Release of leukotriene B4, transforming growth factor-beta1 and microparticles in relation to aortic valve calcification.

BACKGROUND AND AIM OF THE STUDY: Aortic stenosis, the most frequent valvulopathy in the Western world, is characterized by an important extracellular matrix (ECM) remodeling and a process of calcification in the aortic valves. One physiopathological assumption is that transforming growth factor-beta1 (TGF-beta1) acts through ECM remodeling and plays a role in calcification, implicating also microparticles (MPs). Another recent notion is the active involvement of inflammatory mediators in the calcification process of aortic stenosis. METHODS: A total of 105 aortic valves was collected from patients suffering from calcified aortic stenosis with either tricuspid valve (AS) or bicuspid aortic valve (BAV), rheumatic aortic stenosis (RA), endocarditis, or aortic regurgitation (AR). Each valve was incubated for 24 h in culture medium and the supernatants (conditioned media) were used to measure the concentrations of leukotriene B4 (LTB4) and TGF-beta1 and to quantify the number of MPs released. Valvular calcification was evaluated using biphotonic absorptiometry. RESULTS: LTB4 concentrations were significantly higher in media conditioned by AS valves compared to those conditioned by RA and endocarditis valves. In addition, LTB4 concentrations correlated significantly with the calcium content of the aortic valves. In contrast, the concentrations of TGF-beta1 and MPs in the conditioned media did not differ significantly between the various groups of valves, and there was no significant correlation between calcification and either TGF-beta1 or the number of MPs released from the aortic valves. CONCLUSION: Taken together, these results indicate that inflammatory signaling through LTB4 may be more closely linked to calcification and aortic stenosis than signaling through TGF-beta1 and MPs.

Plasmin induces apoptosis of aortic valvular myofibroblasts.

Previous studies have described remodelling of the extracellular substratum by matrix metalloproteinases (MMPs) in aortic valves. However, involvement of the fibrinolytic system has not yet been analysed. We hypothesized that plasminogen and plasminogen activator(s) are present in aortic valves and that plasminogen activation could induce the degradation of adhesive proteins and apoptosis of the valvular myofibroblasts. We employed ELISA, western blotting, fibrin-agar zymography, and immunochemistry to detect components of the plasminogen activation system, in samples of aortic valves and valvular myofibroblasts in primary culture. Using myofibroblast cultures, real-time measurement of plasminogen activation was performed in the absence and presence of inhibitors (amiloride, epsilon-aminocaproic acid, and an MMP inhibitor); the degradation of fibronectin was visualized on western blots; and the apoptotic process was assessed by detection of phosphatidylserine exposure (binding of FITC-annexin V) and DNA fragmentation (TUNEL and ELISA). We demonstrate that a time- and plasminogen concentration-dependent generation of plasmin occurs on the surface of cultured valvular myofibroblasts expressing both u-PA and t-PA. Only u-PA appears to activate plasminogen as t-PA is essentially found in complex with PAI-1. Plasmin-dependent degradation of pericellular proteins, such as fibronectin, leads to cell detachment and apoptosis. In conclusion, various proteins of the fibrinolytic system are synthesized in vitro by cultured myofibroblasts from aortic valves, leading to plasmin-dependent cell detachment-induced apoptosis, a biological process named anoikis. The presence of plasminogen in aortic valves suggests that this process may be operating in vivo and may participate in valvular tissue remodelling, as also suggested by the finding of apoptotic cells in valvular tissue. This is the first demonstration of the presence and potential role of enzymes of the fibrinolytic system in aortic valves.

Expression of uPA, tPA, and PAI-1 in Calcified Aortic Valves.

Purpose. Our physiopathological assumption is that u-PA, t-PA, and PAI-1 are released by calcified aortic valves and play a role in the calcification of these valves. Methods. Sixty-five calcified aortic valves were collected from patients suffering from aortic stenosis. Each valve was incubated for 24 hours in culture medium. The supernatants were used to measure u-PA, t-PA, and PAI-1 concentrations; the valve calcification was evaluated using biphotonic absorptiometry. Results. Aortic stenosis valves expressed normal plasminogen activators concentrations and overexpressed PAI-1 (u-PA, t-PA, and PAI-1 mean concentrations were, resp., 1.69 ng/mL ± 0.80, 2.76 ng/mL ± 1.33, and 53.27 ng/mL ± 36.39). There was no correlation between u-PA and PAI-1 (r = 0.3) but t-PA and PAI-1 were strongly correlated with each other (r = 0.6). Overexpression of PAI-1 was proportional to the calcium content of the AS valves. Conclusions. Our results demonstrate a consistent increase of PAI-1 proportional to the calcification. The overexpression of PAI-1 may be useful as a predictive indicator in patients with aortic stenosis.

[Aortic stenosis and extracellular matrix remodeling]. / Remodelage de la matrice extracellulaire dans les valves atteintes de sténose aortique.

Valvular heart diseases represent an important public health burden. With the decrease in the incidence of rheumatic heart disease, calcific aortic stenosis has now become the most common valvular disease in Western countries. Its prevalence increases with age, such that its affects about 4% of the elderly population and it is the most common motive for valve replacement. Several tissue abnormalities were observed in aortic valves from patients suffering from aortic stenosis: presence of large calcium deposits, inflammatory cells, lipids, and neocapillaries as well as extracellular matrix remodeling. The aortic valves show three characteristic layers: the fibrosa composed mainly of collagen bundles, the spongiosa which consists of a proteoglycan matrix, and the ventricularis which contains several elastic lamellae. The components of the extracellular matrix are synthesized by valvular mesenchymal cells. The turn-over of collagen and elastic fibers is low; the other macromolecules are more rapidly synthesized and hydrolysed. Serine proteases such as enzymes of the fibrinolytic system and matrix metalloproteinases play a role in the remodeling of the extracellular matrix. The hydrolysis of adhesive proteins, such as fibronectin, by plasmin triggers the apoptosis of valvular (myo)fibroblasts, a biological process named anoikis. Cellular events and extracellular matrix remodeling thus participate to the evolution of aortic valves towards aortic stenosis.