The Effect of Stem Cell Mobilization by Granulocyte-Colony Stimulating Factor on Neointimal Hyperplasia and Endothelial Healing After Vascular Injury With Bare-Metal Versus Paclitaxel-Eluting Stents
Hyun-Jai Cho, MD*, , ,
Tae-Youn Kim, BA,
Hyun-Ju Cho, MS,
Kyung-Woo Park, MD*,,,
Shu-Ying Zhang, MD,
Ji-Hyun Kim, MS,
Sung-Hwan Kim, MD*,,,
Joo-Yong Hahn, MD*,,,
Hyun-Jae Kang, MD*,,,
Young-Bae Park, MD*,, and
Hyo-Soo Kim, MD, PhD*,,,*
* Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea
Cardiovascular Center, Clinical Research Institute, Seoul National University Hospital, Seoul, Korea
Cardiovascular Research Laboratory, Clinical Research Institute, Seoul National University Hospital, Seoul, Korea.
View larger version (16K):
[in a new window]
|
Figure 1 Schematic diagram of study design and end points. (A) Experiments to analyze the effect of granulocyte-colony stimulating factor (G-CSF) treatment on neointimal growth or vascular healing of stented artery. (B) Experiments to analyze the progenitor cell mobilization by G-CSF and incorporation into injured artery. BMS = bare-metal stent; d = day; PES = paclitaxel-eluting stent.
|
|
View larger version (63K):
[in a new window]
|
Figure 2 Effects of stem cell mobilization by G-CSF on neointimal hyperplasia after stenting, BMS: EXPRESS versus PES: TAXUS. (A and B) Histology of the iliac artery 60 days after BMS and PES deployment with or without stem cell mobilization by G-CSF. Stem cell mobilization by G-CSF significantly aggravated neointimal formation at BMS. The trend of such aggravation of neointimal formation was also observed at PES. Note that neointimal growth at PES even after G-CSF treatment was less than that at BMS after placebo. Arrowheads indicate internal elastic lamina; BMS, in the placebo group (albumin); PES, in the placebo group; G-BMS, BMS in the G-CSF group; G-PES, PES in the G-CSF group. (C) Digital image-analyzed morphometry of neointimal thickness was presented as mean ± SD BMS versus PES, p < 0.001; BMS versus G-BMS, p = 0.015; BMS versus G-PES, p = 0.036; and PES versus G-PES, p = 0.36. (D) Neointimal area: BMS versus PES, p < 0.001; BMS versus G-BMS, p = 0.053; BMS versus G-PES, p = 0.015; and PES versus G-PES, p = 0.505 by analysis of variance with Bonferroni’s correction. Abbreviations as in Figure 1.
|
|
View larger version (38K):
[in a new window]
|
Figure 3 Mobilization of putative vascular progenitor cells by granulocyte-colony stimulating factor (G-CSF) and in vitro differentiation to vascular smooth muscle cell (SMC) or endothelial cell (EC). Four quadrant analysis of antigen expression in circulating peripheral blood mononuclear cells after 6 days of G-CSF or placebo injection, and serial observation of cell culture with vascular endothelial growth factor (VEGF) or platelet-derived growth factor (PDGF). Endothelial lineage cells were identified by CD31 and VE-Cadherin (VE-CAD) in FACS analysis and PE-labeled-CD31 immunocytochemical staining (red color), and vascular smooth muscle lineage cells were identified by alpha-smooth muscle actin (SMA) in FACS analysis and FITC-alpha-SMA immunostaining (green color). Both endothelial and smooth muscle lineage cells increased with G-CSF treatment in peripheral blood. During culture of peripheral blood mononuclear cells, endothelial lineage cells increased preferentially with VEGF. In contrast, PDGF induced a part of the cells to differentiate into smooth muscle lineage in addition to double positive (CD31+/alpha-SMA+) or endothelial lineage cells.
|
|
View larger version (107K):
[in a new window]
|
Figure 4 Incorporation of mobilized peripheral mononuclear cells into vascular wall and differentiation to vascular smooth muscle cell (SMC). Peripheral blood mononuclear cells were isolated from rabbit after 6 days of granulocyte-colony stimulating factor (G-CSF) or placebo injection and tagged with DiI (red color). In another rabbit, these cells were infused systemically after balloon injury to the iliac artery. Two weeks after injury, frozen sections were stained with alpha-smooth muscle actin (SMA) (green color). Peripheral blood mononuclear cells from placebo-injected animals (A) revealed no double-positive (DiI+/alpha-SMA+) cells, implying that the infused cells did not differentiate to SMC but remained as mono-macrophage lineage cells. But the mobilized mononuclear cells from G-CSF–injected animals were observed as DiI+/alpha-SMA+ double positive cells within both media (B) and neointima (C), implying that the cells mobilized by G-CSF incorporated into vascular wall and differentiated to SMC. Some of the mobilized cells incorporated into neointima but did not differentiate into SMC (D). Such infiltration of the cells mobilized by G-CSF into vascular wall led to the increased neointimal thickness at 2 weeks (B, C, and D) compared with control animals infused with non-mobilized peripheral mononuclear cells (A).
|
|
View larger version (51K):
[in a new window]
|
Figure 5 Differential inhibitory effect of paclitaxel on proliferation of smooth muscle versus endothelial lineage cells. (A) Anti-proliferative effects of paclitaxel on rabbit endothelial progenitor cells (EPCs) and smooth muscle progenitor cells (SPCs) were evaluated by enzyme-linked immunosorbent assays for bromodeoxyuridine (BrdU) incorporation. At the presumable tissue concentration (100 nmol/l to 1 µmol/l) of paclitaxel at vessel with TAXUS stenting, EPC still maintained its proliferative potential, whereas SPCs did not. *EPC versus vascular progenitor cell, p < 0.001; p = 0.002. (B) Human EPCs, mature endothelial cells (ECs), and vascular smooth muscle cells (SMCs) were also evaluated. Paclitaxel caused a dose-dependent inhibition of SMC growth even from a low therapeutic concentration (100 nmol/l). In contrast, EC growth was inhibited only at 1 µmol/l of paclitaxel, a high therapeutic concentration. But proliferative activity of EPCs was not inhibited in the range of therapeutic concentration eluted from TAXUS stent. *EC versus SMC, p = 0.001; EPC versus EC, p = 0.036; #EC versus SMC, p = 0.002 by analysis of variance with Bonferroni’s correction.
|
|
View larger version (120K):
[in a new window]
|
Figure 6 Effects of stem cell mobilization on endothelial recovery after BMS and PES stenting. Gross (A) and scanning electron microscopy (SEM) findings (B) of stented rabbit iliac artery at 28 days. (First row) Endothelium and neointima were fully formed after BMS. (Third row) Aggravation of neointimal growth by G-CSF–mediated stem cell mobilization was reflected as the decreased transparency of gross finding and the disappearance of stent strut silhouette on SEM. (Second row) In case of PES, the evidences of delayed endothelial healing was observed as hemorrhage around PES struts in gross finding and uncovered stent struts in SEM finding. (Fourth row) After stem cell mobilization with G-CSF, however, endothelial layers were covered with cobblestone-shaped endothelial cells without focal hemorrhage, suggesting the enhanced endothelial recovery even on PES (arrowheads = hemorrhage; arrows = stent struts not covered by endothelium, owing to delayed re-endothelialization). Other abbreviations as in Figure 1.
|
|
|
