Beneficial Effects of Reconstituted High-Density Lipoprotein (rHDL) on Circulating CD34+ Cells in Patients after an Acute Coronary Syndrome

Figures

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  Fig 1. Representative example of sequential gating strategy for flow cytometric analysis of endothelial progenitor cells. A modified ISHAGE strategy was applied for EPC quantification. 1) Representative sample stained with CD45-FITC. Region R6 represents lymphocytes. 2) Anti-CD34-PE staining of cells from R1. Region R2 represents CD34+ cells. 3) Region R3 is placed to include the low Side Scatter and low to intermediate CD45 staining. 4) R4 represents all events from regions R1, R2 and R3 displayed on a FSC vs SSC dot plot to confirm that the selected events fall into a lymph-blast region. 5) Displays the events included in regions R1, R2, R3 and R4. A quadrant was positioned to separate the positive and the negative cells for VEGFR2 staining. An appropriate isotype control was used to adequately place the quadrant. Region R5 represents the total EPCs (CD34+/VEGFR2+ cells). 6) Events from region R6. This region is used to set the region R4. 7) All events. This histogram is useful to establish the lower limit of CD45 expression for the CD34+ events. The region R8 is placed in the right top of the histogram to count all Stem-count fluorospheres accumulated for each sample for absolute…

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  Fig 2. Relative preservation of CD34+ cells in patients with acute coronary syndrome following treatment with reconstituted high-density lipoprotein (rHDL) compared to controls. The CD34+ progenitor cells (A and B) and CD34+/KDR+ endothelial progenitor cells (C and D) were quantified in blood samples collected at baseline and at follow-up. The follow-up samples for the CSL-111-treated group were obtained 16 ± 4 days following completion of the 4 weekly rHDL infusions. Each box plot in A and C shows the median, the interquartile range, the maximum and the minimum of the relative change. B and D show absolute numbers of CD34+ (B) and CD34+/KDR+ (D) endothelial progenitor cell count at baseline and at follow-up. p < 0.05 between groups (A and B), p = N.S. (C and D) from Mann-Whitney tests.* one outlier (baseline cell count 1.6/μl, follow-up 0.1/μl) not presented in figure due to axis limits.

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  Fig 3. Decreased levels of serum stromal cell-derived factor-1 (SDF-1) in patients with acute coronary syndrome following treatment with reconstituted high-density lipoprotein (rHDL) compared to controls. Relative changes from baseline in serum SDF-1 in the control group and in the rHDL-treated group. Each box plot shows the median, the interquartile range, the maximum and the minimum. p < 0.05 between groups from Mann-Whitney test.

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  Fig 4. In vitro exposure of circulating progenitor cells to CSL-111. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors (n = 7) and plated on fibronectin-coated plates in the absence or presence of CSL-111 (1 mg/mL) from day 0 to day 4 (D0-4), 4 to 7 (D4-7) or 0 to 7 (D0-7). After 7 days of culture, adherent cells were harvested and analyzed by flow cytometry. (A) All adherent cells were quantified by flow cytometry using cell counting beads for enumeration. (B) CSL-111 treatment increases the total number of CD34+ cells when added to cell culture media at D0-4 and D0-7; CD34+ cells were quantified by flow cytometry. (C) CSL-111 treatment reduces basal apoptosis in eEPCs when added to cell culture media at D0-4 and D0-7. Apoptosis was measured by flow cytometry using Annexin V labeling. Each box plot shows the median, the interquartile range, the maximum and the minimum of the relative change. * indicates p < 0.05 between groups from Wilcoxon signed-rank tests.

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  Fig 5. In vitro studies on the effect of CSL-111 on migratory capacity of eEPC. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors (n = 3) and plated on fibronectin-coated plates in the absence or presence of CSL-111 (1 mg/mL) from day 0 to day 4 (D0-4), 4 to 7 (D4-7) or 0 to 7 (D0-7). On day 7 of culture, adherent cells were harvested and assayed in a modified Boyden chamber for their capacity to migrate along an SDF-1 gradient. Significantly increased migration was observed among cells treated with CSL-111 for day 0 to day 4 (**; p = 0.0003) and 0 to 7 (*; p = 0.0135) compared to controls. Figure shows adjusted mean±standard error of the mean (SEM). p-values are reported from mixed model ANOVA.