Steve Leu1, Hung-I Lu2, Cheuk-Kwan Sun3, Jiunn-Jye Sheu2, Yung-Lung Chen4, Tzu-Hsien Tsai4, Kuo-Ho Yeh4, Han-Tan Chai4, Sarah Chua4, Ching-Yen Tsai5, Hsueh-Wen Chang6, Fan-Yen Lee2, Hon-Kan Yip7. 1. Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan. 2. Division of Thoracic and Cardiovascular Surgery, Department of Surgery, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan. 3. Department of Emergency Medicine, E-Da Hospital, I-Shou University, Kaohsiung, Taiwan. 4. Division of Cardiology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan. 5. Institue of Molecular Biology, Academia Sinica, Taipei, Taiwan. 6. Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan. 7. Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan; Division of Cardiology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan. Electronic address: han.gung@msa.hinet.net.
Abstract
BACKGROUND: This study tested the hypothesis that tissue plasminogen activator (tPA) is crucial for regulating endothelial progenitor cell (EPC) mobilization from bone marrow to circulation in murine critical limb ischemia (CLI) by ligating the left femoral artery. METHODS: Wild-type (C57BL/6) (n=40) mice were equally divided into group 1A (sham control), group 2A (CLI), group 3A [control-tPA (4.0 mg/kg)] and group 4A [CLI-tPA (intravenously at 3 h after CLI)]. Similarly, tPA knock-out (tPA(-/-)) mice (n=40) were equally divided into group 1B (sham control), group 2B (CLI), group 3B [control-tPA (4.0 mg/kg)], and group 4B (CLI-tPA). RESULTS: The circulating levels of EPCs (C-kit/CD31+, Sca-1/KDR+, CXCR4/CD34+) were lower in groups 1B and 2B than in groups 1A and 2A, respectively (all p<0.01), and were reversed after tPA treatment (3B vs. 3A or 4B vs. 4A, p>0.05) at 6 h and 18 h post-CLI. Levels of these biomarkers decreased again 14 days after CLI in tPA(-/-) mice compared to those in wild-type between the respective groups (all p<0.01). Laser Doppler flowmetry showed a higher ratio of ischemic-to-normal blood flow in 2A than in 2B and in 4A than in 4B by day 14 after CLI (all p<0.05). Angiogenesis at protein (CXCR4, SDF-1α, VEGF) and cellular (CXCR4+, SDF-1α+, and CD31+ cells) levels was highest in animals with CLI-tPA, significantly higher in mice with CLI only than in sham controls for both wild-type and tPA(-/-) mice (p<0.01). CONCLUSION: tPA played an essential role in augmenting circulating EPCs, angiogenesis, and blood flow in the ischemic limb in a murine model.
BACKGROUND: This study tested the hypothesis that tissue plasminogen activator (tPA) is crucial for regulating endothelial progenitor cell (EPC) mobilization from bone marrow to circulation in murine critical limb ischemia (CLI) by ligating the left femoral artery. METHODS: Wild-type (C57BL/6) (n=40) mice were equally divided into group 1A (sham control), group 2A (CLI), group 3A [control-tPA (4.0 mg/kg)] and group 4A [CLI-tPA (intravenously at 3 h after CLI)]. Similarly, tPA knock-out (tPA(-/-)) mice (n=40) were equally divided into group 1B (sham control), group 2B (CLI), group 3B [control-tPA (4.0 mg/kg)], and group 4B (CLI-tPA). RESULTS: The circulating levels of EPCs (C-kit/CD31+, Sca-1/KDR+, CXCR4/CD34+) were lower in groups 1B and 2B than in groups 1A and 2A, respectively (all p<0.01), and were reversed after tPA treatment (3B vs. 3A or 4B vs. 4A, p>0.05) at 6 h and 18 h post-CLI. Levels of these biomarkers decreased again 14 days after CLI in tPA(-/-) mice compared to those in wild-type between the respective groups (all p<0.01). Laser Doppler flowmetry showed a higher ratio of ischemic-to-normal blood flow in 2A than in 2B and in 4A than in 4B by day 14 after CLI (all p<0.05). Angiogenesis at protein (CXCR4, SDF-1α, VEGF) and cellular (CXCR4+, SDF-1α+, and CD31+ cells) levels was highest in animals with CLI-tPA, significantly higher in mice with CLI only than in sham controls for both wild-type and tPA(-/-) mice (p<0.01). CONCLUSION:tPA played an essential role in augmenting circulating EPCs, angiogenesis, and blood flow in the ischemic limb in a murine model.