Kinetics and Apoptotic Profile of Circulating Endothelial Cells In Patients Undergoing Autologous Stem Cell Transplantation

Tumor growth and progression are influenced by endothelial cells proliferation and differentiation. In patients with hematological malignancies circulating endothelial cells (CEC) are elevated at the diagnosis and decrease after successful chemotherapy. The other spectrum of interest is evaluation of CEC in the context of their influence on regeneration of hematopoiesis from damage caused by high dose chemotherapy and stem cells transplantation. The aim of our study was to assess the kinetics of CEC and their subsets (mature, progenitor and apoptotic CEC) in patients undergoing autologous hematopoietic stem cells transplantation. Additionally we analyzed the relationship between the number of CEC subsets and clinical course in the post-transplant period and time to engraftment. Methods: CEC were measured in peripheral blood of 39 patients (18 males and 21females, median age 54; range 26–69) scheduled for high dose chemotherapy with autologous stem cell transplantation. The group consisted of 29 multiple myeloma patients and 10 lymphoma patients. All of the patients were in best response to previous treatment before transplantation, 20 in CR and 19 in PR. Conditioning treatment in patients with myeloma was Melphalan 200 mg/m², while in lymphoma patients BEAM. Evaluation of CEC: Blood samples were collected at different time points: 1) at baseline - directly before conditioning; 2) a day after finishing of chemotherapy; 3) at the day “0” just before transplantation of CD34+ HSC and 4) four times after transplantation :1hour and 1 day after HSC transfusion, at the time of the nadir in peripheral blood and at the day of neutrophil engraftment). CEC were evaluated by 4 colour flow-cytometry according to the procedure described previously. Circulating progenitor cells (CEPC) were defined as negative for hematopoietic marker CD45 and positive for the endothelial cell markers CD 34, CD31 and the endothelial progenitor marker CD133. CD105 and CD106 positive CEC were classified as activated CEC (aCEC). Expression of CD36 characterized CEC originating from microvessels. Apoptotic CEC (apoCEC) were defined as CD146 positive and Annexin V positive. Results: The median (Me) CEC number at the baseline was 9, 5/μL and dropped significantly 24 h after chemotherapy (Me 8, 3/μL; p=0, 002). At day “0” before infusion of HSC, the count of CEC continued to decrease (Me 6, 6/ μL, p<0, 0001 when compared to baseline) and remained stable shortly after transplantation (1h and 1day after transplantation Me 6, 2/μL p=0, 0001 and 6, 8/μL, p=0, 0002 respectively). At nadir of WBC count, the nadir of CEC (Me 4, 6/μL) was also observed. CEC number increased at the engraftment period, but did not reach pretreatment values (Me 5, 3/μL, p0, 0001). The number of aCEC and CEPC followed CEC kinetics. In contrast, the count of apoptotic CEC 24h after conditioning (Me 4, 15/μL) was significantly enhanced in comparison to baseline values (Me 3, 1/μL; p=0, 0005) and drop down 1 day after transplantation to pretreatment values (Me 3, 6/μL; p=0, 4), and then continued to decrease (WBC nadir - Me 2, 1/μL, engraftment Me 1, 85 /μL).For the purpose of the study CEC and their subsets were evaluated as dichotomous variables using median value as cut-off to divide patients into low- or high- expressers groups. Patients with low (<Me) expression apoCEC at baseline had significantly shorter time for neutrophil engraftment (Me 13; range11–18 days) than high expressers: (Me15, 5; range 11–21days, p=0, 028). Additionally we observed significant correlation between the higher number of CEPC at 1 h after transplantation with shorter time to neutrophil engraftment (Spearman rank coefficient r= −0, 49, p=0, 008). Similar correlation between the increased count of endothelial cells originating from microvessels measured 1h after transplantation and shorter time to neutrophil engraftment was also observed (r= −0, 39, p=0, 04). Conclusions: Low count of apoptotic CEC at baseline correlates with shorter time to engraftment which may suggest that intact bone marrow microenviroment takes part in effective engraftment. Additionally, our results indicate that progenitor CEC and CEC derived from microvessels could support bone marrow regeneration after HSCT. The role of CEC and their subsets in the context of hematopoietic recovery after high dose chemotherapy and autoHSCT needs further evaluation.

No relevant conflicts of interest to declare.