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The therapeutic effect of vascular endothelial growth factor gene- or heme oxygenase-1 gene-modified endothelial progenitor cells on neovascularization.

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Presentation on theme: "The therapeutic effect of vascular endothelial growth factor gene- or heme oxygenase-1 gene-modified endothelial progenitor cells on neovascularization."— Presentation transcript:

1 The therapeutic effect of vascular endothelial growth factor gene- or heme oxygenase-1 gene-modified endothelial progenitor cells on neovascularization of rat hindlimb ischemia model  Jianting Long, MD, PhD, Sanming Wang, MD, PhD, Yuanqi Zhang, MD, PhD, Xiangxia Liu, MD, PhD, Hui Zhang, MD, PhD, Shenming Wang, MD, PhD  Journal of Vascular Surgery  Volume 58, Issue 3, Pages e2 (September 2013) DOI: /j.jvs Copyright © 2013 Society for Vascular Surgery Terms and Conditions

2 Fig 1 Immunohistochemical staining of endothelial progenitor cells (EPCs). P3 EPCs crawling microscopic slides were prepared for immunohistochemical staining for CD133 (A2 and B2), CD34 (A3 and B3), and Von Willebrand factor (vWF) (A4 and B4). The expressions of CD34 and vWF dramatically increased in P3 cells with human vascular endothelial growth factor (VEGF) induction (B3 and B4), compared with P3 cells without VEGF induction (A2-A4). Representative images of three independent experiments were shown. Journal of Vascular Surgery  , e2DOI: ( /j.jvs ) Copyright © 2013 Society for Vascular Surgery Terms and Conditions

3 Fig 2 Endothelial progenitor cell (EPC) biomarkers expression in P3 cells. P3 cells at the end of the 7-day culture were collected and subjected to EPC biomarkers testing. CD133 and CD34 expressions were quantified as the percentage of cells labeled by fluorescein isothiocyanate (FITC)-conjugated secondary antibodies, which were bound to primary antibodies against these two proteins, data were expressed as the (mean ± standard deviation)% of triplicate readings of each sample in one experiment representative of three performed with similar results (A). At day 7, P3 cells intensively took up 1,1-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (Dil-Ac-LDL) (red) and bound to an endothelial-specific lectin-FITC (green) as revealed by fluorescence microscopy (B). Quantitative analysis of the percentage of cells positive for Dil-Ac-LDL/lectin dual staining (merge) was performed as described in the Methods section. Total RNA from P3 cells were amplified by real-time polymerase chain reaction (RT-PCR) using specific vascular endothelial growth factor (VEGF)-R2 and β-actin primers. Data were expressed as the mean ± standard deviation of three samples of each group in one experiment representative of three performed with similar results (C). Whole cell extracts were subjected to Western blotting, testing protein expression of VEGF-R2. The representative image of three independent experiments was shown. *P < .01 vs no human VEGF induction. Journal of Vascular Surgery  , e2DOI: ( /j.jvs ) Copyright © 2013 Society for Vascular Surgery Terms and Conditions

4 Fig 3 Validation of vascular endothelial growth factor A (VEGFA) and heme oxygenase-1 (HO-1) gene-modified endothelial progenitor cell (EPC). EPCs were examined for transfection efficiency by fluorescence microscopy 48 hours after transfection with the gene of interest and EGFP (A). Total RNA from transfected cells were amplified by real-time polymerase chain reaction (RT-PCR) using specific VEGFA, HO-1, and β-actin primers. Data were expressed as the mean ± standard deviation of three samples of each group in one experiment representative of three performed with similar results (B, upper panel). Whole cell extracts were subjected to Western blotting, testing protein expression of VEGFA, and HO-1. The representative image of three independent experiments was shown (B, lower panel). *P < .001 vs pCMV-Tag 2B/empty vector control. Journal of Vascular Surgery  , e2DOI: ( /j.jvs ) Copyright © 2013 Society for Vascular Surgery Terms and Conditions

5 Fig 4 Transgene expressions and microvessel density changes after transplantation. To test the efficiency of the transplantation of gene-modified endothelial progenitor cells (EPCs), six rats from each group were euthanized at day 7 after transplantation. Muscle tissue from ischemic limb was subjected to total RNA and protein extraction, as described in the Methods section. Total RNA was amplified by real-time polymerase chain reaction (RT-PCR) using specific vascular endothelial growth factor A (VEGFA), heme oxygenase-1 (HO-1), and β-actin primers. Data were expressed as the mean ± standard deviation of six samples from each group (A, upper panel). Total protein was subjected to Western blotting, testing protein expressions of VEGFA and HO-1. The representative image of six blots was shown (A, lower panel). Microvessel density (MVD) was calculated as described in the Methods section at days 7, 14, and 21 after surgery (six rats from each group at each timepoint). Data were expressed as the mean ± standard deviation capillaries/m2 (B). MVD changes were compared among different time points in the same treatment group (B, left panel), and among different treatment groups at the same time point (B, right panel). *P < .05 vs phosphate-buffered saline (PBS); **P < .05 vs PBS or EPC; ***P < .001 vs PBS, EPC, VEGF, or HO-1. Journal of Vascular Surgery  , e2DOI: ( /j.jvs ) Copyright © 2013 Society for Vascular Surgery Terms and Conditions

6 Fig 5 Recovery of perfusion and salvage rate of ischemic limbs after transplantation. Subcutaneous blood flow rate was detected by laser Doppler imager (LDPI). Calculated perfusion was expressed as the percentage ratio of average blood flow in the surgical limb to that in the healthy limb. Data were expressed as the (mean ± standard deviation)% of six samples of each group (A). *P < .001 vs phosphate-buffered saline (PBS); **P < .05 vs PBS or endothelial progenitor cell (EPC); ***P < .001 vs PBS, EPC, vascular endothelial growth factor (VEGF), or heme oxygenase-1 (HO-1). The representative LDPI image set of 18 sets of images with similar results, taken from 18 animals at day 21 after surgery, was shown (B). The degree of ischemic damage in the affected limb evaluated macroscopically was shown (C). Journal of Vascular Surgery  , e2DOI: ( /j.jvs ) Copyright © 2013 Society for Vascular Surgery Terms and Conditions

7 Supplementary Fig 1 (online only)
Morphologic changes of endothelial progenitor cell (EPC). EPCs initially isolated (passage 1, P1) were small, round, and undistinguishable from blood cells (A). P2 cells were more adherent and changed to spindle shape with a tendency to form cluster or colony (B). P3 cells displayed uniformly spindle-shaped with long, dendritic-like protrusions and a tendency to form lines (C). Journal of Vascular Surgery  , e2DOI: ( /j.jvs ) Copyright © 2013 Society for Vascular Surgery Terms and Conditions

8 Supplementary Fig 2 (online only)
BrdU immunohistochemical staining of muscle tissue from ischemic limb. Journal of Vascular Surgery  , e2DOI: ( /j.jvs ) Copyright © 2013 Society for Vascular Surgery Terms and Conditions

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