Patients with bicuspid aortic valve (BAV) may gradually develop significant valve dysfunction, whereas others remain free of dysfunction. Factors that determine the prognosis of BAV remain unclear. Because endothelial progenitor cells (EPCs) have a role in the repair of endothelial surfaces after injury, we hypothesized that EPCs may also be involved in preventing BAV degeneration. Accordingly, we compared EPC level and function in patients with BAV with versus without valve dysfunction. The study group included 22 patients with BAV and significant valve dysfunction (at least moderate aortic regurgitation and/or at least moderate aortic stenosis). The control group included 28 patients with BAV without valve dysfunction. All patients had 1 blood sample taken. Proportion of peripheral mononuclear cells expressing vascular endothelial growth factor receptor 2, CD133 and CD34 was evaluated by flow cytometry. EPC colony-forming units (CFUs) were grown from peripheral mononuclear cells, characterized, and counted after 7 days of culture. The 2 groups had similar clinical characteristics except for higher prevalence of hypertension in the dysfunctional valve group. Number of EPC CFUs was smaller in the dysfunctional valve group (32 CFUs/plate, 15 to 42.5, vs 48 CFUs/plate, 30 to 62.5, respectively, p = 0.01), and the migratory capacity of the cells in this group was decreased. In addition, the proportion of cells coexpressing vascular endothelial growth factor receptor 2, CD133, and CD34 tended to be smaller in the dysfunctional valve group. In conclusion, patients with BAV and significant valve dysfunction appear to have circulating EPCs with impaired functional properties. These findings require validation by further studies.
Bicuspid aortic valves (BAVs) with degeneration have macroscopic features that resemble those seen in “senile” aortic stenosis (e.g., heavily calcified incompetent and/or stenosed valve). These similarities raise the question of whether a shared mechanism of degeneration is involved. The pathogenesis of degenerative aortic stenosis shares certain aspects with atherosclerosis such as endothelial dysfunction and damage to the endothelial layer lining the valve or vessel. Because mature endothelial cells have a limited regenerative capacity, focus has shifted to endothelial progenitor cells (EPCs) as a potential source for endothelial regeneration and repair. Circulating EPCs coexpress CD133, CD34, and vascular endothelial growth factor receptor 2 (VEGFR-2) antigens on their surface and have the potential to differentiate into mature endothelial cells. Recent evidence has suggested these cells participate in the process of vascular repair by promoting re-endothelialization after injury. It is also well documented that the number and function of circulating EPCs are decreased in several atherosclerotic vascular diseases such as stable coronary artery disease, stroke, and peripheral vascular disease. Matsumoto et al recently showed that decreased regenerative capacity of valvular endothelial cells from increased senescence of cells and decreased number and function of circulating EPCs characterizes patients with degenerative severe aortic stenosis (patients with BAV were excluded from this study). We therefore hypothesized that EPCs may have an important role in the process of BAV endothelial layer regeneration and repair, and possibly impaired repair may contribute to development of significant valve dysfunction in patients with BAV. Accordingly, we examined whether patients with BAV who develop significant valve dysfunction have decreased EPC numbers and/or dysfunctional EPCs compared to patients with BAV without significant valve dysfunction.
Methods
Fifty patients with BAV were included in the study. The study group (dysfunctional valve) included 22 patients with BAV and echocardiographic evidence of aortic regurgitation and/or aortic stenosis with at least moderate severity. The control group included 28 patients with BAV without significant valve dysfunction (none or mild). All patients underwent echocardiography at the echocardiography unit of the Rabin Medical Center, Israel from 2009 through 2010. In all patients special notice was given to BAV appearance on 2-dimensional echocardiogram. Thickening of leaflets and presence of echogenic deposits (e.g., calcium deposits) were interpreted as valve degeneration. Exclusion criteria for the 2 groups included other valvular dysfunction (moderate or worse), anemia (hemoglobin <10 g/dl), renal insufficiency (creatinine ≥2.5 mg/dl), history of acute coronary syndrome or revascularization in the previous 3 months, or any type of malignant or hematologic disorder. The study was approved by the investigational review board (ethics committee) of the Rabin Medical Center, Israel, and all subjects provided written informed consent.
After overnight fasting, venous blood was drawn in the morning from an antecubital vein for EPC tests (blood drawn in heparinized tubes). Blood samples were processed within 1 hour of blood collection. Investigators performing the EPC assays were blinded to patient group allocation.
Circulating EPC levels were quantified by measurement of surface markers VEGFR-2, CD34, and CD133 by flow cytometry. Functional aspects of EPCs were evaluated by measurement of colony-forming units (CFUs) and by migration and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays of cultured cells after 7 days of culture. Peripheral mononuclear cells (PMNCs) were fractionated using Ficoll density-gradient centrifugation. PMNCs were isolated and washed with phosphate buffered saline after red cell lysis.
Isolated PMNCs were resuspended with Medium 199 (Invitrogen, Carlsbad, California) supplemented with 20% fetal calf serum (Gibco BRL Life Technologies, Gaithersburg, Maryland). Cells were then plated on 6-well plates coated with human fibronectin at a concentration of 5 × 10 6 cells/well. After 48 hours, nonadherent cells were collected and replated onto fibronectin-coated 24-well plates (10 6 cells/well). EPC colonies were counted using an inverted microscope 7 days after plating. An EPC colony was defined as a cluster of ≥100 flat cells surrounding a cluster of rounded cells. To confirm endothelial cell lineage, indirect immunostaining of randomly selected colonies was performed with antibodies directed against VEGFR-2, CD31 (Becton Dickinson, New Jersey) and Tie-2 (Santa Cruz Biotechnology, Santa Cruz, California), as we previously demonstrated. Results expressed as mean number of CFUs per plate.
Aliquots of PMNCs were incubated with monoclonal antibodies against VEGFR-2 (Fluorescein isothiocyanate (FITC) labeled; R&D, Minneapolis, Minnesota), CD45-CYT5.5 (Dako, Denmark), and CD133 (phycoerythrin labeled; Miltenyi Biotech, Auburn, California), or CD34 (phycoerythrin labeled, Miltenyi Biotech). Isotype-identical antibodies were used as controls. After incubation cells were washed with phosphate buffered saline and analyzed with a flow cytometer (FACSCalibur, Becton Dickinson). Each analysis included 100,000 events after selection for CD45 + cells (including low-intensity CD45 + cells) and exclusion of debris. In the next step gated CD34 + or CD133 + cells were examined for expression of VEGFR-2. Analyses were performed in duplicates. Results are presented as percent PMNCs coexpressing VEGFR-2 and CD133 or VEGFR-2 and CD34.
Migration of EPCs was measured by the modified Boyden migration assay (Greiner Bio-One, Borchen, Germany) in 10 randomly selected patients in each group, as previously described. After 7 days in culture cells were detached using trypsin ethylenediaminetetra-acetic acid, harvested by centrifugation, resuspended in endothelial basal medium, and counted, and 1 × 10 5 cells were placed in the upper part of the chamber. The chamber was placed in a 24-well culture dish containing endothelial basal medium and human recombinant VEGF (50 ng/ml; PeproTech, Asia). After 24 hours of incubation at 37°C, the lower side of the filter was washed with phosphate buffered saline and fixed with 2% paraformaldehyde. For quantification, cell nuclei were stained with 4′-6-Diamidino-2-phenylindole (Santa Cruz Technologies). Migrating cells in the lower chamber were counted manually in 3 random microscopic fields.
The MTT assay was performed in 10 randomly selected patients in each group to evaluate viability of cultured EPCs, as previously described. MTT measures mitochondrial activity in living cells. After 7 days of culture MTT (Sigma, St. Louis, Missouri) 1 mg/ml was added to the EPC medium culture and incubated for an additional 3 to 4 hours. After incubation the medium was removed and cells were solubilized in isopropanol. Amount of dye released from cells was measured with a spectrophotometer at 570 nm and subtracted background at 690 nm. An increase in the number of viable cells results in an increase of MTT formed and therefore in absorbance. Results were corrected for number of EPC CFUs.
EPC parameters (flow cytometrically determined levels, number of EPC CFUs, and results of functional assays) were non-normally distributed (as determined by Shapiro–Wilk normality test). Therefore, EPC data are presented as median (twenty-fifth to seventy-fifth percentiles), and comparisons between the 2 groups were performed by Mann–Whitney–Wilcoxon nonparametric 2-tailed test. Categorical variables were compared using chi-square tests. Analyses were performed using SPSS 15 (SPSS, Inc., Chicago, Illinois), and a p value <0.05 was considered statistically significant.
Results
Clinical characteristics and current medical treatment are presented in Table 1 . The 2 groups had similar characteristics except for a higher prevalence of hypertension in patients with dysfunctional valves. Accordingly, treatment with angiotensin-converting enzyme inhibitors or angiotensin receptor blockers was also more common in patients with dysfunctional valves. Echocardiographic parameters are presented in Table 2 . As determined by group allocation, patients with dysfunctional BAVs had significant (at least moderate) aortic regurgitation or aortic stenosis, whereas none of the patients in the normal function valve group had significant valve dysfunction. Of note 17 patients (77%) in the dysfunctional valve group had significant aortic regurgitation and 7 patients (32%) had significant aortic stenosis. Two patients had aortic regurgitation and stenosis. Patients in the dysfunctional valve group also had larger left ventricular diameters and mass and higher rates of aortic valve leaflet calcification and/or thickening (reflecting valve degeneration) than patients without valve dysfunction.
| Variable | Valve Function | |
|---|---|---|
| Normal (n = 28) | Abnormal (n = 22) | |
| Age (years) | 47 ± 12 | 49 ± 13 |
| Women | 10 (36%) | 5 (23%) |
| Body mass index (kg/m 2 ) | 26 ± 4 | 27 ± 5 |
| Diabetes mellitus | 1 (4%) | 2 (9%) |
| Hypertension ⁎ | 7 (25%) ‡ | 12 (55%) ‡ |
| Dyslipidemia † | 10 (36%) | 7 (32%) |
| Low-density lipoprotein (mg/dl) | 122 ± 25 | 119 ± 22 |
| Smoking (current or previous) | 11 (39%) | 7 (32%) |
| Previous myocardial infarction | 3 (11%) | 1 (5%) |
| Previous coronary bypass grafting | 1 (4%) | 1 (5%) |
| Previous percutaneous coronary intervention | 5 (18%) | 4 (18%) |
| Current medications | ||
| Aspirin | 7 (25%) | 8 (36%) |
| Statins | 9 (32%) | 7 (32%) |
| Angiotensin-converting enzyme inhibitors/angiotensin receptor blockers | 5 (18%) § | 12 (55%) § |
| β Blockers | 4 (14%) | 6 (27%) |
| Calcium blockers | 3 (11%) | 5 (23%) |
⁎ Diagnosis previously made by physician or use of antihypertensive medications.
† Diagnosis previously made by physician or use of lipid-lowering medications.
| Variable | Valve Function | p Value | |
|---|---|---|---|
| Normal | Abnormal | ||
| (n = 28) | (n = 22) | ||
| Significant (at least moderate) aortic regurgitation | 0 | 17 (77%) | <0.0001 |
| Significant (at least moderate) aortic stenosis | 0 | 7 (32%) | 0.001 |
| Aortic root diameter (mm) | 39 ± 7 | 41 ± 6 | 0.25 |
| Calcified and/or thickened aortic valve leaflets | 14 (50%) | 17 (77%) | 0.05 |
| Mitral annular calcium | 2 (7%) | 1 (5%) | 0.7 |
| Left ventricular end-diastolic diameter (mm) | 47 ± 4 | 55 ± 7 | <0.0001 |
| Left ventricular end-systolic diameter (mm) | 28 ± 4 | 35 ± 7 | 0.0003 |
| Ventricular septal diameter (mm) | 10 ± 2 | 12 ± 2 | 0.0002 |
| Left ventricular posterior wall diameter (mm) | 9 ± 2 | 11 ± 2 | 0.0002 |
| Left ventricular mass (g) | 155 ± 61 | 257 ± 71 | <0.0001 |
| Left ventricular mass index (g/m 2 ) | 78 ± 22 | 130 ± 34 | <0.0001 |
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