Murasawa Satoshi, Llevadot Joan, Silver Marcy, Isner Jeffrey M, Losordo Douglas W, Asahara Takayuki
Department of Medicine (Vascular Medicine, Cardiology, Cardiovascular Research), St Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Mass 02135, USA.
Circulation. 2002 Aug 27;106(9):1133-9. doi: 10.1161/01.cir.0000027584.85865.b4.
The regulatory molecule for cell life span, telomerase, was modified by human telomerase reverse transcriptase (hTERT) gene transfer to investigate its effect on regenerative properties of endothelial progenitor cells (EPCs) in neovascularization.
Telomerase activity was enhanced in hTERT-transduced EPCs (Td-TERTs) (1.2-fold versus no transduced EPCs [no-Td] and 1.2-fold versus GFP-transduced EPCs [Td/GFPs] at day 8; 5.2-fold versus no-Td and 4.8-fold versus Td/GFP at day 21, respectively) Mitogenic capacity in Td/TERTs exceeded that in Td/GFPs at day 8 (0.62+/-0.02 versus 0.53+/-0.01, respectively; P<0.01). Vascular endothelial growth factor-induced cell migration in EPCs was markedly enhanced by hTERT overexpression (Td/TERTs versus Td/GFPs, 292+/-12 versus 174+/-6 cells, respectively; P<0.01). hTERT overexpression has rescued EPCs from starvation-induced cell apoptosis, an outcome that was further enhanced in response to vascular endothelial growth factor. The colony appearance of totally differentiated endothelial cells (tdECs) was detected before day 30 only in Td/TERT, whereas no tdEC colonies could be detected in both Td/GFPs and no-Tds. Finally, we investigated in vivo transplantation of heterologous EPCs. Td/TERTs dramatically improved postnatal neovascularization in terms of limb salvage by 4-fold in comparison with that of Td/GFPs; limb perfusion was measured by laser Doppler (0.77+/-0.10 versus 0.47+/-0.06; P=0.02), and capillary density (224+/-78 versus 90+/-40 capillaries/mm2; P<0.01).
These findings provide the novel evidence that telomerase activity contributes to EPC angiogenic properties; mitogenic activity, migratory activity, and cell survival. This enhanced regenerative activity of EPCs by hTERT transfer will provide novel therapeutical strategy for postnatal neovascularization in severe ischemic disease patients.
通过人端粒酶逆转录酶（hTERT）基因转移对细胞寿命的调节分子端粒酶进行修饰，以研究其对新生血管形成中内皮祖细胞（EPCs）再生特性的影响。
在hTERT转导的EPCs（Td - TERTs）中端粒酶活性增强（第8天时，与未转导的EPCs [未Td]相比增加1.2倍，与绿色荧光蛋白转导的EPCs [Td/GFPs]相比增加1.2倍；第21天时，分别与未Td相比增加5.2倍，与Td/GFP相比增加4.8倍）。第8天时，Td/TERTs的促有丝分裂能力超过Td/GFPs（分别为0.62±0.02对0.53±0.01；P<0.01）。hTERT过表达显著增强了血管内皮生长因子诱导的EPCs细胞迁移（Td/TERTs对Td/GFPs，分别为292±12对174±6个细胞；P<0.01）。hTERT过表达使EPCs免于饥饿诱导的细胞凋亡，这一结果在血管内皮生长因子作用下进一步增强。仅在Td/TERT中于第30天前检测到完全分化的内皮细胞（tdECs）的集落出现，而在Td/GFPs和未Td中均未检测到tdEC集落。最后，我们研究了异种EPCs的体内移植。与Td/GFPs相比，Td/TERTs在肢体挽救方面使出生后新生血管形成显著改善了4倍；通过激光多普勒测量肢体灌注（0.77±0.10对0.47±0.06；P = 0.02），以及毛细血管密度（224±78对90±40条毛细血管/mm2；P<0.01）。
这些发现提供了新的证据，表明端粒酶活性有助于EPCs的血管生成特性；促有丝分裂活性、迁移活性和细胞存活。hTERT转移增强的EPCs再生活性将为严重缺血性疾病患者出生后的新生血管形成提供新的治疗策略。
