Abstract
Objective. Endothelial progenitor cells (EPC) are a rare population of circulating cells involved in vascular homeostasis. Our aim was to analyze EPC in patients with mixed cryoglobulinemia (MC).
Methods. EPC were evaluated by cytometry according to guidelines of the International Society for Hematotherapy and Graft Engineering in 17 patients with MC and 36 controls.
Results. Numbers of EPC were significantly increased in MC compared to controls and correlated with cryocrit, but not with clinical manifestations of the disease.
Conclusion. The high number of EPC might indicate an intact vascular repair ability. Alternatively, EPC might be defective in homing ability and their increase may represent the attempt to restore vascular integrity.
The peripheral blood of adults contains a small population of bone marrow-derived endothelial progenitor cells (EPC) that are able to differentiate into mature endothelial cells and thus are involved in endothelial repair and vasculogenesis1. In patients with coronary artery disease, numbers of EPC are inversely correlated with cardiovascular risk factors2, and this has an important prognostic value, that is, to predict the occurrence of cardiovascular events and death from cardiovascular causes3. EPC have been shown to home to sites of vascular damage and to contribute to the revascularization of ischemic areas4.
A role of EPC in vascular repair in patients with vasculitis has also been proposed, but information on this is limited. In antineutrophil cytoplasmic antibody-associated vasculitis5 and in Kawasaki disease6 EPC are significantly increased in patients that, after immunosuppressive therapy, have low disease activity.
We analyzed levels of EPC by flow cytometry in a group of patients with mixed cryoglobulinemia (MC).
MATERIALS AND METHODS
Patients
Seventeen patients (12 women, 5 men, mean age 72 yrs, range 47–85 yrs) with MC followed in the clinical immunology and rheumatology units were recruited consecutively. MC was diagnosed in the presence of Meltzer’s triad (purpura, weakness, and arthralgia) and cryoglobulins in the sera. A full clinical and serological evaluation was performed in all patients, including measurement of complement and cryocrit (percentage of packed cryoglobulins after centrifugation of serum at 4°C).
Thirty-six healthy laboratory personnel (26 women, 10 men, mean age 40 yrs, range 20–56 yrs) served as controls.
Detection of EPC
EPC were assessed by flow cytometry in EDTA-preserved whole blood7–9. Briefly, the blood sample was stained within 2 h after collection by anti-CD34 PerCP-Cy5,5 (Becton Dickinson, Franklin Lakes, NJ, USA); anti-VEGFR2-FITC (R&D Systems, Minneapolis, MN, USA); and anti-CD133-PE (Miltenyi Biotec, Bergisch Gladbach, Germany). After 30 min, erythrocytes were lysed by ammonium chloride and the samples were run in the flow cytometer.
In parallel samples, whole blood (0.1 ml) was added to a tube containing a predefined number of fluorescent beads (Trucount, Becton Dickinson), stained for 30 min with anti-CD34-PE and anti-CD45-FITC (Becton Dickinson), and run in the flow cytometer after erythrocyte lysis. Following modified guidelines of the International Society for Hematotherapy and Graft Engineering (ISHAGE)9,10, the use of defined numbers of fluorospheres allowed measurement of the absolute number of CD34+ cells. The number of EPC was then obtained by calculating the percentage of CD34+ cells that coexpressed CD133 and VEGFR2.
Nonparametric assays were used for statistical analysis (Mann-Whitney test and Spearman correlation coefficient as appropriate).
RESULTS
MC was associated with hepatitis C virus in 16 patients and hepatitis B virus in 1 patient. C4 levels were low in 16 patients and in 8 of them C3 was also reduced. Thirteen patients had liver involvement and 3 developed cirrhosis; renal involvement was diagnosed in 4 patients. Eight patients had sicca syndrome and 3 Raynaud’s phenomenon. Nine patients were treated with low-dosage steroids (< 10 mg prednisone equivalent), in 1 associated with mycophenolate mofetil; 11 patients received antihypertensive drugs (Ca2+ channel blockers, angiotensin-converting enzyme inhibitors) that maintained blood pressure in the normal range.
Levels of EPC were significantly increased in patients with MC compared to controls (median value 84 vs 52 cells/ml; p = 0.032; Figure 1). A direct correlation was observed with cryocrit (p = 0.02; Figure 2), but not with levels of complement. EPC levels did not differ in subgroups of patients with different types of organ involvement or in patients treated with steroids compared to untreated patients, and were not related to disease duration or to the presence of active vasculitis.
Number of EPC/ml blood was measured in controls and MC patients; number of EPC was significantly higher in MC patients (Mann-Whitney test).
Positive correlation between cryocrit (percentage of packed cryoglobulins) and EPC level in MC patients (Spearman rank correlation test).
In particular, the 4 MC patients displaying the highest numbers of EPC were not homogeneous in terms of treatment, disease phenotype, or disease activity, and they did not differ significantly from the remaining patients.
DISCUSSION
Our findings indicate that patients with MC were characterized by high numbers of EPC, which were correlated with cryocrit data and were not related to other serological or clinical disease measures.
Similar results have been reported in other small and large-vessel vasculitides5,6, but comparison with data obtained from patients with other disorders is difficult. Different methods for evaluation of EPC are currently employed, which probably detect distinct subsets of endothelial cell and thus are not highly correlated11. EPC can be detected by functional assays, based on their capacity to form endothelial cell colonies, or by phenotypic characterization, taking advantage of membrane antigen expression. In patients with vasculitis, Nakatani, et al6 detected EPC as CD133-positive cells out of isolated endothelial cells, whereas de Groot, et al5 performed an in vitro functional assay.
In our study, the first report on numbers of EPC in MC, we followed the ISHAGE guidelines for enumeration of stem cells; using a known amount of fluorescent microbeads and flow cytometry, we determined an absolute amount of CD34+ cells coexpressing VEGFR2 and CD133. This combination of surface markers is widely accepted for identification of “true” EPC in humans; but recent data suggest that isolated CD34+ VEGFR2+ CD133+ cells do not generate new endothelial cells, but instead represent hematopoietic-derived cells that indirectly contribute to vascular homeostasis12.
In patients with MC, high numbers of EPC are observed despite long disease duration and advanced age, conditions usually associated with a decline of EPC13,14. In this respect, it is important to stress that the control group we studied had a lower mean age than the MC group (40 vs 72 yrs): thus, a “high” number of EPC was expected in the controls.
The correlation of number of EPC with amount of cryoglobulin is an interesting and unexpected finding: cryocrit, in fact, is not related to the activity or severity of the disease. This correlation may suggest a more extensive endothelial damage directly mediated by cryoglobulins, or due to hyper-viscosity that may induce a higher bone marrow output of EPC; alternatively, higher amounts of cryoglobulins may interfere with the homing of EPC and thus increase their amount in the circulation.
Thus, the high numbers of EPC might indicate an intact vascular repair ability or represent an attempt to counteract a defect in homing. Followup studies have been planned to clarify whether the number of EPC changes with disease activity, and functional assays will be performed to verify the homing ability of EPC in patients with MC.
Footnotes
- Accepted for publication February 9, 2010.
