Circulating endothelial cells in patients with venous thromboembolism and myeloproliferative neoplasms

Abstract

Background: Circulating endothelial cells (CEC) may be a biomarker of vascular injury and pro-thrombotic tendency, while circulating endothelial progenitor cells (CEP) may be an indicator for angiogenesis and vascular remodelling. However, there is not a universally accepted standardized protocol to identify and quantify these cells and its clinical relevancy remains to be established.

Objectives: To quantify CEC and CEP in patients with venous thromboembolism (VTE) and with myeloproliferative neoplasms (MPN), to characterize the CEC for the expression of activation (CD54, CD62E) and procoagulant (CD142) markers and to investigate whether they correlate with other clinical and laboratory data.

Patients and methods: Sixteen patients with VTE, 17 patients with MPN and 20 healthy individuals were studied. The CEC and CEP were quantified and characterized in the blood using flow cytometry, and the demographic, clinical and laboratory data were obtained from hospital records.

Results: We found the CEC counts were higher in both patient groups as compared to controls, whereas increased numbers of CEP were found only in patients with MPN. In addition, all disease groups had higher numbers of CD62E+ CEC as compared to controls, whereas only patients with VTE had increased numbers of CD142+ and CD54+ CEC. Moreover, the numbers of total and CD62+ CEC correlated positively with the white blood cells (WBC) counts in both groups of patients, while the numbers of CEP correlated positively with the WBC counts only in patients with MPN. In addition, in patients with VTE a positive correlation was found between the numbers of CD54+ CEC and the antithrombin levels, as well as between the CD142+ CEC counts and the number of thrombotic events.

Conclusions: Our study suggests that CEC counts may reveal endothelial injury in patients with VTE and MPN and that CEC may express different activation-related phenotypes depending on the disease status.

Figure 1. Data acquisition strategy used to identify and quantify the circulating endothelial cells.

Our acquisition gating strategy assures the quantification of the circulating endothelial cells (CEC) and endothelial progenitor cells (CEP) without creating enormous FCS2.0 data files. Dot plot A illustrates the initial gating based on the FSC/SSC parameters (R1), in order to exclude events with lower FSC/SSC, which comprise mainly debris and platelets; dot plot B shows the events within the gate (R1), and includes a gate for Trucount™ beads (R2) and another gate for mononuclear cells (R3); dot plot C shows the events within the gate R2 and includes a new gate only for the beads (R4); dot plot D shows the events within the gate of mononuclear cells (R3) and contains a new gate only for CD146+ cells, either CD45- or CD45+ (R5). All the events that were inside the R4 (beads) or R5 (CD146+ cells) regions were acquired and stored.

Figure 2. Data analysis strategy used to identify and quantify the circulating endothelial cells.

Our data analysis gating strategy assures the identification and quantification of the circulating endothelial cells (CEC) and endothelial progenitor cells (CEP), among stored CD146+ cells. Using the Paint-a-Gate™ or the Infinicyt™ software we first identified the beads (blue dots) in the CD45 vs. CD146 dot plot (A); after that, we selected the total CD146+ events (green dots) (B, for selection of CD146+ cells; C, showing the light scatter properties of the cells); after excluding the CD146- cells (D to F), we finally were obtained the CEC (CD45-CD146+, yellow dots), the CEP (CD45+lowCD146+, red dots), activated lymphocytes (CD45+highCD146+, green dots), as well as the beads (blue dots), being then able to perform CEC and CEP quantification.