Induction of angiogenesis by lidocaine and basic fibroblast growth factor: a model for in vivo retroviral-mediated gene therapy.
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.
Jejurikar
S S
SS
Department of Surgery, University of Michigan Medical School, Ann Arbor 48109, USA.
Welling
T H
TH
Zelenock
J A
JA
Gordon
D
D
Burkel
W E
WE
Carlson
B M
BM
Messina
L M
LM
D000469:Alkaline Phosphatase / Q000235:genetics; D000818:Animals; D002196:Capillaries / Q000166:cytology / Q000187:drug effects / Q000254:growth & development; D002455:Cell Division / Q000187:drug effects; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects; D018014:Gene Transfer Techniques; D005819:Genetic Markers; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D008012:Lidocaine / Q000494:pharmacology*; D008297:Male; D008954:Models, Biological; D018482:Muscle, Skeletal / Q000098:blood supply / Q000187:drug effects; D018919:Neovascularization, Physiologic / Q000187:drug effects* / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D012190:Retroviridae / Q000235:genetics
D005819:Genetic Markers; D016222:Fibroblast Growth Factor 2; D008012:Lidocaine; D000469:Alkaline Phosphatase