Jejurikar S S, Welling T H, Zelenock J A, Gordon D, Burkel W E, Carlson B M, Messina L M
Department of Surgery, University of Michigan Medical School, Ann Arbor 48109, USA.
J Surg Res. 1997 Feb 1;67(2):137-46. doi: 10.1006/jsre.1996.4989.
A strategy of direct, in vivo retroviral-mediated gene therapy targeting capillary endothelial cells must provide an environment of active angiogenesis. Both lidocaine and basic fibroblast growth factor (bFGF) promote angiogenesis, but the angiogenic response invoked by these substances in normal skeletal muscle has not been fully characterized. We sought to characterize these agents' angiogenic effects in anterior tibialis muscles of male Sprague-Dawley rats. An injection of either 1% lidocaine with 1:100,000 epinephrine or alternate-day injections of bFGF (0.025 or 0.25 microgram) with or without heparin were tested (n = 6 muscles/condition). Rats were sacrificed 4, 7, 10, or 12 days later and muscles were evaluated histologically to determine the number of proliferating cells using 5-bromo-2'-deoxycytidine (BrdC) and evaluated for capillary density using Griffonia simplicifolia I (GSI) lectin. At all time points, lidocaine produced at least 20-fold greater capillary density and cellular proliferation than PBS control (P < 0.0001). Injections of high-dosage bFGF produced more than fivefold greater capillary density than control injections at 7 and 10 days (P < 0.001), and more than twofold greater proliferation at 4, 7, and 12 days (P < 0.001). Capillary density returned to control levels 12 days following bFGF administration, whereas it remained well above control levels for 12 days after lidocaine administration. To confirm that lidocaine can be utilized in gene therapy strategies targeting vascular endothelium and skeletal muscle fibers, concentrated pLJ retrovirus containing cDNA for the heat-stable human placental alkaline phosphatase (hpAP) marker gene was infused into the rat hindlimb vasculature 4 days post-lidocaine administration. Rats receiving pLJhpAP retrovirus demonstrated significant hpAP transgene expression in endothelial cells and myocytes 21 days after the lidocaine injection (n = 6 muscles). In contrast, controls receiving pLJhpAP infusion without prior lidocaine administration failed to demonstrate any hpAP transgene expression. Lidocaine treatment evokes a substantially higher proliferative response than bFGF and, importantly, a durable angiogenic response in skeletal muscle. Thus, lidocaine is an ideal agent to induce angiogenesis in preparation for direct in vivo retroviral-mediated gene therapy targeting vascular endothelium.
一种针对毛细血管内皮细胞的直接体内逆转录病毒介导的基因治疗策略必须提供一个活跃的血管生成环境。利多卡因和碱性成纤维细胞生长因子（bFGF）均可促进血管生成，但这些物质在正常骨骼肌中引发的血管生成反应尚未得到充分表征。我们试图在雄性Sprague-Dawley大鼠的胫前肌中表征这些药物的血管生成作用。测试了注射含1:100,000肾上腺素的1%利多卡因，或隔日注射bFGF（0.025或0.25微克）加或不加肝素的情况（每种情况n = 6块肌肉）。4、7、10或12天后处死大鼠，对肌肉进行组织学评估，使用5-溴-2'-脱氧胞苷（BrdC）确定增殖细胞数量，并使用西非豆凝集素I（GSI）凝集素评估毛细血管密度。在所有时间点，利多卡因产生的毛细血管密度和细胞增殖比PBS对照至少高20倍（P < 0.0001）。高剂量bFGF注射在第7天和第10天时产生的毛细血管密度比对照注射高五倍以上（P < 0.001），在第4、7和12天时产生的增殖比对照高两倍以上（P < 0.001）。bFGF给药后12天毛细血管密度恢复到对照水平，而利多卡因给药后12天毛细血管密度仍远高于对照水平。为了证实利多卡因可用于针对血管内皮和骨骼肌纤维的基因治疗策略，在利多卡因给药4天后，将含有热稳定人胎盘碱性磷酸酶（hpAP）标记基因cDNA的浓缩pLJ逆转录病毒注入大鼠后肢血管系统。接受pLJhpAP逆转录病毒的大鼠在利多卡因注射21天后在内皮细胞和肌细胞中显示出显著的hpAP转基因表达（n = 6块肌肉）。相比之下，未预先给予利多卡因而接受pLJhpAP注射的对照未显示任何hpAP转基因表达。利多卡因治疗引发的增殖反应比bFGF高得多，重要的是，在骨骼肌中引发持久的血管生成反应。因此，利多卡因是一种理想的药物，可用于诱导血管生成，为针对血管内皮的直接体内逆转录病毒介导的基因治疗做准备。
