting of developing aortic stenosis.

Angiogenesis is an essential biological process not only in embryogenesis, but also in the progression of several major diseases, including cancer, diabetes, and inflammation. Excessive vascularization can also contribute to some cardiovascular pathologies, such as atherosclerosis, but contradictory reports still prevail regarding its impact on aortic stenosis. Using immunohistochemical techniques, we assessed the vascular density and distribution of angiogenesis (FVIII) and vascular endothelial growth factor (VEGF) expression as well as the expression of 2 VEGF receptors, Flt-1 and Flk-1, in 55 nonrheumatic and 6 control aortic valves. In the light of the fact that the angiogenic effect of VEGF is mediated by sustained formation of nitric oxide, the samples were also immunostained with 3 nitric oxide synthase (eNOS, iNOS, and nNOS) antibodies. The immunohistochemical findings of VEGF and its receptors were verified by immunoblotting techniques. Vascular density was highest in the cases with moderate valve stenosis, and the mean number of FVIII-positive blood vessels was 1.7 +/- 1.9 vessels/mm(2) in the diseased valves, whereas the normal valves contained no blood vessels. Vascular density was significantly higher in the cases showing chronic inflammation (P = 0.007). Interestingly, the patients receiving statin therapy had significantly lower vascular densities than those not receiving such therapy (P = 0.001). Diseased valves showed distinct VEGF, Flt-1, Flk-1, and eNOS positivity of activated endothelial, stromal fusiform myofibroblastic, and histocytic cells. In contrast, immunoreactivity for iNOS and nNOS was seen only in nonendothelial stromal cells, and their expression was weaker. Enhanced vascular density was significantly associated with increased expression of Flk-1 (P = 0.028 for endothelial and P = 0.009 for stromal cells) and with endothelial eNOS expression (P = 0.024). A similar tendency was also observed for VEGF, but not for Flt-1. Our results show a distinct angiogenic response and the presence of angiogenic factors in nonrheumatic aortic valve stenosis, suggesting that angiogenesis may influence on the evolution of this disease.

Objectives: We sought to assess tenascin-C (TN-C) expression and its possible pathobiological impact in human aortic valve stenosis., Background: Tenascin-C, a large extracellular matrix glycoprotein, has lately been increasingly connected to cardiovascular pathologies. As TN-C is a multifunctional protein implicated in cell proliferation, migration and differentiation, we investigated the pattern of its expression in diseased human aortic valves., Methods: Fifty-five tricuspid, non-rheumatic stenotic aortic valves were collected from patients undergoing aortic valve replacement, and the controls consisted of four normal valves from individuals who had suffered traumatic death and one from a patient operated on because of a noncalcified purely regurgitant valve. A monoclonal mouse antibody to human TN-C (143DB7) was used as the primary antibody in immunostaining. To study the source of TN-C messenger RNA synthesis, some tissue samples were also examined using in situ hybridization. In order to identify smooth muscle cell differentiation, commercially available antibodies against alpha-smooth muscle actin were used, and immunophenotypic analysis of inflammatory cells was carried out by using the monoclonal mouse antibodies UCHL-1, L26 and PGM-1., Results: In normal valves, TN-C expression was associated with the basement membrane beneath the endothelial cells, whereas stenotic valves showed no such expression but rather immunoreactivity in the deeper layers of the valves. This reactivity was associated with the characteristics typical of the stenosing process and the increased mechanical loading caused by hypertension., Conclusions: We hypothesize that the overexpression of TN-C in stenotic human aortic valves may emphasize that this disease is an active rather than a degenerative process.