196. Regional Angiogenesis Mediated by Platelet-Derived VEGF in Response to Thrombopoietin Gene Transfer

• Published in: Molecular therapy Vol. 9; no. S1; pp. S75 - S76
• Main Authors: Amano, Hideki, Hackett, Neil R, Shido, Koji, Tejada, Rafael, Avegilla, Scott, Rafii, Shahin, Crystal, Ronald G
• Format: Journal Article
• Language: English
• Published: Milwaukee Elsevier Inc 01.05.2004; Elsevier Limited
• Subjects: Angiogenesis; Blood platelets; Cytokines; Disease; Gene therapy; Hypoxia; Investigations; Ischemia; Vascular endothelial growth factor; Vectors (Biology); Veins & arteries
• ISSN: 1525-0016; 1525-0024
• DOI: 10.1016/j.ymthe.2004.06.104

Vascular endothelial growth factor (VEGF), a potent mediator of angiogenesis, has been used in gene transfer experiments to enhance blood flow to ischemic tissues. A major source of endogenous VEGF is the platelet, a blood element known to contain substantial reserves of VEGF. In this study we evaluated the hypothesis that gene transfer of thrombopoietin (TPO; the systemic signal for megakaryocyte differentiation) is angiogenic due to an increase in the level of platelets which provide a source of VEGF that mediates local angiogenesis. The impact of quadriceps administration of AdmTPO (E1-E3- adenovirus vector expressing the murine TPO cDNA behind a CMV promoter), on recovery of ischemic hind limb blood flow in mice following excision of a section of external iliac artery was assessed by Doppler scanning. On day 28 after ligation, blood flow to the treated limb, expressed as the flow ratio between the ischemic and control limb, recovered to 0.38 ± 0.10 for the control group treated with 10 8 particle units (pu) of AdNull (identical to AdmTPO but with no transgene) while treatment with 108 pu of AdmTPO mediated recovery to a flow ratio of 0.83 ± 0.06. (p<0.001). The platelet count 2 wk following administration of 108 pu of vector was 2000 ± 97 × 103 /ml for AdmTPO group vs 731 ± 64 x 103 /μl for the AdNull group (p<0.01). Serum VEGF levels measured by ELISA were 181 ± 9 pg/ml for the AdmTPO group and 101 ± 9 pg/ml for the AdNull group (p<0.05). Immunohistochemical staining of the injected muscle for PECAM positive endothelial cells revealed that AdmTPO injected mice had an increased PECAM positive cell per muscle fiber ratio (2.8 ± 0.8) compared to AdNull treated mice (1.5 ± 0.7, p<0.05). If platelets act as a source of VEGF for endogenous angiogenesis, we predicted that TPO knockout (mice which have low platelet levels) would have impaired recovery from hind limb ischemia. Spontaneous recovery of flow ratio in untreated wild type mice after 28 days was 0.56 ± 0.08 for wild type mice versus 0.27 ± 0.07 for TPO knockout mice (p < 0.001). To demonstrate that the observed effects were mediated by VEGF released by the platelets, antibodies that block the interaction of VEGF with the three VEGF receptors were administered 5 times per week following artery excision and treatment by AdmTPO. Recovery from ischemia as assessed by flow ratio at 28 days was 0.88 ± 0.04 for mice treated with a control non-specific IgG while treatment with anti-VEGFR1 gave a flow ratio of 0.37 ± 0.04 (p < 0.001), anti-VEGFR2 gave a flow ratio 0.47 ± 0.06 (p < 0.001) and anti-VEGFR3 gave a flow ratio of 0.78 ± 0.08 (p > 0.08). These results suggest that TPO can induce angiogenesis by elevating platelet levels that act as a local source of VEGF and suggests possible roles of thrombopoietic factors in angiogenic therapy.Dr. Crystal has equity in, is a consultant to, and receives sponsored research funds from, Gen Vec, Inc., Gaithersburg, Maryland, a publicly-traded biotechnology company.