© 1997 by European Society of Cardiology
Copyright © 1997, European Society of Cardiology
Arterial gene transfer of acidic fibroblast growth factor for therapeutic angiogenesis in vivo: critical role of secretion signal in use of naked DNA
Department of Medicine (Cardiology) and Biomedical Research, St. Elizabeth's Medical, Center of Boston, Tufts University School of Medicine, 736 Cambridge St., Boston, MA 02135, USA
* Corresponding author. Tel.: +1 617 7892392; Fax: +1 617 7895029; E-mail: jisner@opal.tufts.edu
Received 17 February 1997; accepted 12 May 1997
 |
Abstract |
|---|
 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionReferences |
|---|
Objective: Previous studies have demonstrated that arterial gene transfer of naked DNA encoding for a secreted protein may permit modulation of the host phenotype despite a low transfection efficiency. Acidic fibroblast growth factor (aFGF) is an angiogenic growth factor, but is not secreted by intact cells. In the current study, we investigated the hypothesis that addition of a hydrophobic leader sequence to achieve active secretion of the gene product would permit therapeutic angiogenesis following arterial gene transfer of naked DNA encoding for aFGF. Methods: Ten days following surgical induction of unilateral hindlimb ischemia, New Zealand white rabbits were randomized to intra-arterial gene transfer with one of three plasmids: p267 (encoding non-secreted aFGF, n = 10), pMJ35 (encoding secreted aFGF) (n = 10), or 500 µg of pGSVLacZ (control, n = 10) (500 µg each). All animals were studied at 30 days post-gene transfer for evidence of therapeutic angiogenesis. Results: pMJ35 transfectants had more angiographically visible collaterals (angiographic score=0.76±0.02) than either p267 (0.55±0.02, p<0.01) or LacZ (0.47±0.02, p<0.001). Limb blood pressure ratio for pMJ35 was 0.88±0.02 vs. 0.68±0.04 for p267 (p<0.01) and 0.57±0.04 for LacZ (p<0.001). Vascular resistance was significantly lower in the pMJ35 group, compared with that in pGSVLacZ group, both in resting state (3.2±0.4 vs. 7.4±1.4 respectively, p<0.05) and after the administration of nitroprusside. Capillary density (per mm2) was also superior in pMJ35 group (274±10) vs. p267 (204±9, p<0.01) and LacZ (177±6, p<0.001). Conclusion: The paracrine effects of a secreted gene product may obviate the need for adjunctive vectors in strategies of arterial gene therapy.
KEYWORDS Acidic fibroblast growth factor (aFGF); Gene therapy; Angiogenesis; Signal peptide
|
1 Introduction |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
|---|
The success of gene therapy as a clinically applicable modality is dependent upon several elements: these include the disease target, the gene product, and the proportion of host cells which can be successfully transfected. The latter, conventionally referred to as ‘transfection efficiency’, has been considered the Achilles’ heel of gene therapy. Because the transfection efficiency associated with the transfer of naked DNA to most tissues is extremely low [1], the development of novel vectors designed to transfect more of the target cells has become the central focus of gene therapy research [2].
Unfortunately, animal and/or clinical testing of most novel vectors developed to date have suggested that improvements in transfection efficiency may come at the price of vector-dependent toxicity. Replication-defective adenovirus, for example, have achieved wide popularity as an effective adjunct for gene therapy, but have been poorly tolerated by a variety of species due to immunologic toxicity [3].
While naked DNA cannot match the transfection efficiency achieved with viral vectors, the virtue of this strategy is that it has thus far proved to be devoid of serious toxicity [4]. Moreover, in certain animal and clinical trials of gene therapy, the use of naked DNA has proved sufficient to achieve phenotypic modulation of the host organism [5–7]. Analysis of these results has suggested that this is due in part to certain features of the protein encoded by the transgene. In particular, studies in our laboratory [8, 9]have documented that genes which encode for secreted proteins — as opposed to proteins which remain intracellular — may yield meaningful biological outcomes due to paracrine effects of the secreted gene product.
Acidic fibroblast growth factor (aFGF), is an endothelial cell mitogen. When administered as recombinant protein, aFGF has been shown to stimulate angiogenesis in vivo [10–12]and promote repair of damaged endothelium [13]. The structure of the aFGF translation product does not possess a classical secretory signal sequence [14, 15]. A secreted form of the aFGF, however, has been engineered by ligation of the signal sequence from the human fibroblast interferon gene to the first encoded residue (methionine) of aFGF [16].
In the current study, we used constructs encoding non-secreted and secreted forms of aFGF to test the hypothesis that the success of naked DNA gene transfer is dependent upon the presence of a signal sequence permitting protein secretion.
|
2 Methods |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
|---|
2.1 Animal model
We used a rabbit ischemic hindlimb model that has been described previously [17], to investigate the development of therapeutic angiogenesis. All protocols were approved by St. Elizabeth's Institutional Animal Care and Use Committee. Thirty male New Zealand White rabbits weighting 4–4.5 kg (Pine Acre Rabbitry, Norton, MA) were anesthetized with a mixture of ketamine (50 mg/kg) and acepromazine (0.8 mg/kg) following premedication with xylazine (2.5 mg/kg). A longitudinal incision was then performed, extending inferiorly from the inguinal ligament to a point just proximal to the patella. The limb in which the incision was performed — right versus left — was determined at random at the time of surgery by the operator. Through this incision, with surgical loops, the femoral artery was dissected free along its entire length; all branches of the femoral artery, including the inferior epigastric, deep femoral, lateral circumflex and superficial epigastric arteries, were also dissected free.
After further dissecting the popliteal and saphenous arteries distally, the external iliac artery as well as all above arteries were ligated with 4.0 silk (Ethicon, Sommerville, NJ). Finally, the femoral artery was completely excised from its from its proximal origin as a branch of the external iliac artery, to the point distally where it bifurcates into the saphenous and popliteal arteries. The excision of the femoral artery results in retrograde propagation of thrombus to the origin of the external iliac artery. Consequently, blood flow to the ischemic limb is dependent upon collateral vessels which may originate from the internal iliac artery.
2.2 Recombinant plasmid
The recombinant expression vector p267 (paFGF), containing the human aFGF cDNA, encodes the 16-kDa aFGF [18]. The cDNA sequence was cloned under the control of the simian virus 40 early promoter (SV40 ep) and the cytomegalovirus (CMV) enhancer, with downstream SV40 regulatory sequences. A similar construct, pMJ35 (pSP-aFGF), containing a heterologous signal peptide (SP) sequence coding for 21 amino acids 5' to the aFGF cDNA was constructed. This construct joins the SP of human fibroblast interferon [19]to the first residue (methionine) of the encoded aFGF (Fig. 1) [16]. These recombinant plasmids, pMJ35 and p267, were the gift of Dr. Michael Jaye (Rhone–Poulenc Rorer Central Research, Collegeville, PA). The plasmid pGSVLacZ (courtesy of Dr. Claire Bonnerot) containing a nuclear targeted β-galactosidase sequence coupled to the simian virus 40 early promoter [20]was used for the control transfection experiment.
|
2.3 Percutaneous arterial gene transfer
An interval of 10 days between the time of surgery and the gene transfer was allowed for post-operative recovery of rabbits and development of endogenous collateral vessels. Beyond this timepoint, studies performed up to 90 days post-operatively [17]have demonstrated no significant collateral vessel augmentation. At 10 days post-operatively (day 0), after performing a baseline angiogram, the internal iliac artery of the ischemic limb was transfected with recombinant plasmid percutaneously using a 2.5 mm hydrogel-coated balloon catheter (Slider with Hydroplus, Boston Scientific, Watertown, MA) [21]. The angioplasty balloon was prepared (ex vivo) by first advancing the deflated balloon through a 5 Fr. Teflon sheath (Boston Scientific), applying of plasmid to the 20 µm-thick layer of hydrogel on the external surface of the inflated balloon, and then retracting the inflated balloon back into the protective sheath. The sheath and angioplasty catheter were then introduced to the lower abdominal aorta using a 0.036 mm (0.014 inch) guide wire (Hi-Torque Floppy II, Advanced Cardiovascular Systems, Temecula, CA) under fluoroscopic guidance. The balloon catheter was then advanced into the internal iliac artery of the ischemic limb, inflated for 3 min at 6 atmospheres, deflated, and withdrawn. Heparin was not administered at the time of transfection or angiography.
A total 30 rabbits were transfected with the following plasmids: 500 µg of pMJ35 (secreted type aFGF DNA) (n = 10); p267 (non-secreted type aFGF DNA) (n = 10); and pGSVLacZ (n = 10). Previous studies performed using this technique of naked DNA arterial gene transfer in this animal model have repeatedly documented a transfection efficiency of <1% [5, 6, 21, 22].
2.4 Reverse transcription-polymerase chain reaction (RT-PCR)
We evaluated the time course of gene expression for human aFGF mRNA using RT-PCR. Rabbit iliac arteries transfected with pMJ35 were harvested at days 3, 7, 14, 21, and 30 post-transfection (n = 2 for each time point). Total cellular RNA was isolated from the sample using ULTRASPEC RNA (Biotech Laboratories, Houston, TX) according to the manufacturer's instructions. Extracted RNA was treated with Dnase 1 (0.5 µl, 10 U/µl, Rnase-free, Message Clean kit, GenHunter, Boston, MA) at 37°C for 30 min to eliminate DNA contamination. The yield of extracted RNA was determined spectrophotometrically by ultraviolet absorbance at 260 nm. One µg of each RNA sample was used to make cDNA in a reaction volume of 20 µl containing 0.5 mM each of deoxynucleotide triphosphate (Pharmacia, Piscataway, NJ), 10 mM dithioyhreitol, 20 units of Rnasin (Promega, Madison, WI), 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 0.5 µg oligo primers (Promega), and 200 units of M-MLV reverse transcriptase (GIBCO BRL). Reactions were incubated at 42°C for 1 h, then 95°C for 5 min to terminate the reaction.
Primers were designed according to conserved regions of the known human aFGF sequence [14]: aFGF-1 (5-AAT TAC AAG AAG CCC AAA CTC-3) and aFGF-2 (5-AGA CTG GCA GGG GGA GAA A-3) are homologous to the human aFGF sequence inserted into the pMJ35 and p267. In GeneAmp reaction tubes cDNA equivalent to 1 µg of total RNA was dissolved in 50 µl containing 1xPCR buffer, 2.5 mM MgCl2, 25 pmoles aFGF-1 and aFGF-2 primer and 1.25 units Taq polymerase (Perkin Elmer, Branchburg, NJ). The PCR was performed on a 9600 PCR system (Perkin Elmer). Amplification included 30 step cycles: denaturing for 60 s at 94°C, annealing for 60 s at 60°C, and extension for 120 s at 72°C. The final incubation was extended to 6 min. The sample was then rapidly cooled to 4°C and kept on ice for analysis by 1.5% agarose gel electrophoresis. DNA bands were visualized under UV illumination after staining with ethidium bromide.
To ensure that negative PCRs were not due to markedly different starting concentration of mRNA, PCR analysis for constitutively expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was performed on serial cDNA used in each PCR.
2.5 Evaluation of angiogenesis in the ischemic limb
Development of collateral vessels in the ischemic limb was serially evaluated by limb blood pressure measurement and internal iliac arteriography immediately prior to transfection (day 0), and then day 30 post-transfection. On each occasion, it was necessary to lightly anesthetize the animal with a mixture of ketamine (10 mg/kg) and acetapromazine (0.16 mg/kg) following premedication with xylazine (2.5 mg/kg). Following the final 30 day follow-up, the animal was sacrificed, and the tissue sections were prepared from the hindlimb muscles in order to perform analysis of capillary density and microsphere analysis. These analyses are discussed in detail below.
2.5.1 Limb blood pressure ratio
Limb blood pressure was measured in both hindlimbs using a Doppler Flowmeter (Model 1050, Parks Medical Electronics, Aloha, OR), immediately prior to transfection (day 0) as well as on day 30. On each occasion, the hindlimbs were shaved and cleaned; the pulse of posterior tibial artery was identified using standard techniques [23]. The limb blood pressure ratio was defined for each rabbits as the ratio of systolic pressure of the ischemic limb to systolic pressure of the ischemic limb to systolic pressure of the normal limb.
2.5.2 Selective internal iliac arteriography
Collateral artery development in this ischemic hindlimb model originates from the internal iliac artery. Accordingly, selective internal iliac arteriography was performed on day 0 (immediately prior to transfection) and on day 30 post-transfection as previously described [23]. A 3 Fr. end-hole infusion catheter (Tracker-18, Target Therapeutics, San Jose, CA) was introduced into the common carotid artery through a small cutdown, and advanced to the internal iliac artery of the ischemic limb using a 0.356 mm guide wire (Hi-Torque Floppy II) under fluoroscopic guidance. The tip of the catheter was positioned in the internal iliac artery at the level of the interspace between the seventh lumbar and the first sacral vertebrae. Following intra-arterial injection of nitroglycerin (0.25 mg, SoloPark Laboratories, Franklin Park, IL), a total of 5 ml of contrast media (Isovue-370, Squibb Diagnostics, New Brunswick, NJ) was then injected using an automated angiographic injector (Medrad, Pittsburgh, PA) programmed to reproducibly deliver a flow rate of 1 ml/s. Serial images of the ischemic hindlimb were recorded on 105-mm spot film at a rate of 1 film/s for at least 10 s. Following completion of arteriography, the catheter was removed and the wound was closed. All of the above-described procedures were completed without the use of heparin.
Morphometric angiographic analysis of collateral vessel development was performed on the 4-s angiogram. To assess the number of collateral vessels, we used a grid overlay comprised of 2.5 mm-diameter circles arranged in rows spaced 5 mm apart. This acetate overlay was placed over the angiogram recorded at the level of the medial thigh. The number of contrast-opacified arteries crossing over circles as well as the total number of circles encompassing the medial thigh area were counted by a single observer blinded to the treatment regimen. An angiographic score was calculated for each film as the ratio of grid intersections crossed by opacified arteries divided by the total number of grid intersections in the medial thigh.
2.5.3 Measurement of blood flow in ischemic hindlimb
Blood flow was assessed on day 30 in the ischemic hindlimb with use of a Doppler guide wire, immediately before selective internal iliac arteriography. As previously described [24–28], a 0.457 mm (0.018 inch) Doppler guide wire (Cardiometrics, Inc., Mountain View, CA) was advanced through the 3F infusion catheter (Tracker-18) to the proximal segment of the internal iliac artery supplying the ischemic limb. The Doppler wire recorded a real-time spectral analysis of the Doppler signal, from which the average peak velocity (APV) (the temporal average of the instantaneous peak velocity waveform) was calculated and displayed on-line. A second 3F perfusion catheter was introduced into the common carotid artery through the same cutdown and advanced under fluoroscopic guidance to the origin of the common iliac artery of the ischemic limb with a 0.356 mm guide wire (Hi-Torque Floppy II). This catheter was used for infusion of sodium nitroprusside (Sigma, St. Louis, MO), for direct measurement of intra-arterial blood pressure via connection to a pressure transducer (model 78534C, Hewlett Packard, Andover, MA), and for selective angiography of the ischemic limb. Blood pressure was monitored before and immediately after sodium nitroprusside administration. Angiography was performed before and after drug administration with 1 ml of contrast media (Isovue-370). Serial images of the ischemic limb were recorded on 105-mm spot film at a rate of two films per second for 5 s. Sodium nitroprusside was administered intra-arterially via a constant infusion pump (1 ml/min) at doses of 1.5 µg kg–1 min–1 for 2 min, as previously reported [29, 30].
The vascular diameter was measured at the site of the Doppler sample volume (5 mm distal to the wire tip [24]). Cross-sectional area was calculated assuming a circular lumen. Doppler-derived flow was calculated as QD=(
d2/4) (0.5xAPV), where QD is Doppler-derived time average flow (ml/min), d is vessel diameter, and APV is time average of the spectral peak velocity [24]. The mean velocity was estimated as 0.5xAPV by assuming a time-averaged parabolic velocity profile across the vessel. Vascular resistance was calculated as RE=PE/QD, where RE is vascular resistance and PE is intra-arterial blood pressure.
2.5.4 Capillary density and capillary/muscle fiber ratio
The angiogenic effect of aFGF at the microvascular level was examined by measuring the number of capillaries in light microscopic sections taken from the ischemic hindlimbs. Tissue specimens were obtained as transverse sections from the adductor muscle and the semimembranosus muscle of both limbs of each animal at the time of sacrifice (day 30 post-transfection). These two muscles were chosen for light microscopic analysis because (1) they are 2 major muscles of the medial thigh, and (2) each was originally perfused by the deep femoral artery, ligated at the time that the femoral artery was excised. Muscle samples were embedded in O.C.T. compound (Miles, Elkhart, IN) and snap frozen in liquid nitrogen. Multiple frozen sections (5 µm in thickness) were then cut from each specimen on a cryostat (Miles), so that the muscle fibers were oriented in a transverse fashion, and two sections were then placed on glass slides. Tissue sections were stained for alkaline phosphatase using an indoxyl-tetrazolium method to detect capillary endothelial cells as previously described, [31]and were then counterstained with eosin. Capillaries were counted under 20x objective to determine the capillary density (mean number of capillaries per mm2). A total of 20 different fields from the two muscles were randomly selected, and the number of capillaries counted. To ensure that analysis of capillary density was not overestimated due to muscle atrophy, or underestimated due to interstitial edema, capillaries identified at necropsy were also evaluated as a function of muscle fibers in histologic section. The counting scheme used to compute the capillary/muscle fiber ratio was otherwise identical to that used to compute capillary density.
2.5.5 Measurement of regional blood flow
To evaluate the regional blood flow [32], colored microspheres (Dye–Track, Triton Technology, San Diego, CA) [29, 33]were injected into the left ventricle at day 30 immediately before Doppler guide wire analysis and angiography. Reference arterial blood samples were collected through the aortic catheter starting 10 s before injection of the microspheres and continuing for 120 s at the rate of 2 ml/min. Two or more tissue specimens were obtained from the adductor muscle, the semimembranosus muscle, and the gastrocnemius muscle at the time of sacrifice. Samples were placed into teflon-sealed 16 ml screw-cap tubes and 7 ml of a 4 molar KOH solution containing 2% Tween 80 were added to each sample for digestion of the tissue. The digested sample solution underwent vacuum filtration using a polyester filter (Triton Technology). The tightly folded filter with entrapped spheres, was placed in a 1.5 ml Eppendorf tube and 150 µl of dimethyl-formamide were added. The container was capped and vortex mixed for 30 s, followed by 3 min of centrifugation at 2000 g. Then the solution was transferred to a spectrophotometer cuvette for photometric absorption analysis (Model 8452A spectrophotometer, Hewlett Packard, Andover, MA). Blood flow was calculated with the following equation:
|
|
2.6 Dose–response of pMJ35-induced angiogenesis
To evaluate the dose–response relationship of pMJ35, we transfected 100, 200, 300, 400, 500, 700, 1000 µg of pMJ35 to the rabbit ischemic model (n = 2 for each dose) and analyzed the limb blood pressure ratio, angiographic score, capillary density, and regional blood flow using colored microspheres at 30 days post-transfection. All procedures followed the same protocols above.
2.7 Histologic assessment of transfected arterial segment
To determine whether transfection of pMJ35 or p267 could affect neointimal thickening, transfected iliac arteries were harvested at necropsy (day 30). The vessels were fixed with methanol and embedded in paraffin. Sections were stained with hematoxylin-eosin and elastic trichrome.
Neointimal thickening was assessed in terms of intima area to medial area ratio (I/M). Histologic sections were projected onto a digitizing board (Summagraphic Corp., Fairfield, CT), and values for intima and media areas were calculated by a technician blinded to treatment regimen, using a computerized sketching program (MACMEASURE, version 1.9, NIMH, Bethesda, MD).
2.8 Statistical analysis
All results are expressed as mean±standard error (m±SE). Statistical comparisons were performed with the use of ANOVA. When a significant difference was detected, multiple-comparison analysis was performed using Scheffe's procedure. A value of p<0.05 was considered to denote statistical significance.
|
3 Results |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
|---|
3.1 Time course of human aFGF gene expression
To determine the time course of human aFGF transgene expression, rabbit iliac arteries transfected with pMJ35 were harvested at days 3, 7, 14, 21, 30 post-transfection (n = 2 for each timepoint), and analyzed for aFGF mRNA using RT-PCR. Human fetus brain was used as a positive control. Human aFGF mRNA expression was observed in the arteries harvested at days 3, 7, 14, 21 post-transfection. In arteries harvested at day 30 post-transfection, however, aFGF gene expression was not observed. No aFGF gene expression was observed in the arteries transfected with pGSVLacZ
(Fig. 2). In other organs, such as liver, heart, lung, spleen, testis, kidney, muscle, and non-transfected artery harvested at day 3 post-pMJ35 transfection, aFGF gene expression was not observed.
|
3.2 Angiographic assessment
Fig. 3 illustrates a representative internal iliac angiogram at day 30, recorded from pMJ35, p267, and pGSVLacZ transfected animals. Serial assessment of number of angiographically visible collateral vessels (angiographic score as described above) showed a progressive increase throughout the follow-up period in all three groups. At baseline (day 0), there was no significant difference in angiographic score among the pMJ35, p267, and pGSVLacZ transfected groups (0.288±0.01 vs. 0.287±0.01 vs. 0.288±0.01, p = ns). By day 30, however, the angiographic score in pMJ35 transfected group (0.76±0.02) was significantly higher than p267 group (0.55±0.02, p<0.01) and pGSVLacZ group (0.47±0.02, p<0.001).
|
3.3 Limb blood pressure ratio
Reduction of the hemodynamic deficit in the ischemic limb following pMJ35 transfection was confirmed by measurement of limb blood pressure ratio (ischemic/normal limb). Limb blood pressure ratio was similar in all three groups prior to transfection (day 0) (0.39±0.02 in pMJ35, vs. 0.35±0.02 in p267, vs. 0.35±0.02 in pGSVLacZ, p = ns). At day 30, the blood pressure ratio for pMJ35 transfected group (0.88±0.02) was significantly higher than p267 group (0.68±0.04, p<0.01) and pGSVLacZ group (0.57±0.04, p<0.001).
3.4 Intravascular Doppler wire measurements of blood flow in the ischemic limb
At day 30, average peak velocity (AVP) and intra-aortic blood pressure were recorded at rest and after administration of sodium nitroprusside. At the same time, serial measurements of angiographic luminal diameter were made in the internal iliac artery at the site from which the Doppler measurements of flow were obtained. From these measurements, blood flow and vascular resistance were calculated. Blood flow at rest was not different among the three groups (24.8±3.6 ml/min in pMJ35, vs. 16.6±2.2 in p267, vs. 14.8±2.9 in pGSVLacZ). However, maximum flow provoked by intra-aortic administration of nitroprusside was significantly higher in the pMJ35 group (49.6±7.6 ml/min) than in the pGSVLacZ group (25.0±5.7 ml/min) (p<0.05) (Fig. 4a).
|
In contrast, vascular resistance was significantly lower in the pMJ35 group, compared with that in pGSVLacZ group both in resting state (3.2±0.4 vs. 7.4±1.4 respectively, p<0.05) and after the administration of nitroprusside (1.5±0.2 vs. 4.3±1.1, respectively, p<0.05) (Fig. 4b).
3.5 Capillary density and capillary/muscle fiber ratio
A favorable effect of pMJ35 transfection was also shown at the capillary level. The medial thigh muscles of the ischemic limbs were examined histologically at day 30 after transfection, as described above. Mean value of capillary density in pMJ35 group (274±10/mm2) was significantly higher than that in p267 group (204±9 mm2, p<0.01) and that in pGSVLacZ group (177±6 mm2, p<0.001) (Fig. 7). Analysis of capillary/muscle fiber ratio disclosed a value of 0.90±0.02 in the pMJ35 group versus 0.66±0.03 in the p267 group (p<0.001) and versus 0.54±0.02 in the pGSVLacZ group (p<0.001).
|
3.6 Measurement of regional blood flow using colored microspheres
Regional blood flow was assessed using colored microspheres described above. In the ischemic adductor muscle, the regional blood flow of the pMJ35 group (6.0±1.7 ml/min/100 g tissue) was significantly higher than that of the p267 group (3.0±0.4, p<0.05), as well as that of the pGSVLacZ group (1.9±0.4, p<0.05). In the ischemic semimembranosus muscle regional blood flow was 4.3±0.7 in pMJ35, vs. 2.7±0.4 in p267, vs. 1.8±0.4 in pGSVLacZ (p<0.05 vs. pGSVLacZ). For the ischemic gastrocnemius muscle, flow was 4.7±0.7 in pMJ35, vs. 1.8±0.3 in p267, vs. 1.4±0.4 in pGSVLacZ (p<0.05 vs. pGSVLacZ) (Fig. 5). No significant differences in normal limb blood flow were measured among the three groups.
|
3.7 Dose-response analysis of pMJ35
Evaluation of angiogenesis as a function of the dose of pMJ35 employed is summarized in Fig. 6. Angiographic score (Fig. 6a), blood pressure ratio (Fig. 6b), capillary density (Fig. 6c), and measurement of regional blood flow by colored microspheres (Fig. 6d) were not significantly increased with low doses (100–300 µg) of pMJ35, compared with LacZ. A statistically significant increase in these indices was, however, demonstrated at a dose of 500 µg. These results were not further augmented at higher doses (700–1000 µg) pMJ35. Although regional blood flow to the gastrocnemius muscle was increased at 1000 µg compared with 500 µg of pMJ35, the magnitude of this increase was not statistically significant.
|
p<0.01 vs. 0 µg, 3.8 Histologic analysis of transfected iliac artery
Histological cross sections of the internal iliac artery were analyzed at day 30 for all three groups. Analysis of I/M ratio disclosed no statistically significant difference among the three groups (Fig. 7).
|
4 Discussion |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
|---|
This study was designed to test the hypothesis that the success of naked DNA gene transfer is dependent upon the presence of a signal sequence permitting protein secretion. We employed a gene encoding a protein, aFGF, which is normally not secreted, and a modified version of the transgene including a signal peptide to permit active secretion from intact cells. Indeed, we observed that this single alteration (regulatory transcriptional elements were unchanged) permitted successful gene therapy with naked DNA.
The evidence of successful gene expression was documented by RT-PCR: similar to previous studies performed with naked DNA in this animal model [6], gene expression was site-specific and limited to <30 days. The fact that rabbit iliac arteries transfected with β-galactosidase gene were negative when examined for human aFGF mRNA and that other organs showed no aFGF mRNA expression at day 3 post-transfection of plasmid encoding aFGF DNA, supports that notion that the primers employed for RT-PCR did not cross-react with endogenous rabbit aFGF mRNA.
Evidence of successful therapy was documented using several parameters describing anatomic and functional evidence of new blood vessel development. Anatomic evidence of augmented collateral artery development was established by quantitative analysis of angiographically visible blood vessels in the ischemic limb. Previous studies in this animal model have established that the extent of natural (spontaneous) collateral development reaches a plateau at 30 days and remains unchanged up to 90 days follow-up [17]. Morphometric studies performed at necropsy also disclosed an increase in blood vessels at the capillary level; previous studies have established that such increases in capillary density are the result of enhanced endothelial cell proliferation [34]. These findings suggest that the range of new collateral vessels included dimensions intermediate between arteries which are angiographically visible and those at the capillary level. This notion is consistent with morphometric analyses of swine collaterals described previously by White et al. [35]and recent in vivo studies performed using synchrotron radiation [36].
Evidence that augmented angiogenesis in the pMJ35-transfected rabbits was utilitarian included improvement in the limb blood pressure ratio, maximum flow recorded in the ischemic limb using the intra-arterial Doppler guide wire, corresponding reduction in vascular resistance, and perfusion of ischemic muscle determined by the use of colored microspheres.
Previous investigators have reported evidence that aFGF is a mitogen for smooth muscle cell proliferation and/or neointimal thickening [10, 11, 37, 38]. In the current study, histologic analysis of the site of arterial gene transfer disclosed no statistically significant impact on neointimal thickening compared to LacZ. The basis for this fortuitous outcome remains enigmatic but may be in part related to the fact that the mitogenic effect of aFGF on endothelial cells has been previously shown to accelerate re-endothelialization following balloon injury [13], and thereby inhibit neointimal thickening. The kinetics of these competing mitogenic effects when aFGF is constitutively expressed in the absence of deep-wall injury merit further study.
These findings are consistent with the results which have been previously reported in this same animal model using an alternative transgene encoding for vascular endothelial growth factor (VEGF) [39–41]. VEGF differs from aFGF in at least two important respects: it is mitogenic for endothelial cells, but not for smooth muscle cells, and all three isoforms include a secretion signal. The lack of cell specificity did not result in obvious phenotypic differences in the present series of rabbits, compared to those previously treated with phVEGF165. While the presence of a classical signal peptide has been presumed to account for the phenotypic modulation observed in animals [5, 6]and patients [7]transfected with naked DNA encoding for VEGF, no experiments have been performed to date using a non-secreted transgene encoding for VEGF. Thus, the current series of experiments constitutes the first demonstration to our knowledge in which the same plasmid — altered only with regard to the presence or absence of the secretion sequence — has been used to isolate the contribution of the signal peptide to the success of naked DNA gene transfer.
While never achieving statistical significance, it is intriguing to note that the results observed with p267 (plasmid not encoding for signal peptide) were generally intermediate between pMJ35 and lacZ. It is possible that these results reflect ‘leakage’ of a small amount of aFGF from transfected cells via a non-classical secretory pathway [42].
We did not test the necessity for performing intra-arterial gene transfer in the ipsilateral ischemic limb. The fact that VEGF is secreted might be interpreted to suggest that gene transfer might have been performed at a site remote from the ischemic limb. Indeed previous studies from our laboratory [30]have shown such an effect in a restenosis model in which the circulating gene product (VEGF) accelerated re-endothelialization in the contralateral limb.
Alternative vectors, including liposomes [37]and adenoviral vectors [43], may also be employed to optimize transfection efficiency. Mühlhauser et al., for example, have shown that angiogenesis can be promoted in a subcutaneous Matrigel plug by adenoviral vectors encoding either secreted or non-secreted aFGF [44]. The use of naked DNA alone, however, simplifies the transfection protocol and obviates concerns regarding the potential toxicity of liposomes, [45]inactivation of liposomes by heparin and/or serum, [46]and immunologic or other toxicities which have been observed in the case of viral vectors [3].
These findings thus challenge certain conclusions of the Verma report [47]. The need for more basic science and novel vectors cannot be disputed. However, the extent to which the gene product itself may determine the success of gene therapy has been largely ignored. The current and previous [48]findings suggest that consideration of certain features of the encoded protein, together with the specific pathology of the host and goals of the intended therapy, may identify circumstances in which the safety of gene transfer can be enhanced and bioactivity perhaps preserved by the use of naked DNA.
Time for primary review 37 days.
|
References |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
|---|
- Mulligan RC. The basic science of gene therapy. Science (1993) 260:926–932.
[Abstract/Free Full Text] - Wilson J. Vehicles for gene therapy. Nature (1993) 365:691–692.[Medline]
- Yang Y, Nunes FA, Berencsi K, Furth EE, Gonczol E, Wilson JM. Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. Proc Natl Acad Sci USA (1994) 91:4407–4411.
[Abstract/Free Full Text] - Ledley FD. Nonviral gene therapy: The promise of genes as pharmaceutical products. Human Gene Ther (1995) 6:1129–1144.[Web of Science][Medline]
- Takeshita S, Weir L, Chen D, et al. Therapeutic angiogenesis following arterial gene transfer of vascular endothelial growth factor in a rabbit model of hindlimb ischemia. Biochem Biophys Res Commun (1996) 227:628–635.[CrossRef][Web of Science][Medline]
- Takeshita S, Tsurumi Y, Couffinhal T, et al. Gene transfer of naked DNA encoding for three isoforms of vascular endothelial growth factor stimulates collateral development in vivo. Lab Invest (1996) 75:487–502.[Web of Science][Medline]
- Isner JM, Pieczek A, Schainfeld R, et al. Clinical evidence of angiogenesis following arterial gene transfer of phVEGF165. Lancet (1996) 348:370–374.[CrossRef][Web of Science][Medline]
- Takeshita S, Losordo DW, Kearney M, Isner JM. Time course of recombinant protein secretion following liposome-mediated gene transfer in a rabbit arterial organ culture model. Lab Invest (1994) 71:387–391.[Web of Science][Medline]
- Losordo DW, Pickering JG, Takeshita S, et al. Use of the rabbit ear artery to serially assess foreign protein secretion after site specific arterial gene transfer in vivo: Evidence that anatomic identification of successful gene transfer may underestimate the potential magnitude of transgene expression. Circulation (1994) 89:785–792.
[Abstract/Free Full Text] - Folkman J, Klagsbrun M. Angiogenic factors. Science (1987) 235:442–447.
[Abstract/Free Full Text] - Thompson JA, Anderson KD, DiPietro JM. Site-directed neovessel formation in vivo. Science (1988) 241:1349–1352.
[Abstract/Free Full Text] - Selke FW, Jianyi L, Stamler A, Lopez JJ, Thomas KA, Simons M. Angiogenesis induced by acidic fibroblast growth factor as an alternative method of revascularization for chronic myocardial ischemia. Surgery (1996) 120:182–188.[CrossRef][Web of Science][Medline]
- Bjornsson TD, Dryjski M, Tluczek J, et al. Acidic fibroblast growth factor promotes vascular repair. Proc Natl Acad Sci USA (1991) 88:8651–8655.
[Abstract/Free Full Text] - Jaye M, Howk R, Burgess W, et al. Human endothelial cell growth factor: Cloning, nucleotide sequence, and chromosome localization. Science (1986) 233:541–545.
[Abstract/Free Full Text] - Abraham JA, Mergia A, Whang JL, et al. Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor. Science (1986) 233:545–548.
[Abstract/Free Full Text] - Jouanneau J, Gavrilovic J, Caruelle D, et al. Secreted or nonsecreted forms of acidic fibroblast growth factor produced by transfected epithelial cells influence cell morphology, motility, and invasive potential. Proc Natl Acad Sci USA (1991) 88:2893–2897.
[Abstract/Free Full Text] - Pu LQ, Jackson S, Lachapelle KJ, et al. A persistent hindlimb ischemia model in the rabbit. J Invest Surg (1994) 7:49–60.[Medline]
- Jaye M, Lyall RM, Mudd R, Schlessinger J, Sarver N. Expression of acidic fibroblast growth factor cDNA confers growth advantage and tumorigenesis to Swiss 3T3 cells. EMBO J (1988) 7:963–969.[Web of Science][Medline]
- Taniguchi T, Ohno S, Fujii-Kuriyama Y, Muramatsu M. The nucleotide sequence of human fibroblast interferon cDNA. Gene (1980) 10:11–15.[CrossRef][Web of Science][Medline]
- Bonnerot C, Rocancourt D, Briand P, Grimber G. A beta-galactosidase hybrid protein targeted to nuclei as a marker for developmental studies. Proc Natl Acad Sci USA (1987) 84:6795–6799.
[Abstract/Free Full Text] - Riessen R, Rahimizadeh H, Blessing E, Takeshita S, Barry JJ, Isner JM. Arterial gene transfer using pure DNA applied directly to a hydrogel-coated angioplasty balloon. Human Gene Ther (1993) 4:749–758.[Web of Science][Medline]
- Isner JM, Walsh K, Rosenfield K, et al. Arterial gene therapy for restenosis. Human Gene Ther (1996) 7:989–1011.[Web of Science][Medline]
- Takeshita S, Zheng LP, Brogi E, et al. Therapeutic angiogenesis: A single intra-arterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hindlimb model. J Clin Invest (1994) 93:662–670.[Web of Science][Medline]
- Doucette JW, Corl PD, Payne HM, et al. Validation of a Doppler wire for intravascular measurements of coronary artery flow velocity. Circulation (1992) 85:1899–1911.
[Abstract/Free Full Text] - Labovitz AJ, Anthonis DM, Cravens TL, Kern MJ. Validation of volumetric flow measurements by means of a Doppler-tipped coronary angioplasty guide wire. Am Heart J (1993) 126:1456–1461.[CrossRef][Web of Science][Medline]
- Isner JM, Kaufman J, Rosenfield K, et al. Combined physiologic and anatomic assessment of percutaneous revascularization using a Doppler guidewire and ultrasound catheter. Am J Cardiol (1993) 71:70D–86D.[CrossRef][Medline]
- Bauters C, Asahara T, Zheng LP, et al. Recovery of disturbed endothelium-dependent flow in the collateral-perfused rabbit ischemic hindlimb after administration of vascular endothelial growth factor. Circulation (1995) 91:2802–2809.
[Abstract/Free Full Text] - Bauters C, Asahara T, Zheng LP, et al. Physiologic assessment of augmented vascularity induced by VEGF in ischemic rabbit hindlimb. Am J Physiol (1994) 36:H1263–H1271.
- Walter DH, Hink U, Asahara T, et al. The in vivo bioactivity of vascular endothelial growth factor/vascular permeability factor is independent of N-linked glycosylation. Lab Invest (1996) 74:546–556.[Web of Science][Medline]
- Asahara T, Chen D, Tsurumi Y, et al. Accelerated restitution of endothelial integrity and endothelium-dependent function following phVEGF165 gene transfer. Circulation (1996) 94:3291–3302.
[Abstract/Free Full Text] - Ziada AM, Hudlicka O, Tyler KR, Wright AJ. The effect of long-term vasodilation on capillary growth and performance in rabbit heart and skeletal muscle. Cardiovasc Res (1984) 18:724–732.
[Abstract/Free Full Text] - Van Belle E, Chen D, Bunting S, Ferrara N, Isner JM. Revascularization achieved by therapeutic angiogenesis is associated with improvement of tissue perfusion. J Am Coll Cardiol (1996) 27:35A. abstract.
- Kowallik P, Schulz R, Guth BD, et al. Measurement of regional myocardial blood flow with multiple colored microspheres. Circulation (1991) 83:974–982.
[Abstract/Free Full Text] - Takeshita S, Rossow ST, Kearney M, et al. Time course of increased cellular proliferation in collateral arteries following administration of vascular endothelial growth factor in a rabbit model of lower limb vascular insufficiency. Am J Pathol (1995) 147:1649–1660.[Abstract]
- White FC, Carroll SM, Magnet A, Bloor CM. Coronary collateral development in swine after coronary artery occlusion. Circ Res (1992) 71:1490–1500.
[Abstract/Free Full Text] - Takeshita S, Isshiki T, Tanaka E, et al. Use of synchrotron radiation microangiography to assess development of small collateral arteries in a rat model of hindlimb ischemia. Circulation 1997;(in press).
- Nabel EG, Yang ZY, Plautz G, et al. Recombinant fibroblast growth factor-1 promotes intimal hyperplasia and angiogenesis in arteries in vivo. Nature (1993) 362:844–846.[CrossRef][Medline]
- Banai S, Jaklitsch MT, Casscells W, et al. Effects of acidic fibroblast growth factor on normal and ischemic myocardium. Circ Res (1991) 69:76–85.
[Abstract/Free Full Text] - Ferrara N, Henzel WJ. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun (1989) 161:851–855.[CrossRef][Web of Science][Medline]
- Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science (1989) 246:1306–1309.
[Abstract/Free Full Text] - Keck PJ, Hauser SD, Krivi G, et al. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science (1989) 246:1309–1312.
[Abstract/Free Full Text] - Mignatti P, Morimoto T, Rifkin DB. Basic fibroblast growth factor, a protein devoid of secretory signal sequence, is released by cells via a pathway independent of the endoplasmic reticulum-Golgi complex. J Cell Physiol (1992) 151:81–93.[CrossRef][Web of Science][Medline]
- Giordano FJ, Ping P, McKirnan D, et al. Intracoronary gene transfer of fibroblast growth factor-5 increases blood flow and contractile function in an ischemic region of the heart. Nature Med (1996) 2:534–539.[CrossRef][Web of Science][Medline]
- Muhlhauser J, Pili R, Merrill M, et al. In vivo angiogenesis induced by recombinant adenovirus vectors coding either for secreted or nonsecreted forms of acidic fibroblast growth factor. Human Gene Ther (1995) 6:1457–1465.[Web of Science][Medline]
- Felgner PL, Gadek TR, Holm M, et al. Lipofection: A highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci USA (1987) 84:7413–7417.
[Abstract/Free Full Text] - Felgner PL, Ringold GM. Cationic liposome-mediated transfection. Nature (1989) 337:387–388.[CrossRef][Medline]
- Wadman M. Hyping results ‘could damage’ gene therapy. Nature (1995) 378:655.[Medline]
- Asahara T, Tsurumi Y, Takeshita S, Isner JM. Naked cDNA encoding secreted proteins for intra-arterial and intramuscular gene transfer. Sem Interv Cardiol (1996) 1:225–232.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. E. Epstein, E. Stabile, T. Kinnaird, C. W. Lee, L. Clavijo, and M. S. Burnett Janus Phenomenon: The Interrelated Tradeoffs Inherent in Therapies Designed to Enhance Collateral Formation and Those Designed to Inhibit Atherogenesis Circulation, June 15, 2004; 109(23): 2826 - 2831. [Full Text] [PDF]
|
|
|
|
 |
|
 J. C Hershey, H. A Corcoran, E. P Baskin, D. B Gilberto, X. Mao, K. A Thomas, and J. J Cook Enhanced hindlimb collateralization induced by acidic fibroblast growth factor is dependent upon femoral artery extraction Cardiovasc Res, October 1, 2003; 59(4): 997 - 1005. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. B. Freedman and J. M. Isner Therapeutic Angiogenesis for Coronary Artery Disease Ann Intern Med, January 1, 2002; 136(1): 54 - 71. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Isner, P. R. Vale, J. F. Symes, and D. W. Losordo Assessment of Risks Associated With Cardiovascular Gene Therapy in Human Subjects Circ. Res., August 31, 2001; 89(5): 389 - 400. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. L. Meyerson, C. L. Skelly, M. A. Curi, and L. B. Schwartz Gene Therapy for Cardiovascular Disease Seminars in Cardiothoracic and Vascular Anesthesia, November 1, 2000; 4(4): 289 - 300. [Abstract] [PDF]
|
|
|
|
|
|
K. A. Vincent, K.-G. Shyu, Y. Luo, M. Magner, R. A. Tio, C. Jiang, M. A. Goldberg, G. Y. Akita, R. J. Gregory, and J. M. Isner Angiogenesis Is Induced in a Rabbit Model of Hindlimb Ischemia by Naked DNA Encoding an HIF-1{alpha}/VP16 Hybrid Transcription Factor Circulation, October 31, 2000; 102(18): 2255 - 2261. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Fernandez, A. Buehler, S. Wolfram, S. Kostin, G. Espanion, W. M. Franz, H. Niemann, P. A. Doevendans, W. Schaper, and R. Zimmermann Transgenic Myocardial Overexpression of Fibroblast Growth Factor-1 Increases Coronary Artery Density and Branching Circ. Res., August 4, 2000; 87(3): 207 - 213. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. O Hiltunen, M. P Turunen, M. Laitinen, and S. Yla-Herttuala Insights into the molecular pathogenesis of atherosclerosis and therapeutic strategies using gene transfer Vascular Medicine, February 1, 2000; 5(1): 41 - 48. [Abstract] [PDF]
|
|
|
|
 |
|
 L. M. Mir, M. F. Bureau, J. Gehl, R. Rangara, D. Rouy, J.-M. Caillaud, P. Delaere, D. Branellec, B. Schwartz, and D. Scherman High-efficiency gene transfer into skeletal muscle mediated by electric pulses PNAS, April 13, 1999; 96(8): 4262 - 4267. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. G. Favaloro Landmarks in the Development of Coronary Artery Bypass Surgery Circulation, August 4, 1998; 98(5): 466 - 478. [Full Text] [PDF]
|
|
|
|
|
|
E. Van Belle, B. Witzenbichler, D. Chen, M. Silver, L. Chang, R. Schwall, and J. M. Isner Potentiated Angiogenic Effect of Scatter Factor/Hepatocyte Growth Factor via Induction of Vascular Endothelial Growth Factor : The Case for Paracrine Amplification of Angiogenesis Circulation, February 3, 1998; 97(4): 381 - 390. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||