TITLE: Circulating Apoptotic Endothelial Cells and Apoptotic Endothelial
Microparticles Independently Predict the Presence of Cardiac Allograft Vasculopathy
AUTHORS: Neha Singh MSc, Eline Van Craeyveld PhD, Marc Tjwa MD PhD,
Agnieszka Ciarka MD PhD, Jan Emmerechts MD PhD, Walter Droogne MD,
Stephanie C. Gordts MSc, Vincent Carlier MSc, Frank Jacobs PhD, Steffen Fieuws
PhD, Johan Vanhaecke MD PhD, Johan Van Cleemput MD PhD, Bart De Geest MD
PhD
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Supplemental Figure 1. Flow cytometry analysis of EPCs. Gating strategy for
EPCs, identified as CD34+ VEGFR-2+ mononuclear cells in whole blood samples (A,
B). The first gate is set on CD34+ cells out of the mononuclear cellular events (A),
which is then displayed on a scatter plot to quantify CD34+ VEGFR-2+ cells (B). The
isotype control for VEGFR-2 is shown in panel C. Representative contour plots of
EPCs in healthy controls, CAV negative transplant recipients, and CAV positive
transplant recipients are shown in panels D, E, and F, respectively.
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Supplemental Figure 2. Quantification of EPC number and EPC function in
healthy controls and transplant recipients. EPC number was quantified as the
number of Dil-acLDL and FITC labeled UEA-1 lectin double positive cells (A) or as
the concentration of circulating CD34+ VEGFR2+ cells (B). EPC function was
evaluated by quantification of EPC migration in modified Boyden chambers (C).
Comparisons were made between healthy controls (n=25) and transplant recipients
(n=52). All data represent means ± SEM.
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Supplemental Figure 3. Enumeration of HPCs in healthy controls and transplant
recipients. Comparison of the number of CFU-E (erythroid colony-forming units)
(A), BFU-E (erythroid burst-forming units) (B), CFU-GEMM (granulocyte,
erythrocyte, macrophage, megakaryocyte colony-forming units) (C), CFU-GM
(granulocyte, macrophage colony-forming units) (D), CFU-M (macrophage colonyforming units) (E), and CFU-G (granulocyte colony-forming units) (F) between
healthy controls (n=25) and transplant recipients (n=52). MNC: mononuclear cells.
All data represent means ± SEM.
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Supplemental Figure 4. Quantification of circulating endothelial cells (CECs) by
flow cytometry in healthy controls and transplant recipients. Circulating
endothelial cells (CECs) (A), apoptotic CECs (B), and viable CECs (C) were
compared between healthy controls (n=25) and transplant recipients (n=52). All data
represent means ± SEM of natural logarithm (ln) transformed values.
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Supplemental Figure 5. Flow cytometry analysis of CECs. Gating strategy for
CECs, identified as CD45- CD31bright VEGFR-2+ mononuclear cells in whole blood
samples (A, B, C). An initial gate is set on CD45- cells out of the mononuclear
cellular events (A). Next, a second gate is set on CD45- CD31bright cells (B), which is
further displayed on a FACS scatter plot to identify CD45- CD31bright VEGFR-2+ cells
(C). The isotype control for VEGFR-2 is shown in panel D. Representative contour
plots of Annexin V positive circulating endothelial cells in healthy controls, CAV
negative transplant recipients, and CAV positive transplant recipients are shown in
panels E, F, and G, respectively.
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Supplemental Figure 6. Determination of circulating endothelial microparticles
by flow cytometry in healthy controls and transplant recipients. Circulating
endothelial microparticles (CEMPs) (A), apoptotic CEMPs (B), and viable CEMPs
(C) were analysed in healthy controls (n=25) and transplant recipients (n=52). All
data represent means ± SEM.
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Supplemental Figure 7. Flow cytometry analysis of circulating endothelial
microparticles. Gating strategy for endothelial microparticles in human plasma,
identified as CD42a- CD144+ microparticles (A). The isotype control for CD144 is
shown in panel B. Representative contour plots for Annexin V positive endothelial
microparticles in healthy controls, transplant recipients without CAV, and transplant
recipients with CAV are shown in panels C, D, and E, respectively.
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