Evaluation of Endothelial Progenitor Cell Characteristics as Clinical Biomarkers for Elderly Patients with Ischaemic Stroke

Figures

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  Fig. 1. Study recruitment, patient pathway and sample processing. modified Rankin Scale (mRS), National Institution of Health Stroke Scale (NIHSS), Barthel Index (BI), acetylated low-density lipoprotein (acLDL), endothelial progenitor cells (EPCs)

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  Fig. 2. Endothelial progenitor cell (EPC) counts. (A-E) EPCs were detected by flow cytometry and analysed using Kaluza Analysis 2.1 software. Compensation controls were used in setting up the flow protocol. Fluorescence minus one and isotype controls were used in gating for positive cells. CD45-FITC, CD34-PECy7, CD133-APC and KDR-PE. Approximately 1 million cells were counted. Cells were first gated on the monocyte and lymphocyte gate [A], then for CD45− CD34+ [B] and CD45− CD133+ [C] cells. Finally, cells were gated for [B] + [C] and KDR+ cells [E]. Counting beads were used to normalise samples to EPC counts/mL [F]. (F) EPC counts per mL of peripheral blood for health volunteers (HV) and ischemic stroke patients at baseline (BL) and days 7, 30 and 90 post-stroke. (G) EPC counts per mL of peripheral blood for ischemic stroke patients stratified into cortical and lacunar sub-groups at BL and days 7, 30 and 90 post-stroke. (H) Median EPC counts for the groups in panels F and G above with p values from either the Mann–Whitney U test (*) or Friedman Test (†) comparing HVs to all stroke patients at each time point, comparing cortical and lacunar strokes within the same timepoint or comparing…

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  Fig. 3. Detection and characterisation of endothelial progenitor cells (EPCs). (A) Colony forming units after seeding remaining mononuclear fraction into 24 well plates for 3 different donors. (B) No significant differences in the number of EPC colonies per cm2 were seen between HV and stroke patients (Mann–Whitney U-test) or between different timepoints post-stroke (Friedman Test). (C) No significant differences in the number of EPC colonies per cm2 were seen between cortical and lacunar stroke patients at any timepoints post-stroke (Mann–Whitney U-test) or between timepoints in the subgroups themselves (Friedman Test). (D) Staining outgrowth endothelial cells with acetylated low-density lipoprotein (ac-LDL) and lectin for 2 different donors. Positive staining indicates endothelial cells. (E) Matrigel tube formation assay from 4 different donors shown with either calcein-AM staining or under phase contrast

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  Fig. 4. Functional properties of outgrowth EPCs from healthy volunteers (HV) and ischemic stroke patients at baseline (BL) and days 7, 30 and 90 post-stroke. (A) The rate of EPC migration was significant between BL and Day 30 post-stroke (Linear mixed model with Bonferroni adjusted P value for post-hoc tests). (B) The rate of EPC proliferation was significant between BL and Day 7, BL and Day 30 and Day 7 and Day 90 post-stroke (Linear mixed model with Bonferroni adjusted P value for post-hoc tests). (C) No significant differences in migration rates were seen between cortical and lacunar stroke patients at any timepoints post-stroke (Student’s t-test). (D) No significant differences in proliferation rates were seen between cortical and lacunar stroke patients at any timepoints post-stroke (Student’s t-test). P values for the statistical tests are shown directly below the plots and n numbers are shown directly above the bars. P values of < 0.05 were considered significant and plotted by a *

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  Fig. 5. Analysis of biological markers in the plasma of ischaemic stroke patients stratified to cortical and lacunar subgroups at baseline (BL) and days 7, 30 and 90 post ischaemic stroke. (A) Total antioxidant capacity (TAC) was detected in plasma using a kit and values are Trolox equivalent capacity. (B) Angiostatin, (C) endostatin, (D) granulocyte colony-stimulating factor (GCSF), (E) platelet-derived growth factor-BB (PDGF-BB), (F) stromal cell derived factor-1 (SDF-1), (G) tumour necrosis factor alpha (TNF-α), (H) thrombospondin-1, (I) thrombospondin-2 and (J) vascular endothelial growth factor (VEGF) levels in plasma were detected using ELISAs. A Mann–Whitney U test was used to compare data from cortical and lacunar sub-groups at the same timepoint and a P value of < 0.05 was considered significant and plotted above. A Friedman Test was used to compare all timepoints of the same subgroups (P values directly under plot) with a post-hoc Wilcoxon signed-rank test where a Bonferroni adjusted P value of < 0.0083 was considered significant. (K) Post-hoc analysis P-values

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  Fig. 6. Predictors of day 90 outcome as measured by the modified Rankin Scale (mRS). (A) Univariate proportional odds logistic regression of baseline (BL) variables to predict outcome at day 90 post ischaemic stroke as measured by the mRS. The day 90 mRS data was categorised into the following bands 0–2, 3–4 and 5–6 to represent mild, moderate and severe disease. (B) All variables which were significant on univariate analysis were entered into the model. (C) Wilcoxon signed-rank tests were used to compared baseline and day 90 data for mRS, NIHSS and BI outcome measures and P values are displayed in the table. (D) Frequency distribution of patients in mRS categories at baseline and day 90. P < 0.05 was considered significant for all tests. Endothelial progenitor cells (EPC), National Institution of Health Stroke Scale (NIHSS), Barthel Index (BI), blood pressure (BP), low density lipoprotein (LDL), high density lipoprotein (HDL), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), white blood cell (WBC), transient ischemic attack (TIA), ischemic stroke (IS) and calcium (Ca2+)