Published online before print March 1, 2007, doi: 10.1161/ATVBAHA.107.139626
© 2007 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Circulating Apoptotic Progenitor Cells
A Novel Biomarker in Patients With Acute Coronary Syndromes
From the Departments of Cardiology and Hematology (V.D.), Tel Aviv Sourasky Medical Center, Tel Aviv, Israel: Tel Aviv University (S.S., S.M.-A., S.K., G.K., J.G.), Sackler School of Medicine, Tel Aviv, Israel.
Correspondence to Jacob George, MD, The Department of Cardiology, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel. E-mail jacobg{at}post.tau.ac.il
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Background— Progenitor CD34 cells are capable of differentiating into endothelial cells and play a role in neoangiogenesis. Circulating CD34+ cells and endothelial progenitor cells are increased in acute coronary syndrome (ACS) patients possibly because of peripheral mobilization. We tested the hypothesis that circulating apoptotic progenitors are detectable in healthy subjects and altered in ACS patients.
Methods and Results— Peripheral blood mononuclear cells were isolated by Ficoll density gradient from 53 patients with ACS undergoing coronary angiography and 27 healthy subjects. Apoptosis in progenitor CD34+ cells was assessed using the Annexin V-PE/7-AAD detection kit, and fluorescence-activated cell sorter analysis was performed with triple staining for CD34, annexin-V, and 7-AAD. The percentage of apoptotic CD34+ progenitors was determined in the 2 subject groups and correlated with clinical characteristics. The percentage of apoptotic CD34+ progenitor cells was significantly increased in patients with ACS as compared with healthy subjects and was associated with the extent of coronary stenosis by angiography. There was no significant correlation between apoptotic progenitor CD34+ cells and the other parameters that we examined (age, smoking, hypertension, hyperlipidemia, diabetes mellitus, ejection fraction, creatinine levels, or taking any of the various medications, including beta blockers, thiazides, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, calcium blockers, nitrates, or statins).
Conclusion— We established for the first time to our knowledge an assay to detect circulating apoptotic progenitor cells using fluorescein isothiocyanate–anti-CD34 MAb, annexin V-PE, and 7-AAD and found that apoptotic CD34+ cells are increased in ACS patients and in patients with more extensive coronary artery disease. This novel assay may shed new light on the factors governing the hemeostasis of progenitor CD34+ cells.
We established for the first time to our knowledge an assay to detect circulating apoptotic progenitor cells using fluorescein isothiocyanate–anti-CD34 MAb, annexin V-PE, and 7-AAD and found that apoptotic CD34+ cells are increased in ACS patients and in patients with more extensive coronary artery disease. This novel assay may shed new light on the factors governing the hemeostasis of progenitor CD34+ cells.
Key Words: progenitor CD34+ stem cells • endothelial progenitor cells • endothelial microparticles • apoptosis • Annexin V
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
CD34+ bone marrow progenitor stem cells comprise only 1.5% of marrow mononuclear cells, with minor numbers of circulating cells in the peripheral blood. These cells contain precursors for all lymphohematopoietic lineages, as evidenced by the finding that CD34+ cells purified from marrow can reconstitute hematopoiesis of primates, humans, or mice undergoing autologous marrow reinfusion after myeloablative therapy.1 Recent studies have shown that expression of the CD34 surface antigen characterizes not only hematopoietic progenitor cells but also endothelial progenitor cells (EPCs) and mature endothelial cells.2
In 1997, Asahara et al3 isolated a circulating angioblast (later referred to as EPCs) from human peripheral blood of adults, with a potential to differentiate in vitro into endothelial cells and to contribute to neoangiogenesis after tissue ischemia. Shi et al4 were the first to show that a subset of transplanted bone marrow-derived CD34+ hematopoietic precursor cells participated in the endothelialization in a canine model of vascular graft healing, and thus have the capacity to transform eventually into mature endothelial cells. Expression of the stem cell marker CD34 is also found on a lower level on mature endothelial cells, and the search for more specific stem cell markers led to the discovery of CD133, which is expressed on immature stem cells but whose expression is lost during the differentiation to mature endothelial cells.5
EPCs are a heterogeneous group of cells that can be characterized by the expression of surface markers, such as CD34, CD133, and VEGFR-2 (KDR or Flk-1) in various combinations and, currently, precise phenotype definition is lacking.6
Circulating numbers of EPCs have been shown to negatively correlate with risk factors for atherosclerosis and with disorders associated with vascular dysfunction.7,8 Acute coronary syndrome (ACS) is associated with elevated numbers of circulating EPCs, suggesting that these cells are possibly mobilized in an attempt to participate in vessel repair after severe ischemia.9–11 Similarly, bone marrow-derived progenitor CD34+ cell numbers have been shown to be increased in ACS and acute myocardial infarction.11,12,13
Endothelial cell damage and apoptosis is associated with the release of small membrane particles, which are called endothelial microparticles.14 Elevated endothelial microparticles levels have been described in conditions of severe endothelial damage, including ACS15 and after myocardial infarction,16 and indicate increased apoptosis of endothelial cells. Furthermore, in patients with coronary artery disease, the number of circulating endothelial microparticles positively correlate with the severity of coronary endothelial dysfunction.14
Because bone marrow-derived CD34+ stem cells and EPCs are mobilized toward the peripheral blood in acute myocardial ischemia, we hypothesized that this condition may be associated also with their increased apoptosis resulting in a similar elevation of circulating apoptotic CD34+ cell levels. In this study, we identified, for the first time to our knowledge, the presence of a unique, yet uncharacterized, population of apoptotic progenitors and evaluated their numbers in patients with ACS.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Study Subjects
We studied a total of 53 patients with acute coronary syndrome who underwent coronary angiography and 27 healthy subjects, aged 25 to 60 years (median age, 35), including 13 males and 14 females. The Table summarizes demographic and clinical characteristics of the patient population. Institutional ethics committee approved the study and informed consent was obtained from all patients.
|
Preparation of Blood Samples
Peripheral blood mononuclear cells were isolated from 30 mL of freshly drawn heparinized blood using Isopaque-Ficoll (Amersham Biosciences, Buckinghamshire, United Kingdom) gradient centrifugation.
Flow Cytometry Evaluation of Early Apoptotic Progenitor CD34+ Cells
Apoptosis in progenitor CD34+ cells was assessed using SouthernBiotech ApoScreen Annexin V Apoptosis detection kit (Annexin V-PE, 7-AAD solution, and Annexin V binding buffer). In apoptotic cells, the membrane phospholipid phosphatidylserine is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing phosphatidylserine to the external cellular environment. Annexin V is a 36 kDa Ca2+-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine, and binds to cells with exposed phosphatidylserine.17 Annexin V-PE staining precedes the loss of membrane integrity, which accompanies the latest stages of cell death resulting from either apoptotic or necrotic processes. Therefore, staining with Annexin V-PE is used in conjunction with 7-AAD, which is a vital dye to identify early apoptotic cells before morphological changes associated with apoptosis have occurred and before membrane integrity has been lost. Cells that are viable are Annexin V-PE and 7-AAD–negative; cells that are in early apoptosis are Annexin V-PE–positive and 7-AAD–negative; and cells that are in late apoptosis or already dead are both Annexin V-PE–positive and 7-AAD–positive.18,19
The percentage of apoptotic CD34+ progenitor cells (out of total circulating progenitor CD34+ cells) was assessed by staining peripheral blood mononuclear cells for 3 color fluorescence-activated cell sorter analysis using fluorescein isothiocyanate–anti-CD34 MAb (IQ products), annexin V-PE, and 7-AAD (SouthernBiotech). After Ficoll gradient separation, peripheral blood mononuclear cells were washed with phosphate-buffered saline, and 106 cells were stained with fluorescein isothiocyanate–anti-CD34 MAb for 30 minutes at 4°C in 100 µL fluorescence-activated cell sorter staining buffer (phosphate-buffered saline and 2% fetal calf serum). The cells were washed again with phosphate-buffered saline and resuspended in 100 µL of Annexin V binding buffer and incubated with 10 µL of Annexin V-PE for 15 minutes at 4°C. Without washing, 380 µL of cold binding buffer and 10 µL of 7-AAD solution were added and 80 000 cells were acquired by flow cytometry (FACSCalibur; Becton Dickinson) and analyzed by CellQuest software (BD Bioscience). All analyses and readings were made by technicians that were blinded to the study questions. Intra-assay variability was never >10%, whereas interassay variability did not exceed 15% (when similar patients had samples performed within the same day).
Statistical Analysis
Comparison between apoptotic progenitor cells in healthy subjects and patients with ACS was performed using 1-way analysis of variance with age as covariant. Linear regression analysis was applied to the data to study the significance of the independent variables (age, smoking, hypertension, hyperlipidemia, diabetes mellitus, ejection fraction, creatinine levels, or taking any of the various medications, including beta blockers, thiazides, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers (ARBs), calcium blockers, nitrates, or statins, and the extent of coronary artery stenosis) on apoptotic progenitor CD34+ cells. Analysis was performed using the SAS for windows version 9.1 program (Chicago, Ill). Level of significance was set at P=0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Progenitor CD34+ cells were gated from the standard saline citrate (SSC)/CD34 dot plot according to the Milan protocol20,21 as shown in Figure 1A. Thereafter, apoptotic percentage of progenitor CD34+ cells was determined by fluorescence-activated cell sorter analysis of annexin V/ 7-AAD staining as shown in Figure 1B. Early apoptotic cells were defined as Annexin V-PE–positive and 7-AAD–negative, and thus consist of the population lying in the lower right quadrant in Figure 1B. The percentage of apoptotic CD34+ progenitor cells ±SEM (out of total circulating progenitor CD34+ cells) was significantly higher in the patients (24.5±3.8%) than in the control group (12.3±2.1%; P<0.05), as shown in Figure 2. Interestingly, we found that the percentage of apoptotic progenitor CD34+ cells correlated with the number of coronary vessels displaying significant stenosis (Figure 3A) and did not correlate with the presence of diabetes (Figure 3B). We did not find any significant correlation between apoptotic progenitor CD34+ cells and the other parameters that we examined (age, smoking, hypertension, hyperlipidemia, diabetes mellitus, ejection fraction, creatinine levels, or taking any of the various medications, including beta blockers, thiazides, angiotensin-converting enzyme inhibitors, ARBs, calcium blockers, or statins).
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Previous studies have shown that ACS results in mobilization of progenitor cells, including endothelial progenitor cells, as well as with an increase in circulating endothelial microparticles representing mature endothelial cell damage and apoptosis. In this study, we have established an assay in which apoptotic progenitor cells were identified and quantified in the peripheral blood. We have found that apoptotic progenitor CD34+ cells are increased in ACS patients in comparison with healthy volunteers. Although control subjects and ACS patients were not age-matched, when multivariate statistical analysis was performed, age was not found to be an independent influencing factor on the percentage of apoptotic CD34+ progenitor cells in the patient group. Furthermore, we have found that the percentage of circulating apoptotic CD34+ progenitors correlated with the number of coronary vessels displaying significant atherosclerotic narrowing evident by coronary angiography and with smoking status in ACS patients.
Progenitor cells are important precursors of mature endothelial cells that are essential components of angiogenesis and vasculogenesis. These cells that are mobilized from the bone marrow appear to be destroyed by apoptosis and their circulating numbers appear to be controlled by these 2 processes of mobilization versus apoptotic cell death. Factors favoring peripheral apoptosis are expected to result in reduction in circulating CD34+ cell numbers with subsequent attenuation in the angiogenic capacity that is responsible for the replenishment of dysfunctional endothelium. This could explain a potential causal effect of the increased apoptotic progenitors on the destabilization of the atheroma resulting in ACS. Alternatively, increased oxidative stress known to be present in ACS could be responsible for the increased apoptosis of progenitor CD34+ cells.
Apoptosis is indeed a major determinant of atherosclerotic plaque vulnerability and thus a key player in ACS induction.22,23 Thus, our findings can be explained by increased pro-apoptotic mediators in ischemic heart disease including reactive oxygen species,24,25 oxidized low-density lipoprotein,25,26 high levels of nitric oxide,27,28 and inflammatory cytokines produced by the activated macrophages and T-lymphocytes (including tumor necrosis factor-
, interferon-
, and IL-1β).23 In particular, oxidative stress and cigarette smoke extracts were found to increase Fas ligand and caspase-3–mediated endothelial cell apoptosis.29,30
Regardless of whether apoptotic CD34+ numbers represent a predisposition for plaque destabilization or are byproducts of increased oxidative stress, the finding and identification of this cell population shed new light on the mechanisms governing progenitor cell turnover. It should be mentioned, however, that the relatively small number of circulating CD34+ cells still poses a question of the true functional importance of these cells. In several recent studies,7–11 small numbers (in the range of 0.01% of peripheral mononuclear cells) of circulating endothelial progenitors have been associated with reduced angiogenic capacity. These reports support the importance of even trivial numbers of progenitors, yet additional research should lend proof to this assumption.
In summary, we describe a novel assay to detect and quantify circulating apoptotic CD34+ cells in healthy subjects and in patients with ACS. This assay may help elucidate the factors that control peripheral progenitor cell hemostasis.
|
Acknowledgments |
|---|
Disclosures
None.
|
Footnotes |
|---|
S.S. and V.D. contributed equally to this study.
Original received October 28, 2006; final version accepted February 9, 2007.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Krause DS, Fackler MJ, Civin CI, May WS. CD34: structure, biology, and clinical utility. Blood. 1996; 87: 1–13.
2. Zammaretti P, Zisch AH. Adult "endothelial progenitor cells." Renewing vasculature. Int J Biochem Cell Biol. 2005; 37: 493–503.[CrossRef][Medline] [Order article via Infotrieve]
3. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997; 275: 964–967.
4. Shi Q, Rafii S, Wu MH, Wijelath ES, Yu C, Ishida A, Fujita Y, Kothari S, Mohle R, Sauvage LR, Moore MA, Storb RF, Hammond WP. Evidence for circulating bone marrow-derived endothelial cells. Blood. 1998; 92: 362–367.
5. Peichev M, Naiyer AJ, Pereira D, Zhu Z, Lane WJ, Williams M, Oz MC, Hicklin DJ, Witte L, Moore MA, Rafii S. Expression of VEGFR-2 and AC133 by circulating human CD34(+) cells identifies a population of functional endothelial precursors. Blood. 2000; 95: 952–958.
6. Hristov M, Weber C. Endothelial progenitor cells: characterization, pathophysiology, and possible clinical relevance. J Cell Mol Med. 2004; 8: 498–508.[Medline] [Order article via Infotrieve]
7. Vasa M, Fichtlscherer S, Aicher A, Adler K, Urbich C, Martin H, Zeiher AM, Dimmeler S. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res. 2001; 89: E1–7.[Medline] [Order article via Infotrieve]
8. Hill JM, Zalos G, Halcox JP, Schenke WH, Waclawiw MA, Quyyumi AA, Finkel T. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med. 2003; 348: 593–600.
9. Shintani S, Murohara T, Ikeda H, Ueno T, Honma T, Katoh A, Sasaki K, Shimada T, Oike Y, Imaizumi T. Mobilization of endothelial progenitor cells in patients with acute myocardial infarction. Circulation. 2001; 103: 2776–2779.
10. George J, Goldstein E, Abashidze S, Deutsch V, Shmilovich H, Finkelstein A, Herz I, Miller H, Keren G. Circulating endothelial progenitor cells in patients with unstable angina: association with systemic inflammation. Eur Heart J. 2004; 25: 1003–1008.
11. Massa M, Rosti V, Ferrario M, Campanelli R, Ramajoli I, Rosso R, De Ferrari GM, Ferlini M, Goffredo L, Bertoletti A, Klersy C, Pecci A, Moratti R, Tavazzi L. Increased circulating hematopoietic and endothelial progenitor cells in the early phase of acute myocardial infarction. Blood. 2005; 105: 199–206.
12. Leone AM, Rutella S, Bonanno G, Abbate A, Rebuzzi AG, Giovannini S, Lombardi M, Galiuto L, Liuzzo G, Andreotti F, Lanza GA, Contemi AM, Leone G, Crea F. Mobilization of bone marrow-derived stem cells after myocardial infarction and left ventricular function. Eur Heart J. 2005; 26: 1196–1204.
13. Wojakowski W, Tendera M, Michalowska A, Majka M, Kucia M, Maslankiewicz K, Wyderka R, Ochala A, Ratajczak MZ. Mobilization of CD34/CXCR4+, CD34/CD117+, c-met+ stem cells, and mononuclear cells expressing early cardiac, muscle, and endothelial markers into peripheral blood in patients with acute myocardial infarction. Circulation. 2004; 110: 3213–3220.
14. Werner N, Wassmann S, Ahlers P, Kosiol S, Nickenig G. Circulating CD31+/annexin V+ apoptotic microparticles correlate with coronary endothelial function in patients with coronary artery disease. Arterioscler Thromb Vasc Biol. 2006; 26: 112–116.
15. Bernal-Mizrachi L, Jy W, Jimenez JJ, Pastor J, Mauro LM, Horstman LL, de Marchena E, Ahn YS. High levels of circulating endothelial microparticles in patients with acute coronary syndromes. Am Heart J. 2003; 145: 962–970.[CrossRef][Medline] [Order article via Infotrieve]
16. Boulanger CM, Scoazec A, Ebrahimian T, Henry P, Mathieu E, Tedgui A, Mallat Z. Circulating microparticles from patients with myocardial infarction cause endothelial dysfunction. Circulation. 2001; 104: 2649–2652.
17. Raynal P, Pollard HB. Annexins: the problem of assessing the biological role for a gene family of multifunctional calcium- and phospholipid-binding proteins. Biochim Biophys Acta. 1994; 1197: 63–93.[Medline] [Order article via Infotrieve]
18. Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood. 1994; 84: 1415–1420.
19. Martin SJ, Reutelingsperger CP, McGahon AJ, Rader JA, van Schie RC, LaFace DM, Green DR. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med. 1995; 182: 1545–1556.
20. Cabezudo E, Querol S, Cancelas JA, Garcia J. Comparison of volumetric capillary cytometry with standard flow cytometry for routine enumeration of CD34+ cells. Transfusion. 1999; 39: 864–872.[CrossRef][Medline] [Order article via Infotrieve]
21. Siena S, Bregni M, Brando B, Belli N, Ravagnani F, Gandola L, Stern AC, Landsdorp PM, Bonadonna G, Gianni AM. Flow cytometry for clinical estimation of circulating hematopoietic progenitors for autologous transplantation in cancer patients. Blood. 1991; 77: 400–409.
22. Mitra AK, Dhume AS, Agrawal DK. "Vulnerable plaques"–ticking of the time bomb. Can J Physiol Pharmacol. 2004; 82: 860–871.[CrossRef][Medline] [Order article via Infotrieve]
23. Stoneman VE, Bennett MR. Role of apoptosis in atherosclerosis and its therapeutic implications. Clin Sci (Lond). 2004; 107: 343–354.[Medline] [Order article via Infotrieve]
24. Galle J. [Atherosclerosis and arteriitis: implications for therapy of cardiovascular disease]. Herz. 2004; 29: 4–11.[CrossRef][Medline] [Order article via Infotrieve]
25. de Nigris F, Lerman A, Ignarro LJ, Williams-Ignarro S, Sica V, Baker AH, Lerman LO, Geng YJ, Napoli C. Oxidation-sensitive mechanisms, vascular apoptosis and atherosclerosis. Trends Mol Med. 2003; 9: 351–359.[CrossRef][Medline] [Order article via Infotrieve]
26. Rusinol AE, Thewke D, Liu J, Freeman N, Panini SR, Sinensky MS AKT/protein kinase B regulation of BCL family members during oxysterol-induced apoptosis. J Biol Chem. 2004; 279: 1392–1399.
27. Chung HT, Pae HO, Choi BM, Billiar TR, Kim YM. Nitric oxide as a bioregulator of apoptosis. Biochem Biophys Res Commun. 2001; 282: 1075–1079.[CrossRef][Medline] [Order article via Infotrieve]
28. Patel RP, Levonen A, Crawford JH, Darley-Usmar VM. Mechanisms of the pro- and anti-oxidant actions of nitric oxide in atherosclerosis. Cardiovasc Res. 2000; 47: 465–474.
29. Suzuki M, Aoshiba K, Nagai A. Oxidative stress increases Fas ligand expression in endothelial cells. J Inflamm (Lond). 2006; 3: 11–19.[CrossRef][Medline] [Order article via Infotrieve]
30. Wang J, Wilcken DE, Wang XL. Cigarette smoke activates caspase-3 to induce apoptosis of human umbilical venous endothelial cells. Mol Genet Metab. 2001; 72: 82–88.[CrossRef][Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  D. Tang, J. Lu, J. P. Walterscheid, H.-H. Chen, D. A. Engler, T. Sawamura, P.-Y. Chang, H. J. Safi, C.-Y. Yang, and C.-H. Chen Electronegative LDL circulating in smokers impairs endothelial progenitor cell differentiation by inhibiting Akt phosphorylation via LOX-1 J. Lipid Res., January 1, 2008; 49(1): 33 - 47. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N.-K. Hsieh and H. I. Chen Interacting Leukocytes Predict Atherosclerosis and Restenosis Stroke, December 1, 2007; 38(12): e162 - e163. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||