|Year : 2018 | Volume
| Issue : 4 | Page : 171-178
Role of circulating endothelial cells and platelet microparticles as markers of angiogenesis in chronic myeloid leukemia
Asmaa Nafady1, Mostafa F. Mohammed Saleh2, Hanaa Nafady-Hego3, Mohammed M Wahman4, Khalid A Nasif5, Wael F Sedik5
1 Department of Clinical Pathology, Faculty of Medicine, Assiut University, Assiut; Department of Clinical and
Chemical Pathology, South Valley University, Qena, Egypt
2 Clinical Hematology Unit, Internal Medicine Department, Faculty of Medicine, Assiut University, Assiut, Egypt
3 Department of Microbiology and Immunology, Faculty of Medicine, Assiut University, Assiut, Egypt
4 Department of Clinical Oncology, Qena Faculty of Medicine, Qena University Hospital, South Valley University, Qena, Egypt
5 Department of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Minia University, Minia, Egypt
|Date of Submission||06-Jun-2018|
|Date of Acceptance||18-Jul-2018|
|Date of Web Publication||10-Apr-2019|
Clinical Pathology Department, Assiut University, Assiut 71526
Source of Support: None, Conflict of Interest: None
Background Circulating endothelial cells (CECs) and platelet microparticles (PMPs) are proposed as useful biosensors for angiogenesis and membrane damage in cancer. Moreover, PMPs can modulate cellular and humoral immunity.
Objective To measure CEC and PMP levels in patients with chronic myeloid leukemia (CML) with and without imatinib therapy.
Patients and methods Peripheral blood samples were obtained from 30 patients with CML at diagnosis (group A), 30 patients with CML on imatinib therapy of at least 1 year (group B), and 20 healthy controls (group C). Flow cytometry techniques were used to quantify CEC and PMP levels.
Results PMP percentage significantly increased in groups A and B when compared with group C (P=0.001 and 0.001, respectively). Mean±SEM of groups A, B, and C was 48.67±2.88, 42.50±2.82, and 22.70±1.18, respectively. There was an increased number of CECs in group A and B when compared with controls (P=0.001 and 0.001, respectively). Mean±SEM of groups A, B, and C was 149.33±23.82, 70.96±9.58, and 22.70±1.18, respectively. Patients with advanced phase or higher risk disease had slightly more PMPs and CECs than patients with chronic phase or low risk. Patients on imatinib therapy who achieved a complete molecular response at 1 year showed fewer PMPs and CECs.
Conclusion Higher PMPs and CECs number in patients with CML at diagnosis could indicate their pathogenic role as angiogenesis markers. However, their role of being prognostic factors and predictors of response to therapy in CML needs larger prospective studies.
Keywords: angiogenesis, chronic myeloid leukemia, circulating endothelial cells, platelet microparticles
|How to cite this article:|
Nafady A, Saleh MF, Nafady-Hego H, Wahman MM, Nasif KA, Sedik WF. Role of circulating endothelial cells and platelet microparticles as markers of angiogenesis in chronic myeloid leukemia. Egypt J Haematol 2018;43:171-8
|How to cite this URL:|
Nafady A, Saleh MF, Nafady-Hego H, Wahman MM, Nasif KA, Sedik WF. Role of circulating endothelial cells and platelet microparticles as markers of angiogenesis in chronic myeloid leukemia. Egypt J Haematol [serial online] 2018 [cited 2020 Mar 31];43:171-8. Available from: http://www.ehj.eg.net/text.asp?2018/43/4/171/255869
| Introduction|| |
Chronic myeloid leukemia (CML) is a chronic leukemia of the hematopoietic stem cells that results from a translocation of the Philadelphia chromosome in stem cells of the bone marrow. The latter causes the fusion of a BCR-ABL gene encoding for fusion proteins of tyrosine-kinase activity involving the phosphorylation of several substrates activating multiple signal-transduction cascades involved in cell proliferation and differentiation .
Angiogenesis and suppression of the T-cell-mediated immunity play a crucial role in the pathogenesis of malignancies through enhancement of tumor growth, metastasis, and invasion . Moreover, higher levels of proangiogenic factors have been associated with a poor outcome in those malignancies. In addition, returning of higher marrow vascularity to their normal levels was associated with better response to therapeutics in some of these illnesses .
Platelets play a central role in the pathogenesis of CML, as we suppose, owing to many reasons. First, they are the main regulators of hemostasis, and second, platelets can modulate the immune responses through production of intracellular mediators as cytokines, chemokines, and surface receptors (e.g. integrins, P-selectin, CD40L, junctional adhesion molecule-A, intercellular adhesion molecule-2, CD44, Toll-like receptors, and chemokine receptors). These suggest that platelets not only control innate immunity but also control adaptive immunity through cell-mediated immunity signals that control and enhance the humoral immunity. Taking all together, platelets represent a bridge between immunity and coagulation ,,. To perform their function, tiny vesicles of plasma membrane known as platelet-derived microparticles (PMPs) shed from cells after their activation and circulate in the blood. These PMPs can act as a messenger for platelets as they are found to express and transfer the previously mentioned functional receptors from platelet membranes to the circulation. In addition, PMPs increase the release of cytokines and stimulate the expression on adhesion molecules on cells, which in turn activate intracellular signaling pathways; and cause induction of angiogenesis and alteration of vascular reactivity. All together, PMPs can be involved in metastasis formation . Although recently discovered to be positively correlated with aggressive tumors and a poor prognosis, the role of PMPs in cancer development is still elusive .
Another marker that is thought to play a role in the pathogenesis of malignancies is circulating endothelial cells (CECs), which are mature cells that have been released from the intimal layer after trauma, hemodynamics abnormality or infection. An increase in the number of CECs has been reported in different hemato-oncologic diseases . High levels of CECs were correlated with more aggressive disease and shorter time to first treatment in chronic lymphocytic leukemia, and also related to response to therapy and disease progression in acute myeloid leukemia .
In vitro, PMPs have similar proangiogenic and prometastatic potential as live platelets and increase EC migration and tube formation as well as promote tumor cell matrix metalloproteinases production and invasion through Matriagel (BD Biosciences. Trevigen, Inc.) .
The aim of this study was to evaluate PMP and CEC levels in different phases of CML, including in patients who did not start treatment and patients who are on therapy; all the findings were compared with those of healthy participants. Our hypothesis was that PMPs and CECs as angiogenic and immune modulating models are related to CML development, with a potential prognostic role in CML and predictive value for a response to treatment.
| Patients and methods|| |
Following the approval of the Ethics Board of Faculty of Medicine, Assiut University, 60 patients with CML and 20 healthy volunteers were enrolled in the study after providing written informed consents.
This study included three study groups: group A of 30 patients with newly diagnosed untreated CML, group B of 30 patients with CML who received imatinib 400 mg as a first-line treatment for at least 1 year with available records of their BCR-ABL monitoring by RQ-PCR international scale, and group C of 20 age-matched and sex-matched healthy controls.
Patients were recruited from the Clinical Hematology Unit, Internal Medicine Department, Assiut University, Assiut, from May 2015 to March 2018. All patients were diagnosed and classified according to the WHO classification of myeloproliferative disorders after evaluation of peripheral blood, bone marrow aspirate, bone marrow biopsy, karyotyping, and molecular analysis. Response to therapy in treated group after 1 year of imatinib 400 mg was evaluated according to European Leukemia Net recommendations for management of CML, with complete molecular response (CMR) defined as ‘undetectable’ BCR-ABL by RQ-PCR .
Approximately 5 ml of venous blood was withdrawn from both patients and controls under complete aseptic conditions then divided into two types of tubes (K EDTA tube and Citrate tube); 3 ml in K3 EDTA anticoagulant tubes for complete blood count, peripheral blood smears, and flow cytometric imunophenotyping analysis.
Platelet microparticles detection
Two milliliter is inserted into a 5 ml tube containing 3.2% citrate. To isolate the PMPs, cells were removed by centrifugation for 20 min at 1550g at 20°C within 15 min after collection. Then, 250 μl of plasma was centrifuged for 30 min at 18800g at 20°C. After centrifugation, the supernatant was removed and the pellet was resuspended in phosphate buffer saline (PBS) and centrifuged for 30 min at 18800g at 20°C. The supernatant was removed again, and MPs pellet was resuspended in PBS .
Flow cytometric analysis was used to quantify and characterize PMPs. Overall, 5 μl of PMP sample was diluted in 35 μL of PBS containing 2.5 mmol/L calcium chloride. The samples were then incubated for 20 min at room temperature in the dark with 5 μl of anti-CD41a-phycoerythrin (PE), purchased from Becton Dickinson Pharmingen (San Diego, CA). PBS and red latex beads measuring 1.0 mm in diameter (Sigma-Aldrich, Gillingham, UK) were added to the sample immediately before flow cytometric analysis. PMPs were defined as particles less than the mean diameter of the latex beads and showing positive binding to anti-CD41a. PMPs were reported as a percentage of the total platelet count .
Circulating endothelial cells detection
Overall, 50 UL of EDTA- blood was incubated with fluorescein 6-isothiocyanate conjugated anti- CD45 and phycoerythrin conjugated anti-CD146 purchased from Becton Dickinson Pharmingen (San Diego, CA), for 15 min at 4°C in the dark. The lysing solution was added, the tube was recentrifuged, and the supernatant was discarded. Then PBS was added and another centrifugation performed, the supernatant was discarded, and 0.5–1 ml of PBS was added. An unstained control was also processed in a similar manner to exclude autofluorescence. CECs were defined as cells that are CD45 negative and CD146 positive: gating criteria were set with isotype controls .
The clinical and laboratory data were categorized and processed by statistical package for the social sciences (SPSS: An IBM Company, version 16.0, IBM Corporation, Armonk, NY, USA), version 16, software package. The results were expressed as the mean±SEM and as number and percentages. For statistical evaluation, one-way analysis of variance, the Bonferroni test, was used to compare continuous variables among the three groups. Categorical data were analyzed by the χ2-test. P value less than 0.05 was considered statistically significant.
| Results|| |
[Table 1] outlines the characteristics of the three groups tested in this study. In group A, mean age was 41±2.9 years, mean WBC count was 206.74±29.04×109/L, the mean blast percentage was 6.35±0.93%, and mean platelet count was 391.50±63.43×109/L. In group B, mean age was 39±1.5 years, mean WBC count was 10.5±0.5×109/L, mean blast percentage was 1.1±0.02%, and mean platelet count was 391.50±63.43×109/L. In group C, mean age was 37±1.72 years, mean WBC count was 6.44±0.43×109/L, blast percentage was 0%, and mean platelet count was 231.25±15.70×109/L.
In group A, 23 (76.6%) patients were in chronic phase and five (16.6%) and two (6.8%) patients were in accelerated and blastic crisis, respectively. According to Sokal score, in group A, nine (30%) patients were classified into low risk, 15 (50%) patients into intermediate risk, and six (20%) patients into high risk. Among the patients in group B who were treated by imatinib 400 mg, CMR at 1 year was achieved in 10 (33.3%) patients ([Table 1]).
The percentage of PMPs was elevated in patients with CML (both groups A and B) compared with healthy controls. The mean±SE percentage of PMPs was 22.70±1.18% in controls and significantly higher in group A (48.67±2.88%, P=0.001) and group B (42.50±2.82%, P=0.001). No difference was noted between groups A and B (P=0.139), as shown in [Figure 1].
|Figure 1 Frequency of Platelet microparticles (PMPs) in CML patients on diagnosis, and after therapy and healthy controls.|
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There was an increased number of CECs in group A (149.33±23.82) compared with group B (70.96±9.58; P=0.025) and compared with normal controls (22.70±1.18; P=0.001). Moreover, CECs were statistically significantly higher in group B than in group C (P=0.001) ([Figure 2]).
|Figure 2 Circulating endothelial cells numbers in CML patients on diagnosis, and after therapy and healthy controls.|
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As shown in [Figure 3] and [Figure 4], patients with advanced phase or higher risk disease (intermediate and high Sokal scores) had more PMPs and CECs than patients with chronic phase or low risk, but this was not statistically significant. Patients on imatinib therapy who achieved CMR at 1 year showed fewer PMPs and CECs compared with those who did not; however, this was not statistically significant ([Figure 5]).
|Figure 3 Analysis of PMPs and CECs in newly diagnosed CML patients according to different disease phases.|
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|Figure 4 Analysis of PMPs and CECs in newly diagnosed CML patients according to different Sokal risk score phases.|
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|Figure 5 Analysis of PMPs and CECs in CML patients in relation to therapy response.|
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| Discussion|| |
Despite the marked improvement that has been achieved in the last few decades in the management of CML with the introduction of tyrosine kinase inhibitors (TKIs), disease progression and development of TKIs resistance are evolving issues, indicating different pathophysiologic mechanisms contributing to disease biology still not uncovered yet.
The main finding of our study, elevation of PMPs and CECs in CML patients compared with controls, suggests a possible contribution of PMPs and CECs in CML biology through enhancement of angiogenesis.
In this study, the percent of PMPs was significantly higher in patients with CML at diagnosis compared with healthy controls, whereas no difference was detected between patients who attained CMR after imatinib therapy and those who did not. In accordance with our finding, Zhu et al.  found the expression of BCR-ABL in microparticles by PCR in 29 patients with CML decreased in patients with CMR compared with patients with the major molecular response and complete cytogenetic effect. In addition, patients with CML with the CMR, BCR-ABL levels in microparticles but not in the peripheral cells were decreased in patients treated with imatinib compared with patients after stem cell transplantation.
Patients with CML had significantly higher angiogenesis parameters by measuring microvascular density and a number of different sizes-related and shape-related morphometric parameters of microvessels of bone marrow biopsy when compared with controls. Prognostic significance of the degree of angiogenesis had been demonstrated for the clinical outcome and identified angiogenic predictive factors for achieving optimal response on TKIs therapy .
Platelets play a crucial role in the development of cancer, either by inhibition of immune system or by induction of angiogenesis in the tumors. The increasing incidence of TKI resistance, particularly, imatinib resistance, increases the need for new therapeutics that can target angiogenesis in CML .
In the present study, we found that CECs are higher in CML at diagnosis compared with controls and patients on therapy. No difference was noted between different disease phases or between those who achieved CMR versus those who did not . A group of researchers conducted quantification of CECs in peripheral blood of 84 patients with CML and showed that patients in the blastic phase had significantly higher percentages of CECs than the control and accelerated phase groups. However, the CEC percentages were comparable among chronic phase, active phase, and the healthy participants, which was similar to our finding .
Several reports suggest that endothelial cells in CML could belong to the leukemic clone. CML cells release MPs that are able to stimulate the process of vascularization by human vascular endothelial cells, stimulating the expression of both intercellular adhesion molecule-1 and VCAM-1 adhesion molecules. Interestingly, MPs released by CML cells can mediate the transfer of BCR-ABL mRNA to endothelial cells .
Such findings by our group and others indicate a potential role of PMPs and CECs with possible interaction between them in inducing angiogenesis in patients with CML and as a part of disease biology. Extensive studies are going on evaluating the possible prognostic effect and predictive value for TKIs therapy.In CML, bone marrow angiogenesis was recently linked to TKI resistance that favors tumor growth and is associated with poor outcome . Hence, several therapeutic strategies are focusing on the MPs as a novel target. Inhibition of exosomal transport has been shown to enhance the anticancer efficacy of chemotherapeutic drugs. Studies have shown that these dendritic cells, which presents cancer-associated antigens to the cytotoxic T cells, upon being loaded with leukemic MPs, enhance the cytotoxicity of T cells against cancer cells . Moreover, it is important to stimulate CD4-dependent humoral immunity and subsequent production of tumor-specific IgG, the formation of germinal center, and production of B memory cells .
In a recent study, the proangiogenic activity of exosomes released by CML cells can be pharmacologically modulated; thus, the anticancer drug curcumin considerably attenuates the exosome’s ability to promote an angiogenic phenotype, by preferentially shutting MicroRNA-21 (miR-21) in these microvesicles .
| Conclusion|| |
A better understanding of the mechanisms controlling angiogenesis in CML is warranted to define an efficient strategy to therapeutically inhibit the tumoral angiogenesis mediated by PMPs and CECs.
The authors acknowledge all participants in this work.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Jabbour E, Kantarjian H. Chronic myeloid leukemia: 2018 update on diagnosis, therapy and monitoring. Am J Hematol
O’Byrne KJ, Dalgleish AG, Browning MJ, Steward WP, Harris AL. The relationship between angiogenesis and the immune response in carcinogenesis and the progression of malignant disease. Eur J Cancer
Li WW, Hutnik M, Gehr G. Antiangiogenesis in haematological malignancies. Br J Haematol
Iannacone M. Platelet-mediated modulation of adaptive immunity. Semin Immunol
Sprague DL, Elzey BD, Crist SA, Waldschmidt TJ, Jensen RJ, Ratliff TL. Platelet-mediated modulation of adaptive immunity: unique delivery of CD154 signal by platelet-derived membrane vesicles. Blood
Varon D, Shai E. Platelets and their microparticles as key players in pathophysiological responses. J Thromb Haemost
Ball S, Nugent K. Microparticles in hematological malignancies: role in coagulopathy and tumor pathogenesis. Am J Med Sci
Gendron N, Smadja DM. Circulating endothelial cells: a new biomarker of endothelial dysfunction in hematological diseases. Ann Biol Clin (Paris)
Testa U, Saulle E, Castelli G, Pelosi E. Endothelial progenitor cells in hematologic malignancies. Stem Cell Investig
Yan M, Jurasz P. The role of platelets in the tumor microenvironment: from solid tumors to leukemia. Biochim Biophys Acta
Baccarani M, Deininger MW, Rosti G, Hochhaus A, Soverini S, Apperley JF et al.
European LeukemiaNet recommendations for the management of chronic myeloid leukemia: 2013. Blood
Van Beers EJ, Schaap MC, Berckmans RJ, Nieuwland R, Sturk A, van Doormaal FF et al.
Circulating erythrocyte-derived microparticles are associated with coagulation activation in sickle cell disease. Haematologica
Schmitz G, Rothe G, Ruf A, Barlage S, Tschope D, Clemetson KJ et al.
European Working Group on Clinical Cell Analysis: consensus protocol for the flow cytometric characterisation of platelet function. Thromb Haemost
Starlinger P, Brugger P, Reiter C, Schauer D, Sommerfeldt S, Tamandl D et al.
Discrimination between circulating endothelial cells and blood cell populations with overlapping phenotype reveals distinct regulation and predictive potential in cancer therapy. Neoplasia
Zhu X, Li Q, Zeng C, Zhong Z, You Y, Zou P. Detection of leukemia-derived microparticles in the monitoring of chronic myeloid leukemia. Zhonghua Xue Ye Xue Za Zhi
Cojbasic I, Macukanovic-Golubovic L, Mihailovic D, Vucic M, Cojbasic Z. The significance of angiogenesis for predicting optimal therapeutic response in chronic myeloid leukaemia patients. Pol J Pathol
Repsold L, Pool R, Karodia M, Tintinger G, Joubert AM. An overview of the role of platelets in angiogenesis, apoptosis and autophagy in chronic myeloid leukaemia. Cancer Cell Int
Godoy CR, Levy D, Giampaoli V, Chamone DA, Bydlowski SP, Pereira J. Circulating endothelial cells are increased in chronic myeloid leukemia blast crisis. Braz J Med Biol Res
Schmidt T, Carmeliet P. Angiogenesis: a target in solid tumors, also in leukemia? Hematology Am Soc Hematol Educ Program
Taverna S, Fontana S, Monteleone F, Pucci M, Saieva L, De Caro V et al.
Curcumin modulates chronic myelogenous leukemia exosomes composition and affects angiogenic phenotype via exosomal miR-21. Oncotarget
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]