Cells of primarily extravalvular origin in degenerative aortic valves and bioprostheses

Dirk Skowasch1,*, Stephanie Schrempf1, Nicolas Wernert2, Martin Steinmetz1, Alexander Jabs1, Izabela Tuleta1, Ulrich Welsch3, Claus J. Preusse4, James A. Likungu4, Armin Welz4, Berndt Lüderitz1 and Gerhard Bauriedel1

1Department of Cardiology, Heart Center University of Bonn, Bonn, Germany
2Institute of Pathology, University of Bonn, Bonn, Germany
3Institute of Anatomy II, University of Munich, Munich, Germany
4Department of Heart Surgery, Heart Center University of Bonn, Bonn, Germany

Received 29 March 2005; revised 20 July 2005; accepted 22 July 2005; online publish-ahead-of-print 22 August 2005.

* Corresponding author. Tel: +49 228 287 6670; fax: +49 228 287 4983. E-mail address: dirk.skowasch{at}ukb.uni-bonn.de


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
Aims We assessed aortic valves from patients with non-rheumatic aortic valve stenosis (AS) and with degenerative aortic valve bioprostheses (BP) for the presence of progenitor cell and leukocyte subtype-specific markers.

Methods and results Diseased valve probes from a total of 87 patients (60 AS and 27 BP) were studied. We assessed presence and localization of endothelial progenitor cells (EPCs: CD34, CD133), dendritic cells (DCs: S100), T-lymphocytes (CD3), and macrophages (CD68) by immunohistochemical and morphometric analyses. In the majority of valves, we detected cell-bound signals of CD34 (48% of AS, 74% of BP, respectively), CD133 (58%/81%), S100 (58%/93%), CD3 (62%/81%), and CD68 (78%/93%). Labelled cells were predominantly localized within the valvular fibrosa. As key results, frequency of EPCs, DCs, macrophages, and lymphocytes was found significantly higher in BP when compared with AS (CD34: 19.2±23.2 vs. 5.7±13.0%; CD133: 13.7±12.4 vs. 5.5±8.3%; S100: 15.2±12.2 vs. 5.7±8.9%; CD3: 3.3±2.7 vs. 1.1±1.4%; CD68: 35.3±26.6 vs. 3.4±4.1%; each P≤0.001).

Conclusion EPCs and DCs were detected in a large collective of degenerative aortic valves, more frequently in bioprostheses than in native cusps. Aortoluminal presence of these primarily extravalvular cells co-localized with inflammatory cells is a novel key feature involved in aortic valve degeneration.

Key Words: Aortic valve prostheses • Degenerative aortic stenosis • Dendritic cells • Endothelial progenitor cells • Inflammation


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
Although calcific aortic stenosis is recognized as the most common form of valvular heart disease in the elderly, its underlying pathophysiology is still poorly understood,1 also with regard to the specific cell types involved herein.24 Valvular degeneration is preceded by focal endothelial damage at the fibrosa, with subsequent inflammatory cell infiltration and cellular transformation into an osteogenic phenotype, making these cells capable to initiate mineralization.59 Specifically, T-lymphocytes and macrophages indicating chronic inflammation aggregate in aortic valve stenosis (AS), similar to vascular atherosclerosis.6,7,1017 Clinically, the definite therapy of advanced, degenerative AS is prosthetic valve replacement.1 Herein, biological tissue valves possess superior thromboresistant and haemodynamic properties compared with mechanical valves. However, cusp degeneration and calcification again limit their long-term outcome,18 which device-failure occurs in one-third of valvular bioprostheses in elderly recipients within 10 years.19,20

Interestingly, current concepts of cardiovascular biology and disease stress, the importance of specific cells that are mobilized from bone-marrow and other sources to the circulation in response to injury. Indeed, recent studies have demonstrated the presence of endothelial progenitor cells (EPCs) and other bone-marrow derived cells, including dendritic cells (DCs), in different types of atherosclerosis.2129 Recently, adhesion molecules including CD34, also used as an EPC marker, were found in allogeneic and xenogeneic aortic valve prostheses.30

Therefore, the hypothesis of the present study was that bone-marrow derived and inflammatory cells are involved in the degeneration of both native and bioprosthetic valves. We assessed (i) the presence and frequency of primarily extravalvular cells such as EPCs and DCs in both types of aortic valve degeneration, (ii) quantified inflammatory cells such as T-lymphocytes and macrophages, and thereby (iii) evaluated degenerative native vs. porcine bioprosthetic valves for differences in their expression profiles.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients and valvular specimens
In the present retrospective study, we assessed a series of surgically removed, diseased aortic valves. Tissue probes were obtained from a total of 60 patients with native calcific AS that underwent aortic valve replacement between 1999 and 2001 (n=55) and in 2004 (n=5). Specific exclusion criteria were evidence of rheumatic disease, endocarditis, bicuspid valves, and other co-existing valvular diseases. Of 118 patients with first aortic valve replacement between 1999 and 2001, inclusion criteria and informed consent were given in 55 patients (47% of the eligible patients). Thirty-two were excluded because of exclusion criteria and eight because of refused consent. For technical and logistic reasons, specimens were not obtained from 23 patients. The diagnosis of AS was based on detailed history and physical examination of the patients, as well as echocardiographic and invasive findings. In addition, 27 severely degenerative porcine aortic bioprostheses were obtained during surgical replacement (12 Carpentier–Edwards and 15 Hancock/Hancock II devices; mean duration of implant 13±4 years). These tissue valves were placed between 1983 and 1990. Time of explantation of 22 valves was 1999–2001 (50% of 44 eligible valves; 12 exclusion criteria, two refused consent, and eight technical and logistic reasons); five bioprostheses were obtained in 2004. Porcine bioprosthetic valves were removed because of aortic valve degeneration and incompetence. Relevant patient characteristics are listed in Table 1. To provide a control group with sclerotic aortic valves, additional valves (n=5) were harvested at autopsy; herein, macroscopic disease was classified as mild-to-moderate (opaque leaflets with local areas of thickening and increased stiffness but no significant obstructions), according to recent studies of Otto et al.11 None of these subjects had a history of clinical aortic stenosis (three men, two women; mean age 57±9; two with arterial hypertension, two with obesity; no cardiovascular medication). In addition, non-stenotic aortic valve specimens (n=5) were obtained as controls from surgical patients affected by aortic regurgitation due to aneurysms of the ascending aorta (three men, two women; mean age 59±4; two with arterial hypertension, two with ACE-inhibitors, two with aspirin). In addition, four non-implanted bioprostheses (Hancock II) were regarded. Informed consent for the subsequent analysis of valve tissue was given by each patient and the local Ethics Committee had approved the study.


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Table 1 Patient characteristics
 
Immunohistochemistry
Valvular probes were fixed in 4.5% buffered formaldehyde and embedded in paraffin. Serial sections (4 µm) were taken vertically through the diseased cusp and sinus near the centre of the leaflet adjacent to calcified areas for immunohistochemical analysis. The specimens were not decalcified before sectioning to optimize antigen preservation. After proteolysis with 3% citrate buffer (pH 6.0), non-specific binding sites were blocked by fetal calf serum at a dilution of 1:25 or by rabbit serum 1:5. Thereafter, either the monoclonal antibodies anti-CD34 (1:500; clone 581; Dianova, Hamburg, Germany), anti-CD133 (1:200; clone AC133/1; Miltenyi Biotec, Bergisch-Gladbach, Germany), anti-CD3 (1:100; clone M7193; Dako, Hamburg, Germany), and anti-CD68 (1:100; clone KP1; Dako) or the polyclonal rabbit anti-S100 (1:500; catalog number S2644; Sigma, Deisenhofen, Germany) was applied. For polyclonal antibodies, AffiniPure mouse anti-rabbit IgG (code number 211-005-109; Dianova) was applied for 30 min at room temperature following primary antibody incubation. Colour reaction was done with APAAP marking (Dianova) according to standard protocols13,17,21,26 with Fast Red (Sigma) as a chromogenic substrate. Nuclei were counterstained with haematoxylin. Negative controls included omission of the antibodies and non-specific antibody treatment (mouse monoclonal IgG1; clone NCG01; Dianova and ChromPure rabbit IgG; code number 011-000-003; Dianova, respectively) in the concentrations used for primary antibodies.

Histologic analysis
Haematoxylin-stained histological sections were used to assess cell density, i.e. nuclei per area (0.04 mm2), and to calculate cell density with a computer-assisted morphometric system (VFG 1 graphic card/VIBAM 0.0. software).13,17,21,26 Photomicrographs were obtained by an Optiphot-2 microscope (Nikon, Düsseldorf, Germany) and a downstream KP-C 553 CCD video camera (Hitachi, Rodgau, Germany). Five randomly selected areas were assessed per each distinct leaflet layer, i.e. fibrosa, spongiosa, and ventricularis. The percentage of immunostained cells was determined as the number of positive cells per total number of cells within each layer. All morphometric data were evaluated by two independent examiners.

Statistical analysis
Data are reported as presence (percentage of determinant-positive valves per total number of valves) and as expression (percentage of stained cells per total cell count in five randomly chosen high-power fields; mean±SD). Valves without signals were counted with 0%. Group differences were assessed by the use of {chi}2 test for categorical variables. Probability was calculated with the two-sided Mann–Whitney U test for differences in cell density and expression of valvular determinants. Two-tailed bivariate correlations were determined by the Pearson's coefficient. P values of <0.05 were considered to be statistically significant.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
Diseased valve probes from a total of 87 patients undergoing aortic valve replacement were examined for the presence of endothelial progenitor, dendritic, and inflammatory cells. Sixty valves assessed were of native origin (AS) and 27 were porcine bioprostheses (BP). Patient characteristics, such as age, gender, cardiovascular risk factors, and medication did not differ between both groups and are presented in Table 1.

EPCs and DCs were found in the majority of degenerative valves. Representative examples of both AS and BP that demonstrate the presence of these specific cell types are illustrated by Figure 1. Specifically, CD34 was found in 29/60 (48%) AS and in 20/27 (74%) BP (P=0.025), CD133 in 35/60 (58%) AS and in 22/27 (81%) BP (P=0.036), and S100 in 35/60 (58%) AS and in 25/27 (93%) BP (P=0.001). Positive cells were strictly confined to the fibrosa of AS and the superficial regions of BP. Quantitative immunohistochemical data for these compartments are summarized in Figure 2. As key findings, the percentage of cells labelled by EPC- and DC-markers was two- to three-fold increase in BP when compared with AS (CD34: P=0.001; CD133: P=0.001; and S100: P<0.001).



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Figure 1 Immunohistochemical analysis of native degenerative aortic valves (AS) (A, C, and E) when compared with degenerative bioprostheses (BP) (B, D, and F). Note that signals are present in valvular fibrosa, while no staining of CD34, S100, and CD68 is found in the valvular interstitium. Also, note the higher percentage of cell-bound signals for these markers in degenerative bioprosthetic valves with a typically decreased cellularity. Bar=25 µm.

 


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Figure 2 Quantitative analysis of indicated determinants comparing both subgroups of aortic valve degeneration. Presence of EPCs (CD34, CD133), DCs (S100), macrophages (CD68), and T-lymphocytes (CD3) was significantly increased in diseased bioprostheses ({blacksquare}) when compared with native valves ({square}).

 
As to inflammation, CD3+ lymphocytes were present in 37/60 (62%) of AS and in 22/27 (81%) of BP (P=0.067), whereas staining of CD68 indicative for macrophages was seen in 47/60 (78%) of AS and in 25/27 (93%) of BP (P=0.103). CD3 and CD68 signals were predominantly localized in the subendothelial layer of the valvular fibrosa. Only a few, if any, signals were detected in the regions of the spongiosa (Figure 1E and F). Again, in BP, expression of CD3 and CD68 were markedly increased when compared with AS (each P<0.001) (Figure 2), whereas overall cell density was significantly lower (651±484 vs. 1406±777 cells/mm2, P<0.001). Quantitatively, both inflammatory markers showed a significant correlation (r=0.54, P<0.001). Likewise, we found positive correlations between CD68 and EPC or DC markers (CD34: r=0.46, P<0.001; CD133: r=0.30, P=0.005; S100: r=0.52, P<0.001).

Mild-to-moderate sclerotic aortic valves carried each of the markers studied, whereas the degree of labelling was similar to that in clinically stenotic valves (1.8±1.7% CD34 expression, 2.9±3.2% CD133, 5.0±3.2% S100, 0.6±0.8% CD3, and 3.2±2.2% CD68). Non-degenerative native valves, as well as non-implanted bioprostheses, did not reveal any signal and served as negative controls. We did not find significant relationship between either the frequency or the overall expression levels of specific cell type, cardiovascular risk factor, and/or medication of the patient. In addition, there was no significant difference between cellularity and cell marker expression profiles of Carpentier–Edwards or Hancock bioprosthesis, as well as of bioprostheses obtained in 1999–2001 and those from 2004.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
In the present study, we (i) identify bone-marrow derived EPCs and DCs as novel cell types in degenerative aortic valves, (ii) show their co-localization with inflammatory infiltrates, and (iii) find these primarily extravalvular cell types markedly increased in degenerative prostheses when compared with native diseased valves.

Our findings clearly demonstrate the presence of EPCs in aortic valve degeneration, particularly in aortoluminal regions of injured cusps (Figure 1A and B), whereas non-diseased valves were free of EPCs. Therefore, the presence of EPCs appears to be a novel biological hallmark in end-stage calcified AS, among others such as inflammatory infiltration, heat shock protein 60 homologues, and neoangiogenesis.616 Indeed, our present data show strong co-localization of EPCs with inflammatory cells in a large collective of severely degenerative valves. Quantitatively, 6% of all valvular cells revealed CD34 or CD133 expression. Interestingly, recent data on human in-stent restenosis (ISR) showed a similar expression level for both markers (7%), whereas primary atheroma revealed only 1% for each EPC marker.26 Given these parallel findings for two tissue types, the presence of EPCs in aortic stenosis may indicate a response to injured integrity, analogous to ISR. However, as outlined in Figure 1, presence of both EPCs and inflammatory cells were increased in degenerative valves, whereas ISR contained relatively high EPC levels and only sparse inflammation.17,26 These findings may be plausible by pro-inflammatory stimuli that are continuously present on susceptible cusps, because they impede with permanent blood flow and its pathogenic burden, while ISR rather reflects a circumscript lesional response following a single trauma due to stent implantation. In addition, co-localization and correlation of inflammatory cells with EPCs suggest that inflammation may promote EPC mobilization and/or homing.28 Although the present study cannot definitively answer the role of EPCs, the concept of EPCs that participate in tissue repair is supported by a body of experimental and clinical work in the field of atherosclerosis.23,26,28,29,31,32

We demonstrate, for the first time, the presence of S100+ DCs in the context with aortic valve degeneration (Figure 1C and D). Interestingly, DCs also differentiate from CD34+ haematopoietic progenitors,33 pointing to a common progenitor cell that differentiates to endothelial and dendritic precursors. In the present study, expression of S100 in severely diseased native cusps was 6% when compared with that of 10% recently found in human ISR specimens removed 6 months after stent placement.26 In a response to injury model, DCs constituted approximately half of the cellular content in incipient neointima 4–7 days following balloon trauma.21 Therefore, DCs may be active participants in early vascular and valvular repair processes, and the concept of vascular-associated lymphoid tissue indicated by DCs in vascular walls34,35 may also be adapted to active valve degeneration.

Degenerative valves contain both CD3+ T-lymphocytes and CD68+ macrophages lining the aortic border in a pattern similar to EPCs and DCs (Figure 1E and F). It is known that inflammation plays an important role in the development and/or progression of native valve degeneration.3638 Remarkably, all participating cells were predominantly localized in the superficial valvular fibrosa. Although lymphocytes and macrophages enter the valve from the circulation in response to endothelial injury,39 the origin of the progenitor-like cells remains uncertain. The localization of primarily extravalvular cells close to the aortoluminal border (Figure 1) per se suggests circulating progenitor cells as the source of these cells, as recently shown by longitudinal animal work in traumatized rat carotid arteries.21 This is also supported by our observation that glutaraldehyde-treated bioprosthetic valves, typically devoid of intact cells at the time of implantation, showed a de novo cellularity of ~650 cells/mm2 at explantation.

Of course, observations on human valves are limited to specific time points of valve replacement and therefore cannot assess the progression from sclerosis to end-stage aortic valve disease or functional aspects known from longitudinal animal studies.38 Another limitation is that we have compared native valves to the older generation of porcine valves available since 1980s. Importantly, there are newer generations of prosthetic valves, including porcine valves both stented and stentless and pericardial valves. However, the newer valves have a higher rate of freedom from explantation40 and, by necessity, represent a really rare tissue for analysis until now.

In porcine valve tissue, specific staining of CD34 was increased three-fold and that of the more immature EPC marker CD133 two-fold compared with those of corresponding native valves (Figure 2). Likewise, frequencies of DCs, inflammatory T-lymphocytes, and macrophages were increased in degenerative prostheses (Figure 2), whereas native degenerative valves and sclerotic autopsy valves without stenosis revealed a lower presence of those cells and undiseased control valves did not show specific staining. These graded findings in end-stage degenerative native and bioprosthetic valves suggest not only a unifying pathogenic mechanism that underlies both types of valvular degeneration, but also an even more important role of primarily extravalvular cells in the case of prosthesis degeneration. Recently, increased circulating EPCs were shown to be associated with statin treatment in patients with stable coronary artery disease.32 Likewise, statin therapy apparently delays the haemodynamic progression of mild-to-moderate and severe aortic stenosis in retrospective studies;41 however, failed in a prospective trial.42 In our present work on high-grade aortic stenosis, we did not find a significant relationship between EPC markers and statins, aspirin or ACE-inhibitors that may also exert beneficial effects.43 This lack of correlation could be due, in part, to individually variable time periods until the occurrence of end-stage valve degeneration. Nevertheless, our present work highlights primarily extravalvular cells, among them EPCs that become tissue-resident within diseased valves, in particular in bioprostheses. If the concept is true that mobilization of EPCs contributes to the clinical benefit in patients with aortic valve degeneration, statins may be particularly helpful to prevent the degeneration of bioprostheses. Future clinical trials targeting prosthetic valve failure due to degenerative processes are mandatory to prove this attractive concept.


    Acknowledgements
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors gratefully acknowledge the expert technical assistance of Nicole Kuhn and of Sabine Herzmann as well as the statistical help of Dr R. Fimmers, Institute of Medical Biometry, Informatics and Epidemiology, University of Bonn. The present work was supported partly by grants to G.B. of the Deutsche Forschungsgemeinschaft (DFG Ba 1076/2-2).

Conflict of interest: none declared.


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 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
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