European Heart Journal Advance Access originally published online on January 9, 2006
European Heart Journal 2006 27(12):1495-1504; doi:10.1093/eurheartj/ehi706
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Possible role for mast cell-derived cathepsin G in the adverse remodelling of stenotic aortic valves
1 Wihuri Research Institute, Kalliolinnantie 4, FIN-00140 Helsinki, Finland
2 Division of Cardiology, Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland
3 Minerva Institute for Medical Research, Helsinki, Finland
4 Division of Cardiothoracic Surgery, Department of Surgery, Helsinki University Central Hospital, Helsinki, Finland
Received 25 July 2005; revised 2 December 2005; accepted 8 December 2005; online publish-ahead-of-print 9 January 2006.
* Corresponding author. Tel: +358 9 681 411; fax: +358 9 637 476. E-mail address: ken.lindstedt{at}wri.fi
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Abstract |
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Aims Aortic stenosis (AS) is characterized by extensive remodelling of the valves, including infiltration of inflammatory cells, extracellular matrix degradation, and fibrosis. The molecular mechanisms behind this adverse remodelling have remained obscure. In this article, we study whether cathepsin G, an angiotensin II (Ang II)-forming elastolytic enzyme, contributes to progression of AS.
Methods and results Stenotic aortic valves (n=86) and control valves (n=17) were analysed for cathepsin G, transforming growth factor-ß1 (TGF-ß1), and collagens I and III with RT–PCR and immunohistochemistry. Valvular collagen/elastin ratio was quantified by histochemistry. In stenotic valves, cathepsin G was present in mast cells and showed increased expression (P<0.001), which correlated positively (P<0.001) with the expression levels of TGF-ß1 and collagens I and III. TGF-ß1 was also present in mast cell-rich areas and cathepsin G induced losartan-sensitive TGF-ß1 expression in cultured fibroblasts. Collagen/elastin ratio was increased in stenotic valves (P<0.001) and correlated positively with smoking (P=0.02). Nicotine in cigarette smoke activated mast cells and induced TGF-ß1 expression in cultured fibroblasts. Fragmented elastin was observed in stenotic valves containing activated cathepsin G-secreting mast cells and in normal valves treated with cathepsin G.
Conclusion In stenotic aortic valves, mast cell-derived cathepsin G may cause adverse valve remodelling and AS progression.
Key Words: Angiotensin • Aortic stenosis • Cathepsin G • Elastin • Fibrosis • Mast cell
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Introduction |
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Non-rheumatic aortic stenosis (AS) is an active inflammatory process, which has many similarities to atherosclerosis of the arterial wall. The initial pathological changes associated with AS include valve thickening, accumulation of irregular fibrocalcific masses, disruption of basement membranes, lipid deposits, and infiltration of inflammatory cells, notably macrophages, T lymphocytes, and mast cells.1–3 Therefore, AS has also been called the ‘atherosclerotic’ aortic valve disease.4 The similarities between AS and atherosclerosis extend to clinical risk factors, including smoking, hypertension, hypercholesterolaemia, diabetes, and male sex.5,6 As the degree of stenosis progresses, the natural homeostasis of the extracellular matrix turnover is disrupted. This leads to collagen and elastin fibre disarray and accumulation of collagen with ensuing stiffening of the valves. Indeed, the fibroblasts present in the stenotic aortic valves express characteristics of smooth muscle cells and show signs of cellular senescence in vitro, suggesting that they are in a state of chronic activation similar to that observed in the lesions of fibromatosis and scleroderma.7
A role for angiotensin II (Ang II), a powerful profibrotic and proinflammatory mediator, in the pathogenesis of AS seems obvious, as two Ang II-generating enzymes, angiotensin-converting enzyme (ACE) and chymase, a mast cell-derived protease, are significantly upregulated in stenotic aortic valves.3,8 Increased production of Ang II, in turn, may mediate collagen gene transcription by stimulating the production of transforming growth factor-ß1 (TGF-ß1).9 In addition to ACE and chymase, the neutral protease cathepsin G is also capable of generating Ang II.10 Furthermore, cathepsin G has potent elastolytic activity and may play a role in tissue remodelling.11,12 Cathepsin G was recently shown to be associated with the formation of atheromas in human carotid arteries,13 but its association with the calcific aortic valve disease has not been studied. The aim of our present study was to determine whether cathepsin G is present in stenotic aortic valves, where it may contribute to the local Ang II-forming potential, and so participate in the profibrotic progression of the valves. Furthermore, we sought to determine experimentally whether this potent elastolytic enzyme degrades elastin in the valves, and thus further accelerates stiffening of the valves, which is a key feature in the pathogenesis of AS.
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Samples and study population
Stenotic aortic valves were obtained from 86 patients undergoing valve replacement surgery between August 2000 and January 2003. A detailed description of the patient recruitment has recently been published in this Journal.14 The sample size was based on the time constraint because of the limited duration of our study period. Importantly, all patients had isolated AS, in that patients with more than mild aortic regurgitation or mitral valve disease were excluded, as were patients with either history of myocardial infarction or any proximal coronary artery stenosis exceeding 50% of the luminal diameter at angiography. Individuals with complicated diabetes and renal insufficiency (serum creatinine >170 µmol/L) were also excluded, as were patients with endocarditis. The clinical characteristics of the patients are summarized in Table 1. Control non-stenotic valves were obtained from patients undergoing cardiac transplantation due to dilated (n=12) or ischaemic (n=1) cardiomyopathy or from organ donors without cardiac disease whose hearts could not be used as grafts (n=4) due to advanced age, suspected ischaemia, or resuscitation. All organ donors had died either of cerebrovascular accident or of trauma. Only valves without any visible disease were accepted as controls. Of the 13 patients undergoing transplantation, 12 had been on ACE-inhibitor (n=9) or angiotensin receptor antagonist (n=3) therapy for prolonged periods and one patient had started ACE-inhibitor 2 days before the transplantation. The main characteristics of the control group are presented in Table 2. The investigation conforms with the principles outlined in the Declaration of Helsinki. The protocol was approved by the Ethics Committee of Helsinki University Central Hospital. All participants signed an informed consent document.
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Conventional and competitive RT–PCR
Total RNA was isolated from the aortic valves of 84 patients (RNA isolation from two patient samples failed because of a high degree of calcification) and 17 control subjects, and RT–PCR was performed as described previously.3 The primers were as follows: cathepsin G: 5'-CTCAATATAATCAGCGGACC (F), 5'-CCAGCAGTTTGAAGCTTCTC (R); TGF-ß1: 5'-CAACACATCAGAGCTCCGAGAAGC (F), 5'-TTGCAGTGTGTTATCCCTGCTGTC (R); collagen I: 5'-GACCGATGGATTCCAGTTCG (F), 5'-TGTGACTCGTGCAGCCATCG (R); collagen III: 5'-AGATGTCCTTGATGTGCAGC (F), 5'-CCACCAATGTCATAGGGTGC (R); and GAPDH: 5'-ACCACAGTCCATGCCATCAC (F), 5'-TCCACCACCCTGTTGCTGTA (R). The competitor DNA for TGF-ß1 was constructed by inserting a 129 bp external DNA fragment into the SmaI site and for collagens I and III by inserting a 129 bp external DNA fragment into the DraII site. The PCR products were verified, by DNA sequencing, to represent the corresponding target. The competitive RT–PCR assay was standardized to the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The PCR products were quantified with a Gel Doc 2000 gel documentation system (Bio-Rad), and the logarithm of the competitor-to-target ratio was plotted against the logarithm of the competitor DNA molecules.15
Immunohistochemistry of cathepsin G, TGF-ß1, and TßRII
Frozen aortic valve leaflets were divided into four sections from base to tip. Immunohistochemistry of cathepsin G was performed in a randomly selected subpopulation of the stenotic valves (26 patients) and in all normal valves (n=17) using a commercially available polyclonal rabbit-anti-human cathepsin G antibody (22 µg/mL, Dako, Glostrup, Denmark). TGF-ß1 was detected with a monoclonal mouse-anti-human TGF-ß1 antibody (3.3 µg/mL, Serotec, Hanar, Germany) and mast cells with a monoclonal anti-tryptase antibody (0.11 µg/mL, Dako) as described previously.3 TGF-ß receptor type II (TßRII) was detected with a goat-anti-human TßRII antibody (2.5 µg/mL, R & D Systems, UK). No staining was detected when primary antibodies were substituted with irrelevant rabbit, mouse, or goat isotype-specific immunoglobulin G (Serotec). Cathepsin G-positive cells were counted by light microscopy, and the areas of the sections were measured by computer-assisted morphometry (Image-Pro Plus, version 4.5).
Double immunofluorescence staining
Double immunofluorescence staining (26 stenotic and 17 control valves) was performed using a commercially available rabbit-anti-human polyclonal cathepsin G antibody (22 µg/mL, Dako) and a monoclonal anti-tryptase antibody (2.2 µg/mL, Dako). Alexa goat-anti-rabbit 546 IgG (red) and Alexa goat-anti-mouse 488 IgG1 (green) (Molecular Probes Europe BV, Leiden, The Netherlands) were used as secondary antibodies at a concentration of 10 µg/mL each. The slides were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma, St Louis, MO, USA) before mounting. An irrelevant rabbit immunoglobulin G served as a negative control. Subcellular distributions of tryptase and cathepsin G in mast cells were visualized, using confocal microscopy (Ultra View, Perkin Elmer), with a section thickness of 
0.5 mm. Double-labelled specimens were serially excited with a krypton/argon laser, and images were recorded with a cooled CCD camera (Perkin Elmer) as reported earlier.16 Merged images were generated using the Adobe Photoshop computer program.
Analysis of collagen and elastin fibres
All stenotic (n=86) and normal (n=17) valves were stained for elastin and collagen fibres with elastic stain, using Van Gieson's solution as counterstain (Accustain Elastic stain kit, Sigma-Aldrich Co., St Louis, MO, USA). Relative proportions of collagen and elastin were determined, using computer-assisted morphometry (Image-Pro Plus, version 4.5). Moreover, the integrity of the elastin fibres was evaluated qualitatively by inspection of the images, and the level of valvular elastin degradation was analysed by semi-quantitative grading (1, no elastin degradation or mild degradation; 2, moderate; 3, moderate to severe; and 4, severe elastin degradation).
To determine whether cathepsin G can degrade aortic valvular elastin fibres in vitro, cryostat sections of normal aortic valves were treated with 1 µM cathepsin G (Calbiochem, Germany) in the presence or the absence of EDTA (5 mM). The sections were incubated in a moist chamber at 37°C for 24 h, rinsed with phosphate-buffered saline (PBS), and fixed in absolute ethanol, followed by staining with Van Gieson's elastic stain.
Mast cell culture and activation with tobacco smoke in vitro
Human cord blood-derived CD34+ progenitor cells (Cambrex Bio Science, Walkersville, MD, USA) were cultured as described previously,17 with some modifications. The cultured mast cells were viable (>95%), contained 4 pg histamine/cell, and stained positive for tryptase and chymase. Cell cultures were tested negative for mycoplasma with the MycoAlert Detection Kit (Roche). Cigarette smoke-treated PBS (Ca2+–Mg2+-free) was prepared by connecting a burning cigarette attached to a piece of plastic tubing to a 50 mL syringe containing 10 mL of PBS. After repeated cycles (seven times) of cigarette smoke suction into the syringe, with vigorous mixing (15 s) between each cycle, the PBS containing the water-soluble components of cigarette smoke was used in experiments. Mast cells (106 cells/sample) were incubated with cigarette smoke-treated PBS (800 µL), nicotine (0.05–100 µg/mL), or acetaldehyde (0.1–1 mM) in 24-well plates for 30, 60, and 120 min at 37°C. Histamine was determined fluorometrically (Beckman Ratio Fluorometer Beckman Instruments, Fullerton, CA, USA) by registering the fluorescence generated by the o-phtaldehyde complex, according to the method of Shore et al.18 Cytotoxicity Detection Kit (Roche) was used to exclude possible cytotoxic effects caused by experimental conditions on mast cells.
Stimulation of cultured human skin fibroblasts
Human skin fibroblasts were grown in monolayer and maintained in a humidified atmosphere (5% CO2) at 37°C in 25 cm2 stock flasks containing 5 mL of standard growth medium [RPMI 1640 supplemented with L-glutamine (1%), penicillin–streptomycin (1%), and non-essential amino acids (1%)]. The cells (fifth and sixth passages) were incubated either with cathepsin G (50 nM, Calbiochem) in the presence or the absence of losartan (10 µM) or with cigarette smoke-treated media (2.5%), nicotine, or acetaldehyde, for 24 h at 37°C. Total RNA was isolated and the mRNA expression levels of TGF-ß1 and collagens I and III were analysed by RT–PCR.
Statistics
Group differences were analysed using Student's t-test for normally distributed data and the Mann–Whitney U test for skewed data distribution (Figure 1). The group data are summarized as mean values±SD or as medians±SEM. For correlations, Spearman's coefficients were calculated. The analyses were done using either SPSS (version 11.0) or SYSTAT (version 9.0) software. Two-sided nominal unadjusted P-values less than 0.05 were considered statistically significant.
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mRNA expression levels in normal and stenotic valves
RT–PCR analyses revealed that the expression levels of cathepsin G mRNA were higher in stenotic than in control valves (P<0.001). Similarly, competitive RT–PCR analyses showed a significant increase in the mRNA expression levels of TGF-ß1, collagen I, and collagen III (P<0.001) in the stenotic aortic valves when compared with control valves (Figure 1 and Table 3). A positive correlation appeared between the mRNA expression levels of cathepsin G and TGF-ß1 (r=0.300; P=0.002). Moreover, both cathepsin G and TGF-ß1 mRNA expression levels correlated positively with those of collagen I (r=0.320, P=0.001 and r=0.319, P=0.001, respectively) and collagen III (r=0.302, P=0.002 and r=0.275, P=0.005, respectively). As shown in Figure 1, the mRNA expression levels of all investigated parameters in the AS group consisted of normal, moderately elevated, and highly elevated values. The characteristics of patients with highly elevated values are presented in Table 4. The clinical parameters did not differ significantly between outliers and other patients, but regarding histopathological features, all these outliers had more severe calcification of the aortic valves.
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The mRNA expression levels were similar in the analysed samples of both tricuspid and bicuspid aortic valves. A subpopulation analysis of subjects receiving ACE-inhibitor or angiotensin receptor antagonist therapy confirmed that the mRNA expression levels of cathepsin G, TGF-ß1, collagen I, and collagen III were, despite the medication, still significantly higher in the stenotic than in the control valves (P=0.004 for cathepsin G; P<0.001 for TGF-ß1; P<0.001 for collagen I; and P=0.012 for collagen III).
Immunohistochemical detection of cathepsin G
Cathepsin G-positive cells (reddish-brown staining) were present in both normal (Figure 2A) and stenotic aortic valves (Figure 2B), and their number was significantly increased in stenotic valves. An accumulation of cathepsin G-positive cells in stenotic valves was evident throughout the valves, i.e. from base to tip of the leaflet, but most prominent in the two middle sections of the stenotic valves when compared with control valves (9.2±6.4 vs. 3.8±1.6 cathepsin G-positive cells/mm2, P<0.001). The distribution of cathepsin G-positive cells was similar in the leaflet base (6.6±4.7 vs. 3.1±1.5 cells/mm2, P=0.003) and tip (6.4±5.0 vs. 3.6±2.2 cells/mm2, P=0.02).
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In the control valves, the cathepsin G-positive cells were localized mainly to the subendothelial space, whereas in the stenotic valves, they were distributed throughout the leaflet and were associated especially with the fibrocalcific lesions.
Colocalization of cathepsin G and mast cells
Double immunofluorescence staining of cathepsin G and tryptase, a mast cell-specific protease, revealed that the majority of cathepsin G-positive cells in the stenotic valves was activated mast cells (Figure 2C–E). However, in normal aortic valves, some resting (i.e. not degranulated) mast cells were also found to be cathepsin G-negative (Figure 2F–H). Examination of the sections with confocal microscopy demonstrated a granular staining pattern of both tryptase (Figure 3A) and cathepsin G (Figure 3B), suggesting that cathepsin G was stored in the mast cell granule compartment (Figure 3C). Indeed, in stenotic leaflets, the granules that had been released into the extracellular space by the activated mast cells stained positively for cathepsin G (Figure 3D).
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Immunohistochemical detection of TGF-ß1 and type II TGF-ß receptors
Immunohistochemistry of TGF-ß1 showed positive staining in both normal and stenotic aortic valves, the staining being stronger in the stenotic valves. TGF-ß1 was present in the endothelium lining the valves, around the neovessels in the stenotic valves, as well as in the deeper regions of the valves, where it was found either bound to the extracellular matrix or confined to the cellular structures. Using adjacent sections of stenotic aortic valves, cathepsin G-positive mast cell-rich areas (Figure 4A) were found to accumulate TGF-ß1 (Figure 4B). In addition, the type II TGF-ß serine/threonine kinase receptor (TßRII), which binds active TGF-ß1 and together with TßRI triggers collagen gene expression, was present in such areas (Figure 4C). Furthermore, in the presence of cathepsin G, cultured human skin fibroblast showed a significant increase (P=0.04) in the expression of TGF-ß1 mRNA and a small increase in collagens I and III mRNAs, which was inhibited by losartan, a specific Ang II type 1 receptor (AT-1R) antagonist (Figure 4D).
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Analysis of collagen and elastin fibres in normal and stenotic valves
When compared with control valves, stenotic valves contained more collagen and less elastin fibres (Table 3). This finding was present throughout the valves, i.e. from base to tip of the leaflet. The valvular cathepsin G mRNA levels and the number of cathepsin G-positive cells in the leaflets correlated positively with the collagen/elastin ratio (r=0.317, P=0.001 for cathepsin G mRNA and r=0.587, P<0.001 for cathepsin G-positive cells). In control valves, the elastin fibres were intact (Figure 5A, arrows and rectangle), whereas a striking pattern of elastin degradation and disarray was found in the stenotic leaflets (Figure 5B, rectangle). In adjacent sections of the stenotic valves, activated cathepsin G-positive mast cells (Figure 5C) were found to colocalize to areas in which elastin fibre degradation was prominent (Figure 5B). The elastin degradation could be mimicked in vitro by incubating sections of control aortic valves (Figure 5D) with cathepsin G (Figure 5E), which decreased the valvular elastin content from 48.5±1.6 to 17.5±1.1% (P=0.002). Semi-quantitative grading (grades 1–4) of the level of elastin degradation in the aortic valves (Figure 5F) revealed that in the majority of the stenotic aortic valves, the degree of elastin degradation was moderate to severe throughout the valve leaflet (grades 2–4), the most prominent alterations being observed in the two middle sections of the valves. No elastin degradation was observed in the control valves (grade 1, from leaflet base to tip).
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The collagen/elastin ratio was significantly higher in the stenotic relative to the non-stenotic valves (P<0.001) and increased in current smokers relative to non-smokers (P=0.02) (Figure 6). Also the difference in the collagen/elastin ratio between the stenotic and non-stenotic valves remained significant (P<0.001) when only patients and controls using ACE-inhibitors or angiotensin receptor antagonists were included in the analysis. No differences were found in the collagen/elastin ratios between the bicuspid and the tricuspid stenotic aortic valves.
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Cigarette smoke activates mast cells and fibroblasts
In the presence of cigarette smoke-treated PBS, cultured human mast cells released up to 45% of their histamine content in a time-dependent manner, reflecting cigarette smoke-induced mast cell activation and degranulation (Figure 7A). Among the individual mediators of cigarette smoke tested, nicotine (100 µg/mL) was found to induce mast cell activation, whereas acetaldehyde (0.1–1 mM) had no effect (Figure 7A). Nicotine and acetaldehyde were both capable of directly inducing TGF-ß1 mRNA expression in cultured human skin fibroblasts (Figure 7B).
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Discussion |
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This is the first study to show that cathepsin G, a potent Ang II-forming enzyme with elastolytic activity, is present in normal aortic valves and that its expression is significantly increased in stenotic aortic valves. More importantly, activated valvular mast cells were identified as the major source of cathepsin G. Thus, in addition to ACE and mast cell chymase, which are significantly increased in stenotic aortic valves,3 cathepsin G may increase the overall Ang II-forming potential in the stenotic valves. We have previously shown that the AT-1Rs, which mediate the adverse remodelling effects of Ang II, are upregulated in stenotic valves when compared with control valves.3 By activating the AT-1Rs, Ang II increases the expression of TGF-ß1, a profibrotic cytokine responsible for connective tissue formation, such as collagen synthesis, during physiological and pathophysiological processes of remodelling.9,19 Analogously, in the present work, cultured human skin fibroblasts treated with cathepsin G significantly induced their expression of TGF-ß1 by a mechanism that was inhibited by losartan, a specific AT-1R antagonist. Thus, cathepsin G was capable of triggering an Ang II-dependent profibrotic response in cultured human fibroblasts, suggesting that the observed increase in cathepsin G in stenotic valves may be of relevance for the adverse remodelling. As several Ang II-generating enzymes (i.e. ACE, chymase, and cathepsin G) are present at low levels also in normal aortic valves, Ang II may regulate the physiological homeostasis of extracellular matrix turnover and repair in ageing normal valves, which are exposed to cyclic mechanical stress throughout life. However, if Ang II-formation in aortic valves becomes chronically elevated, valve fibrosis and calcification with progressive AS may ensue.
In addition to being profibrotic, cathepsin G-mediated TGF-ß1 formation may also contribute to the progression of AS by initiating calcification in the aortic valve.20 Recent studies have shown that calcification of the aortic valves is an actively regulated process21,22 which is associated with a poor outcome and rapid haemodynamic progression of the disease.23 Interestingly, in the present study, we also detected moderate to severe calcification in the aortic valves of the patients with the highest mRNA expression levels of cathepsin G, TGF-ß1, and collagens I and III (Table 4). These findings suggest that cathepsin G-mediated TGF-ß1 formation may be associated with both fibrosis and calcification, the two major pathogenic mechanisms of AS.
In the present study, in areas with prominent elastin degradation, elevated cathepsin G expression in the stenotic valves was accompanied with accumulation of cathepsin G-positive cells, notably, mast cells. Accordingly, the accumulation and activation of mast cells in stenotic valves may perturb the natural homeostatic mechanisms of valve repair by locally increasing the cathepsin G levels and therefore result in adverse remodelling of the valves. This suggestion is further supported by the present in vitro observation that exogenous cathepsin G was able to degrade elastin fibres in thin tissue sections of aortic valves. However, as only moderate correlation between the cathepsin G expression and the collagen/elastin ratio in the valves was observed, it is conceivable that also other elastolytic enzymes contribute to the elastin degradation found in the stenotic aortic valves. These include matrix metalloproteinases, which are upregulated in the stenotic aortic valves and may participate in the adverse remodelling of the valves.24 The possible role of other elastolytic cathepsins in the pathogenesis of AS is at present unknown. Interestingly, cathepsins S and K are markedly elevated at sites of elastin and collagen degradation in advanced rupture-prone atherosclerotic plaques and in aortic aneurysms, where their endogenous inhibitor, cystatin C, is reduced.25,26 Moreover, the activities of cathepsins G and D are significantly increased in aortic aneurysms, suggesting a role for cathepsins in adverse tissue remodelling at least in this disease.27
Recent epidemiological evidence suggests that the traditional risk factors of atherosclerosis hasten the development of AS.5,6 Indeed, we found a positive correlation between smoking and an increased collagen/elastin ratio in the stenotic valves. Although nicotine stimulates fibronectin expression by cultured lung fibroblasts and decreases collagenase activity of cultured cardiac fibroblasts,28,29 the molecular mechanisms of tobacco-related enhancement of the valvular collagen/elastin ratio have remained enigmatic. Here, we show that nicotine and acetaldehyde, two major components in cigarette smoke, induce TGF-ß1 mRNA expression in cultured human skin fibroblasts. The present data also provide an alternative explanation for the association between smoking and an increased collagen/elastin ratio in stenotic aortic valves, namely, smoking-induced mast cell activation. Activation of mast cells with ensuing degranulation is critical for the release of the contents of the cytoplasmic granules, such as cathepsin G. Thus, in the present work, we found that a water-soluble component of cigarette smoke, i.e. nicotine, directly activated cultured human mast cells in vitro. However, nicotine may not be the only mediator in cigarette smoke capable of activating human mast cells, and the presence of other potential mast cell activators in cigarette smoke remains to be shown. As mast cells in the inflamed stenotic valves were activated and the expelled mast cell granules contained cathepsin G, the prerequisites for extracellular remodelling by valvular mast cells were fulfilled.
Currently, no approved medical therapy for AS exists. Thus, there is an urgent need to understand the cellular and molecular mechanisms that contribute to its development. Our previous and present findings, showing an upregulation of three Ang II-forming enzymes in stenotic aortic valves, suggest that blocking the effects of Ang II by ACE-inhibitors or AT-1R antagonists may retard AS progression. Owing to the presence of ACE-independent Ang II-forming mechanisms, the use of AT-1R antagonists may be superior to ACE-inhibitors. However, recent reports on the adverse effects of AT-1R antagonists30,31 may call for alternative treatments. Inhibition of mast cell activation and degranulation, or direct inhibition of chymase and cathepsin G activity in aortic valves with advancing stenotic changes, may be suitable for additional or even alternative modes of pharmacological prevention of AS.
Study limitations
We report here increased expression of cathepsin G and TGF-ß1 in stenotic aortic valves and show that cathepsin G was able to induce losartan-sensitive TGF-ß1 expression in cultured human fibroblasts. However, owing to the low number of patients (n=6) and controls (n=3) receiving AT-1R antagonists, we were unable to draw any conclusion concerning the influence of this medication on the valvular TGF-ß1 expression in the two subgroups. Although the investigated parameters did not differ significantly between the subjects with or without ACE-inhibitor or AT-1R antagonist therapy, we cannot fully exclude the possibility that the medication has affected the results. Furthermore, to define statistically significant differences between the study groups, we used unadjusted P-values. Although our analyses were pre-planned, the number of tests performed was considerable, and therefore, the possibility of type I errors should be taken into account in the interpretation of our data. Finally, as normal, fresh surgical samples of aortic valves are rarely available, the control group in the present study consisted of both organ donors without cardiovascular diseases and patients undergoing cardiac transplantation. However, the aortic valves in both groups were both macroscopically and microscopically normal, and no statistically significant differences in any of the investigated parameters were observed between these two control subgroups.
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This work was supported by the Finnish Foundation for Cardiovascular Research (S.H.), by the Sigrid Juselius Foundation, and by the EVO research funds of Helsinki University Central Hospital. Wihuri Research Institute is maintained by the Jenny and Antti Wihuri Foundation. We also thank Mrs Liisa Blubaum, Mrs Jaana Tuomikangas, Mrs Elina Kaperi, and Ms Suvi Mäkinen for excellent technical assistance.
Conflict of interest: none declared.
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