Reduced number and function of endothelial progenitor cells in patients with aortic valve stenosis: a novel concept for valvular endothelial cell repair

European Heart Journal (2009) 30, 346–355 doi:10.1093/eurheartj/ehn501 CLINICAL RESEARCH Valvular and congenital heart disease Reduced number and function of endothelial progenitor cells in patients with aortic valve stenosis: a novel concept for valvular endothelial cell repair 1 Department of Cardiology, Heart Center Leipzig, University of Leipzig, Strümpellstrasse 39, D-04289 Leipzig, Germany; and 2Department of Cardiac Surgery, Heart Center Leipzig, University of Leipzig, Strümpellstrasse 39, D-04289 Leipzig, Germany Received 13 June 2008; revised 27 September 2008; accepted 16 October 2008; online publish-ahead-of-print 13 November 2008 Aims Endothelial destruction and calcification primarily occur at the aortic side of the calcified aortic valves (AVs). This study investigated whether degenerative AV stenosis (AS) is associated with the presence of valvular endothelial senescence and a reduction in the number and function of endothelial progenitor cells (EPCs). ..................................................................................................................................................................................... Methods Fifteen patients with severe AS and 18 age-matched control subjects were enrolled. Senescence-associated band results galactosidase activity was mostly localized on the valvular endothelial cells (ECs) of the explanted AVs and highly coincided with the calcified lesion at the aortic side. The number (9.3 + 8.3 vs. 20.5 + 9.0 cells per 106 mononuclear cells; P , 0.01) and the migratory capacity (107.8 + 54.6 vs. 185.0 + 68.8 cells per 1000 cells; P , 0.01) of EPCs evaluated by FACS analysis or migration assay were significantly reduced in AS when compared with control. As possible mechanisms of alterations in EPCs, caspase-3 activity was significantly increased, whereas telomere-repeating factor-2 expression quantified by western blot was significantly reduced in EPCs from AS when compared with control. ..................................................................................................................................................................................... Conclusion Reduced regenerative capacity of valvular ECs due to senescence and decreased levels of EPCs might be, at least in part, a pathological link for the destruction of valvular ECs, resulting in progression of degenerative AS. ----------------------------------------------------------------------------------------------------------------------------------------------------------Keywords Aortic valve stenosis † Apoptosis † Endothelial progenitor cells † Senescence † Ageing Introduction Degenerative aortic valve (AV) stenosis (AS) is the most common valvular disease and increases in prevalence with age.1,2 However, no established therapy to prevent development and progression of AS is currently available despite recent promising results with statins or angiotensin-converting enzyme-inhibitors (ACE-I).3 – 6 The pathogenesis of AS shares a number of similarities with atherosclerosis, such as endothelial dysfunction, increased leucocyte adhesion/infiltration, and calcification.7 – 9 The surfaces of valve leaflets are covered with endothelial cells (ECs), which are critical in maintaining a non-thrombogenic surface and for the transduction of mechanical and biochemical signals.10 Notably, the EC layer of calcified AV appears to be damaged especially on the aortic side.11 Mature ECs possess limited regenerative capacity.12 Thus, there is growing interest in circulating endothelial progenitor cells (EPCs), especially in their role for the maintenance of endothelial integrity and function.13,14 Insufficient numbers of EPCs are related to endothelial dysfunction15 and adverse clinical events,16 suggesting that endothelial injury in the absence of sufficient repair by circulating EPCs promotes the progression of vascular disease or valve disorder. It is well documented that the number and function of circulating EPCs are reduced in several atherosclerotic vascular diseases such * Corresponding author. Tel: þ49 341 865 1671, Fax: þ49 341 865 1461, Email: Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2008. For permissions please email: . Yasuharu Matsumoto 1, Volker Adams1*, Claudia Walther 1, Caroline Kleinecke 1, Peter Brugger 1, Axel Linke1, Thomas Walther 2, Friedrich Wilhelm Mohr 2, and Gerhard Schuler 1 347 Impaired valvular endothelial cell repair in AS as stable coronary artery disease (CAD), stroke, and peripheral occlusive vascular disease.17 In addition, EC senescence appears to be an important factor contributing to the pathogenesis of atherosclerosis.18 However, the presence of senescent valvular ECs and the role of circulating EPCs remain unknown in the pathogenesis of valvular heart disease, particularly in patients with AS. Therefore, the aim of this study was (i) to evaluate the presence of senescent valvular ECs in explanted calcified AVs, (ii) to elucidate the amount and function of EPCs in patients with AS, and (iii) to investigate possible molecular mechanisms responsible for alterations in EPCs. Methods All the patients were recruited in our outpatient clinic between September and December 2007. Thirty-three individuals ,75 years were enrolled in this study after assessment by Doppler echocardiography as well as cardiac catheterization and classified into two groups: (i) patients with severe AS (n ¼ 15) from 65 screened patients scheduled for AV replacement; patients with congenital bicuspid AV, severe mitral valve disease, CAD, and chronic dialysis were excluded; (ii) control subjects (n ¼ 18) from 40 screened patients with angina-like symptom; valvular heart disease including AS and CAD were excluded (Figure 1). The absence of CAD was defined as lack of or minimal coronary atherosclerosis by angiography. AS was excluded by direct measurement of the AV pressure gradient during catheterization.19 Detection of senescence-associated b-galactosidase activity and immunohistochemical staining Senescence-associated b-gal activity was examined in the explanted AV tissue as described previously.21 Briefly, the samples were incubated for 24 h at 378C (no CO2) in freshly prepared b-gal staining solutions containing 40 mmol/L citric acid/sodium phosphate, 1 mg/mL 5-bromo-4-chloro-3-indolyl b-D-galactopyranoside (X-gal), 5 mmol/L potassium ferrocyanide, 5 mmol/L potassium ferricyanide, 150 mmol/L NaCl, 2 mmol/L MgCl2 adjusted to pH 6.0. After the stained tissue samples were photographed, they were fixed with 1% glutaraldehyde and embedded in paraffin. For immunohistochemical analysis, paraffin sections (3 mm) were prepared and the expression of CD31 and Krüppel-like factor-2 (KLF-2) was visualized with a polyclonal anti-CD31 antibody (Dako, Hamburg, Germany) or a polyclonal anti-KFL-2 antibody (Santa Cruz, Heidelberg, Germany) as described.22 Figure 1 Flow chart of th (...truncated)