Aortic stenosis (AS) is the most common valvular disease in many Western countries and represents a major healthcare burden [
In the fibrinolytic system, plasminogen activators, tissue-type plasminogen activator (t-PA), and urokinase-type plasminogen activator (u-PA) are responsible for the conversion of the abundant extracellular zymogen plasminogen into the active proteinase plasmin, the key enzyme of fibrinolysis [
Plasminogen activators (PAs) appear to be mainly involved in pericellular proteolysis [
The aim of this study is, first, to determine u-PA, t-PA, and PAI-1 levels in the culture media incubated with aortic stenosis valves using different ELISA kits and in a second step to investigate whether there is any correlation between the different actors of the fibrinolytic system and then to correlate their values with the valvular calcium content.
Sixty-five aortic stenosis valves were collected immediately after cardiac surgery from patients in Bichat-Claude Bernard Hospital in Paris (France) in accordance with French ethical laws (L 1235-2 and L 1245-2 of Public Health legislation) and as part of an ongoing prospective study (GENERAC-ClinicalTrials.gov,
Valves were incubated in DMEM culture medium with antibiotics (100 U/mL penicillin, 100 ng/mL streptomycin, and 0.05 ng/mL amphotericin B) (3 mL/g tissue (wet weight)) for 24 hours at 37°C in a 5% CO2-95% air atmosphere. The conditioned media and the valves were then frozen at −20°C.
Concentrations of urokinase- and tissue-plasminogen activators (u-PA, t-PA) as well as plasminogen activator inhibitor-1 (PAI-1) were measured in the valve-conditioned media, using ELISA assays according to the manufacturer’s instructions (Hyphen Bio Med and Technoclone). Because of the absence of a control group, the levels of plasminogen activator and inhibitor obtained in the conditioned media of the pathological valves were compared to the normal range of each protein according to the device of ELISA used. Normal human plasma range of u-PA, t-PA, and PAI-1 is ≤5 ng/mL, ≤10 ng/mL, and ≤25 ng/mL, respectively.
The DEXA (“Dual-Energy X-Ray Absorptiometry”) is a noninvasive technique for determining the human or animal bodies’ composition. The calcium content of each valve was measured,
Results are reported as means ± standard deviation. Correlation analyses between the levels of u-PA, t-PA, and PAI-1, in the conditioned media with aortic stenosis valves, were performed by calculating the coefficient of Spearman. Only Spearman Rho (
Sixty-five aortic valves were collected immediately after cardiac surgery from patients suffering from aortic stenosis. The average age of the patients operated on is 75 years; 62% of the patients are male and 38% are female. The mean BMC in the AS valves is
Characteristics of patients (pts) and calcium content of the valves with AS. Calcium content of the valves was evaluated using biphotonic absorptiometry; results are expressed as mean ± SD.
Total no. | Female | Male | |
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Number of pts | 65 | 22 | 43 |
Mean age (years) | 75 | 80 | 72 |
Calcium content (g/g wet weight) |
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Samples of each conditioned medium with AS valves were analysed in the plasminogen activators microtiter ELISA, for u-PA and for t-PA. The u-PA and t-PA measured levels differ slightly between the patients with aortic stenosis and were ranged from 0.33 to 3.75 ng/mL and 0.84 to 6.93 ng/mL, respectively. The mean concentrations of u-PA and t-PA of the whole studied conditioned media are 1.69 ng/mL ± 0.80 and 2.76 ng/mL ± 1.33, respectively.
All the studied valves showed normal u-PA and t-PA concentrations (Figures
Concentrations of u-PA, t-PA, and PAI-1 in the conditioned media with aortic stenosis valves. All the aortic stenosis valves showed normal u-PA (a) and t-PA (b) concentrations, and forty-five valves showed elevated PAI-1 levels (c) when compared to the normal human plasma range according to the u-PA, t-PA, and PAI-1 microtiter ELISA device (the upper limit of which is shown as the red horizontal line at 5 ng/mL, 10 ng/mL, and 25 ng/mL, for u-PA, t-PA, and PAI-1 resp.). AS: aortic stenosis.
All the conditioned media with AS valves were also analyzed in the PAI-1 microtiter ELISA. The conditioned media level of PAI-1 differs significantly between the patients with aortic stenosis and was between 3.47 and 176.19 ng/mL. The mean concentration of PAI-1 in the whole studied conditioned media is 53.27 ng/mL ± 36.39.
Forty-five of the sixty-five pathological valves (69%) showed elevated PAI-1 levels (Figure
A weak correlation was found between u-PA and PAI-1 (
u-PA or t-PA and PAI-1 levels in conditioned media with aortic stenosis valves. Correlation between u-PA and PAI-1 measured levels (a) and the t-PA and PAI-1 measured levels (b) in the conditioned media obtained with 65 aortic stenosis valves. The search for correlation was obtained by Spearman test (
There was no significant difference in the average concentration of u-PA and t-PA between the different groups of the BMC valves but the mean concentration of PAI-1 increased in function of the level of calcification of the valve. For the valves with AS in which the calcium content was lower than 0.2 (g/g wet weight), the mean concentration of PAI-1 was 42.47 ng/mL ± 32 and increases to 78.75 ng/mL ± 36 when a calcium concentration is higher than 0.4 (Table
Description of the studied valves with AS. Calcium content of the valves and concentrations of plasminogen activators and inhibitor of the fibrinolytic system released during 24 hours by the AS valves into the incubation medium (valve-conditioned medium) were measured. Results are expressed as mean ± SD.
Number of valves | Calcium content (g/g wet weight) | u-PA (ng/mL) | t-PA (ng/mL) | PAI-1 (ng/mL) |
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65 |
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31 | <0.2 |
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31 | 0.2–0.4 |
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3 | >0.4 |
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u-PA: urokinase-type plasminogen activator; t-PA: tissue-plasminogen activator; PAI-1: plasminogen activator inhibitor 1.
Aortic stenosis (AS) is the most frequent valvulopathy in the Western countries and is the second cause of cardiac surgery after coronary bypasses. 65 human aortic valves suffering from aortic stenosis were analysed in this study. The mean age of the operated patients with AS is 75 years, this is probably related to the slow evolution of this pathology. This result confirmed the high prevalence of the AS in the elderly. Sixty-two percent of the operated patients are male, results in accordance with previous clinical studies associated with calcific aortic valve disease which show that the male gender is associated with a twofold increased risk [
Valves with AS are characterised by calcification and ECM remodelling. The ECM remodelling depends on the activation of different types of proteases including plasmin, a key enzyme of the fibrinolytic system [
Fibrinolytic system has an inactive zymogen called plasminogen. The latter can be activated by plasminogen activators u-PA or t-PA and be converted into plasmin. Plasmin can degrade both the fibrin and the ECM directly and protect the tissues from fibrosis [
In this present study, we demonstrate that studied AS valves released enzymes of the fibrinolytic systems u-PA, t-PA, and PAI-1 in the conditioned media after 24 h of incubation. These valves expressed normal plasminogen activators concentrations but overexpressed PAI-1 (the mean concentrations of u-PA, t-PA, and PAI-1 in all the studied conditioned media were, resp., 1.69 ng/mL ± 0.80, 2.76 ng/mL ± 1.33 and 53.27 ng/mL ± 36.39). Although u-PA and PAI-1 were not correlated (
A limitation of the present study was that we did not have control valves; thus the levels of plasminogen activators and inhibitor obtained in the conditioned media of pathological valves were compared to the normal range of each proteins according to the device of ELISA used.
Forty-five of the sixty-five pathological valves (69%) showed elevated PAI-1 levels. Several groups have reported excess PAI-1 in atherosclerotic plaques in humans [
AS valves are characterised by an important calcification and ECM remodelling with inflammatory process [
Although, several studies have reported an increased level of PAI-1 in different pathological conditions [
In conclusion, our results demonstrate a consistent increase of PAI-1 content in relation to the calcification and the severity of AS valves. The overexpression of PAI-1 may be useful as an important predictive prognostic indicator in patients suffering from aortic stenosis.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Najlah Kochtebane received a research Grant from INSERM-DGRSRT code 08/M11. Abdullah Mossa M. Alzahrani and Aghleb Bartegi received a Grant from KFU Deanship of Scientific Research (130259). The authors would like to thank Professor JB Michel for receiving Najlah Kochtebane in his laboratory and Dr. M. P. Jacob for her help.