Gene transfer to intact mesenteric arteries by electroporation - PubMed (original) (raw)

Gene transfer to intact mesenteric arteries by electroporation

J B Martin et al. J Vasc Res. 2000 Sep-Oct.

Abstract

The purpose of the present study was to develop a rapid, reproducible method of nonviral gene transfer to the intact vasculature. Male Sprague-Dawley rats were anesthetized, a midline abdominal incision was made and segmental branches of the superior mesenteric artery were dissected free of surrounding mesentery. A specially designed electroporation probe was placed around the neurovascular bundle and the electroporation chamber filled with a solution containing the firefly luciferase expressing plasmid (pCMV-Lux-DTS) or the green fluorescent protein expressing plasmid (pEGFP-N1). Vessels were electroporated with eight 10-ms pulses of 200 V/cm. Sixty seconds after electroporation, the DNA solution was removed, the intestine returned to the abdomen and the abdominal wall closed with suture and metal wound clips. Six hours to 5 days later, rats were sacrificed and electroporated vessels were recovered. Luciferase activity of the blood vessels was monitored. Gene expression was detected as early as 6 h postelectroporation, peaked at 1-3 days with levels up to 1 ng of reporter gene product per vessel segment and returned towards baseline by day 5. Histological analysis of blood vessel segments revealed green fluorescent protein-positive cells throughout the thickness of the vessel wall (endothelial cells to adventitia). Responses of electroporated vessels to vasoconstricting stimuli were indistinguishable from those of control vessels at either 2 or 40 days posttreatment. The results of this study provide evidence that electroporation is an effective means for introducing naked DNA into the blood vessel wall and form the basis for future studies on targeted gene therapy to the intact vasculature.

Copyright 2000 S. Karger AG, Basel

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Figures

Figure 1

Figure 1. Cartoon of vascular electrode

Nickel wires were set 3 mm apart from each other, flanking a channel cut into the top of the electrode in which the artery to be electroporated is placed. The total volume of the electrode’s chamber is 55 μl.

Figure 2

Figure 2. Voltage-dependency and time course of gene transfer and expression

Rat mesenteric arteries were placed into the electrode and incubated with pCMV-Lux-DTS (2 mg/ml) for several seconds prior to electroporation. (A) Square wave electroporations were performed using a train of 8 pulses of 10 millisecond duration each at field strengths of 0 to 400 V/cm. The arteries were removed 2 days later and luciferase activity was measured as described in Materials and Methods. (B) Vessels were electroporated with plasmid pCMV-Lux-DTS (2 mg/ml; 200 V/cm, 8 pulses, 10 millisecond duration). Arteries were removed at the indicated times and luciferase activities were determined. The bars represent the average expression of all arteries and the triangles represent the individual arteries. Vessels receiving no DNA are shown as “control”.

Figure 3

Figure 3. Luciferase expression in mesenteric arteries two days post-electroporation

Electroporations of pCMV-Lux-DTS (2 mg/ml) were performed using the standard settings (200 V/cm, 8 pulses, 10 millisecond duration). Control values represent arteries that received no DNA. In all experiments, arteries were electroporated in order from distal to proximal with respect to the ileocecal junction. Two days post-electroporation, vessels were removed and luciferase activities were measured as described in Materials and Methods. Differing numbers of arteries were electroporated in each animal, depending on the anatomy of the animals. Results from independent experiments are shown along the y-axis.

Figure 4

Figure 4. Distribution of gene expression in electroporated arteries

A mixture of pCMV-LUX-DTS and pEGFP (2mg/ml and 0.5 mg/ml, respectively) were transferred to mesenteric arteries with (A) or without (B) electroporation. After two days incubation, the arteries were removed and embedded for cryosectioning. Ten micron sections were prepared and GFP was visualized by fluorescence microscopy before the sections were stained with hemotoxylin and eosin. (C) GFP expression in successive 10 micron sections of an electroporated artery. All photographs were taken at an optical magnification of 25×, before computer analysis. Bars = 100 μm.

Figure 5

Figure 5. Vasoconstriction of electroporated arteries

Plasmid pCMV-LUX-DTS was transferred to rat mesenteric arteries by electroporation (8 pulses of 10 msec each; 200 V/cm). At 2 (A) or 40 (B) days post-transfer, the diameters of electroporated (filled bars) and untreated (“control”, open bars) vessels were determined by intravital microscopy using video calipers. Diameters are reported relative to baseline for the individual vessels, which ranged from 194 μm to 300 μm, n = 10 vessels. Average relative diameters ± standard deviations are shown for the vessels at each indicated concentration of phenylephrine. Vessels from 3 rats were used for measurements at day 2 and those from 2 rats were used for measurements at day 40.

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