Roles of endogenous monocyte chemoattractant protein-1 in ischemia-induced neovascularization
Hiroshi Niiyama, MD*,
Hisashi Kai, MD, PhD*,*,
Tomoka Yamamoto, BS*,
Toshifumi Shimada, MD*,
Ken-Ichiro Sasaki, MD, PhD*,
Toyoaki Murohara, MD, PhD*,
Kensuke Egashira, MD, PhD and
Tsutomu Imaizumi, MD, PhD, FACC*
* Third Department of Internal Medicine and Cardiovascular Research Institute, Kurume University, Kurume, Japan
Cardiovascular Medicine, Kyushu University Graduate School of Medicine, Fukuoka, Japan
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Figure 1 Expression of transfected 7ND in the ischemic adductor muscle. Western blot analysis using an anti-FLAG M2 monoclonal antibody showed a transient expression of FLAG-fusion 7ND protein in the muscle. A total of 50 µg of the total protein was added in each lane. The similar results were obtained from three independent experiments.
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Figure 2 Endogenous monocyte chemoattractant protein-1 (MCP-1) induction in the ischemic hindlimb. Pooled data of temporal changes in endogenous MCP-1 content in the thigh adductor muscle in the ischemic hindlimb (open bars). Solid bars = sham mice. Bar = 1 x SD (n = 8).
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Figure 3 Laser Doppler blood flow (LDBF) analysis was performed serially in each mouse to determine the blood flow in unilateral ischemic hindlimb. (A) Representative LDBF images of the ischemic (arrow heads) and nonischemic hindlimb in 7ND-transfected and mock-treated mice. (B) Pooled data of temporal changes in ischemic/nonischemic LDBF ratio. The recovery of LDBF ratio was deteriorated by 7ND overexpression in a dose-dependent manner. *p < 0.05 and **p < 0.01 vs. mock-treated mice. Bar = 1 x SD (n = 12).
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Figure 4 (A) Representative microphotographs of hematoxylin-eosin–stained sections of ischemic thigh adductor muscle obtained from mock- and 7ND-treated mice at days 3 and 14. (B, top) Representative immunohistostainings for F4/80, a macrophage-specific marker, of the adductor muscles at day 3. The serial sections were subjected to the experiments for A (day 3) and B. (B, bottom) Pooled data showing the number of infiltrated macrophages in mock- and 7ND-transfected mice at day 3. Bar = 1 x SD (n = 8).
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Figure 5 Pooled data demonstrating the effects of 7ND gene transfer on vascular endothelial growth factor (VEGF) induction and tumor necrosis factor (TNF)-alpha induction in ischemic thigh adductor muscle at days 3 and 7. Bar = 1 x SD (n = 8). Solid bars = 7ND-transfected mice; open bars = mock-treated mice; open squares = sham-operated mice without any gene transfection. *p < 0.05 and **p < 0.01 versus sham mice. Analysis of variance followed by Scheffé's F test was performed for statistical comparison.
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Figure 6 Effects of 7ND overexpression on ischemia-induced neovascularization. (A) Postmortem angiograms at day 21; 7ND overexpression eliminated the development of angiographically visible collateral vessels (top), and angiographic score was significantly lower in 7ND-treated mice as compared with mock-transfected mice (bottom). Bar = 1 x SD (n = 12). (B) Capillary density of the thigh adductor muscle at day 21. Immunohistostaining for CD31, an endothelial cell marker (stained as brown), was performed to determine the capillaries in the ischemic muscle (top). Capillary density at day 21 was significantly reduced by 7ND gene transfer in the ischemic hindlimb (bottom). Bar = 1 x SD (n = 12).
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