SDF-1/CXCR4 システムを利用したより治療効果の高い血管新生療法の開発

Transcription

1 Online publication ugust 20, 2010 総 説 第 49 回総会パネルディスカッション 3 どの血管新生療法が臨床で最も有効か SDF-1/CXCR4 システムを利用したより治療効果の高い血管新生療法の開発 要旨 : SDF-1α CXCR4 SDF-1/CXCR4 SDF-1/CXCR4 J Jpn Coll ngiol, 2010, 50: Key words: cytokines, implantation, ischemia, paracrine effect, regeneration はじめに TCT Theapeutic ngiogenesis by Cell Transplantation ml 2 Granulocyte Colony-stimulating Factor; G-CSF Macrophage Colony-stimulating Factor; M-CSF 3 5 CXC chemokine receptor 4 + CXCR4 + Stromal cell-derived factor-1 SDF-1α CXCL12 SDF-1/CXCR4 SDF-1α CXCR4 IDS HIV-1 SDF-1/CXCR SDF-1/CXCR THE JOURNL of JPNESE COLLEGE of NGIOLOGY Vol. 50,

2 SDF-1/CXCR4 Figure 1 SDF-1α expression in hindlimb ischemia. : Total RN was extracted from the ischemic and non-ischemic adductor muscles 24 h after the induction of hindlimb ischemia. Real-time RT-PCR was performed to evaluate SDF-1α mrn expression. Data are expressed as means ± SEM (n=4). p<0.05. : The ischemic and non-ischemic (sham) adductor muscles were excised 7 days after the ischemia induction and immunohistochemically stained with anti-sdf-1α antibody. Scale bar represents 100 mm. 虚血組織における SDF-1α の発現 10 SDF-1α real-time RT-PCR SDF-1α Fig. 1 SDF-1α desmin CD31 SDF-1α SDF-1α hypoxia inducible factor-1 HIF-1 SDF-1α HIF-1 単純培養による CXCR4 発現の増加 SDF-1α CXCR4 G-CSF M-CSF CXCR4 + SDF-1α 3, 5 CXCR4 SDF-1 CXCR FCS /DMEM CXCR CXCR ± ±0.9 Fig CCR7 Gr-1 CD3 Mac-1 CD34 Sca-1 Flk-1 CXCR4 Fig. 1 Gr-1 Mac-1 CD3 Fig. 1C D 24 CXCR4 Cul CXCR4 Fr マウス下肢虚血モデルにおける CXCR4 + 骨髄細胞移植の効果 CXCR4 Fr wild-type CX- CR4 Cul wild-type 324 脈管学 Vol. 50, 2010

3 2 Figure 2 Upregulation of CXCR4 in cultivated MCs. : MCs were collected and incubated at 37 for the indicated periods and stained with anti-cxcr4 antibody. The mean fluorescence intensity (MFI) of the CXCR4 expression was analyzed by flow cytometry. Data are expressed as mean ± SEM (n=3 for each). p<0.01. : MCs were collected and incubated at 37 for 24 h. Freshly isolated (thin line) and cultivated MCs (bold line) were stained with various antibodies against CXCR4, CCR7, L-selectin, Gr-1, CD3, Mac-1, CD34, CD31, Flk-1, Sca-1 and c-kit and analyzed by flow cytometry. Irrelevant IgG was used as a negative control. Representative data are shown (n=3). C and D: Freshly isolated MCs (MC Fr ) and cultivated MCs (MC Cul ) were stained for CXCR4 and Gr-1, CD3 or Mac-1 and analyzed by flow cytometry. Representative data (C) and quantitative analysis (D) are shown. Data are expressed as means ± SEM (n=3). C D Control phosphate buffered saline; PS CXCR4 Fr CXCR4 Cul Fig. 3 CD31 VE-cadherin CXCR4 Cul Fig. 3C D CXCR4 CXCR4 +/- CXCR4 -/- wild-type CXCR4 Cul CXCR4 +/- CXCR4 Cul Fig. 3E CXCR4 高発現細胞移植による血流改善の機序 CXCR4 Cul 2 1 CXCR4 SDF-1α 2 CXCR4 Cul 脈管学 Vol. 50,

4 SDF-1/CXCR4 Figure 3 Neovascularization by MC Fr or MC Cul implantation. MC Fr ( cells), MC Cul, or PS (control) was injected into the site of hindlimb ischemia 24 h after the surgery. lood flow perfusion was measured using a laser Doppler perfusion imaging system. : Representative photographs of blood flow imaging on day 21 after the surgery are shown. : Quantitative analysis of the blood flow ratio was performed. Data are expressed as mean ± SEM (n=9 for each). p<0.05 vs. control, # p<0.05 vs. MC Fr. C: The adductor muscles were excised 21 days after the surgery and immunohistochemically stained with antibodies against CD31 and VE-cadherin. Representative photographs are shown (n=3). Scale bar represents 100 mm. D: Quantitative analysis of CD31 + endothelial cells was performed. Data are expressed as mean ± SEM (n=3 for each). p<0.05 vs. wild-type MC Cul. E: Wild-type MC Cul ( cells) or CXCR4 +/ MC Cul was injected into the site of hindlimb ischemia 24 h after the surgery. lood flow perfusion was measured using a laser Doppler perfusion imaging system. Quantitative analysis of the blood flow ratio was performed. Data are expressed as mean ± SEM (n=6 for each). p<0.05 vs. wild-type MC Cul. C D E Green fluorescent protein GFP CXCR4 Cul GFP + CXCR4 Cul CXCR4 Fr 1 Fig. 4 GFP CXCR4 interleukin-1β IL-1β vascular endothelial growth factor- 326 脈管学 Vol. 50, 2010

5 2 Figure 4 Fate of MC Fr or MC Cul injected into the ischemic hindlimb. : GFP + /MC Fr ( cells) or GFP + /MC Cul ( cells, 24 h of cultivation) were injected into the site of hindlimb ischemia. The adductor muscles were excised on day 1 after implantation and digested by treatment with collagenase. The MCs, peripheral blood cells and splenocytes in the mice were also isolated. The implanted GFP + cells were analyzed by flow cytometry. Representative data are shown (n=3). : Quantitative analysis of the implanted GFP + /MC Fr or GFP + /MC Cul on day 1 (n=6), day 3 (n=4) and day 21 (n=4) was performed. Data are expressed as mean ± SEM. p<0.05. VEGF- mrn real-time RT-PCR Sham Control CXCR4 Fr IL-1β CXCR4 Cul IL-1β VEGF- Sham CXCR4 Fr Fig. 5 IL-1β 3 VEGF Fig. 5 IL-1β VEGF- ELIS Fig. 5C monocyte chemoattractant protein-1 MCP-1 IL-6 IL-1β VEGF- CXCR4 +/- CXCR4 おわりに SDF-1/CXCR4 SDF-1/ CXCR4 CXCR4 CXCR4 11 CXCR4 SDF-1α CXCR4 IL-1β VEGF- 脈管学 Vol. 50,

6 SDF-1/CXCR4 Figure 5 Cytokine mrn expression in vivo and in vitro. : Total RN was extracted from the adductor muscles of the sham-operated mice and mice implanted with or without MC Fr, MC Cul and PS (control) 24 h after the surgery. Real-time RT-PCR was performed to evaluate mrn expression of IL-1b and VEGF-. Data are expressed as means ± SEM (n=6 for each). p<0.05 vs. sham, # p<0.05 vs. control, $ p<0.05 vs. MC Fr. and C: MCs were collected from the wild-type (gray) and CXCR4 +/ (black) mice, incubated at 37 for the indicated periods, and analyzed by real-time RT-PCR () and ELIS (C). Data are expressed as mean ± SEM (n=5 for each). p<0.05 vs. 0 h, # p<0.05 vs. wild-type MCs. C SDF-1/ CXCR4 謝辞 GFP CXCR4 +/- 文献 1 Tateishi-Yuyama E, Matsubara H, Murohara T et al: Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial. Lancet, 2002, 360: Jia L, Takahashi M, Yoshioka T et al: Therapeutic potential of endothelial progenitor cells for cardiovascular diseases. Curr Vasc Pharmacol, 2006, 4: Yoshioka T, Takahashi M, Shiba Y et al: Granulocyte colonystimulating factor (G-CSF) accelerates reendothelialization and reduces neointimal formation after vascular injury in mice. Cardiovasc Res, 2006, 70: Shiba Y, Takahashi M, Yoshioka T et al: M-CSF accelerates neointimal formation in the early phase after vascular injury in mice: the critical role of the SDF-1-CXCR4 system. rterioscler Thromb Vasc iol, 2007, 27: Morimoto H, Takahashi M, Shiba Y et al: one marrow-derived CXCR4+ cells mobilized by macrophage colony-stimulating factor participate in the reduction of infarct area and improvement of cardiac remodeling after myocardial infarction in mice. m J Pathol, 2007, 171: Tachibana K, Hirota S, Iizasa H et al: The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract. Nature, 1998, 393: Schober, Karshovska E, Zernecke et al: SDF-1alphamediated tissue repair by stem cells: a promising tool in cardiovascular medicine? Trends Cardiovasc Med, 2006, 16: Salcedo R, Oppenheim JJ: Role of chemokines in angiogenesis: 328 脈管学 Vol. 50, 2010

7 2 CXCL12/SDF-1 and CXCR4 interaction, a key regulator of endothelial cell responses. Microcirculation, 2003, 10: Schober. Chemokines in vascular dysfunction and remodeling. rterioscler Thromb Vasc iol, 2008, 28: Shiba Y, Takahashi M, Ikeda U: Models for the study of angiogenesis. Curr Pharm Des, 2008, 14: Shiba Y, Takahashi M, Hata T et al: one marrow CXCR4 induction by cultivation enhances therapeutic angiogenesis. Cardiovasc Res, 2009, 81: Development of New Therapeutic ngiogenic Strategy Using SDF-1/CXCR4 System Masafumi Takahashi, Yuji Shiba, and Uichi Ikeda Department of Cardiovascular Medicine, Shinshu University Graduate School of Medicine, Nagano, Japan Division of ioimaging Sciences, Center for Molecular Medicine, Jichi Medical University, Tochigi, Japan Key words: cytokines, implantation, ischemia, paracrine effect, regeneration Recent studies demonstrated that implantation of bone marrow cells successfully promotes therapeutic angiogenesis. We found that upregulation of CXCR4, a receptor for chemokine SDF-1α, on bone marrow cells significantly enhances therapeutic angiogenesis in a murine model of hindlimb ischemia. These findings suggest that the SDF-1/CXCR4 system may serve as a useful source of MCs for accelerating therapeutic angiogenesis in ischemic cardiovascular diseases. (J Jpn Coll ngiol, 2010, 50: ) 脈管学 Vol. 50, 2010 Online publication ugust 20,