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(Hypertension. 1995;26:1046-1050.)
© 1995 American Heart Association, Inc.

Articles

Heterogeneity of Adenovirus-Mediated Gene Transfer in Cultured Thoracic Aorta and Renal Artery of Rats

Aqing Yao; Donna H. Wang

From the Department of Internal Medicine, Hypertension and Vascular Research Laboratories, University of Texas Medical Branch, Galveston.

Correspondence to Donna H. Wang, MD, The University of Texas Medical Branch, Department of Internal Medicine, Hypertension and Vascular Research Laboratories, 8.104 Medical Research Bldg, Galveston, TX 77555-1065. E-mail dwang%intmedS1@mhost.utmb.edu.

	Abstract

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Abstract Replication-deficient recombinant adenovirus vectors have been used to transfer foreign genes effectively to a wide variety of cell types in vivo and in vitro. We have now used adenovirus containing either the Escherichia coli ß-galactosidase (ß-gal) gene (AdHCMVsp1LacZ) or the firefly luciferase gene (Ad5-luc3) to test the hypothesis that efficiencies of adenovirus-mediated gene delivery into organ cultures of smooth muscle differ according to the anatomic origin of the muscle. Thoracic aorta and renal artery were isolated from 9-week-old male Sprague-Dawley rats and exposed to adenovirus after 16 hours of incubation with serum-free medium (Dulbecco's modified Eagle's medium). With the use of histochemical methods, ß-gal staining was noted in both endothelial and adventitial cells but not in the muscular media of thoracic aorta and renal artery exposed to AdHCMVsp1LacZ. The efficiency of the transfection, assessed either by counting of ß-gal–stained cells in intact vessels or by measurement of ß-gal activity in tissue extracts, was higher in renal artery than thoracic aorta (P<.05). Consistent with this result, luciferase activity in renal artery exposed to Ad5-luc3 (15.9±2.1x10⁶ relative light units per milligram protein) was higher than that in thoracic aorta (8.3±2.0x10⁶, P<.05). To determine whether increased efficiency of adenovirus-mediated gene transfer into renal artery is a function of the replication status of vessels, we assessed [³H]thymidine incorporation. [³H]Thymidine uptake by thoracic aorta was only 63% of that in renal artery (P<.05), indicating that more proliferating cells are present in renal artery. We conclude that the efficiency of adenovirus-mediated gene transfer into cultured renal artery is enhanced compared with that into thoracic aorta and propose that the increase in efficiency is related to the higher proliferative activity of renal artery.

Key Words: gene transfer • dependovirus • organ culture • aorta • renal artery

	Introduction

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Gene therapy is not only a medical intervention that alters the genetic program of cells for therapeutic purposes but also is a powerful tool for biological research in which the function of a specific gene can be studied.¹ Previous in vivo and in vitro experiments using liposomal or retroviral methods to transfer genes into vascular endothelial or smooth muscle cells have demonstrated that the efficiencies of the transduction into these tissues are very low.^{2 3} Retrovirus can transfect only proliferative cells in order to express the newly integrated gene.⁴ In contrast, replication-deficient recombinant adenovirus vectors can transfer foreign genes to both replicating and nonreplicating cells and be efficient agents for gene transfer.^{5 6 7 8 9 10 11 12 13}

It has been shown that exogenous genes can be successfully transferred to rat aortic smooth muscle cells⁶ as well as intact or injured carotid arteries by adenovirus.^{5 11 12} Although transfection efficiencies are high, no studies have compared the efficiency of transfection between aorta and the smaller arteries. Considering the critical role of the kidney in the pathogenesis of clinical and experimental hypertension,^{14 15 16 17} this tissue is a reasonable target for gene transfection both at a basic science level and as a target for gene therapy. To provide information about the utility of gene transfer in the renal artery, we used adenovirus containing either firefly luciferase gene (Ad5-luc3) or Escherichia coli ß-galactosidase (ß-gal) gene (AdHCMVsp1LacZ) to determine whether the efficiencies of adenovirus-mediated gene delivery into organ cultures of thoracic aorta and renal artery are different.

	Methods

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Adenovirus Vectors
Two adenoviruses (kindly provided by Dr Frank L. Graham, McMaster University, Canada) were used in this study. One was Ad5-luc3,¹⁸ which contains the firefly luciferase gene flanked by simian virus 40 regulatory sequences inserted in the early region 3 (E3) of Ad5 genome. The other was AdHCMVsp1LacZ,¹⁹ which encodes for the histochemical marker gene ß-gal derived from Escherichia coli. The ß-gal gene was placed under the control of human cytomegalovirus promoter and inserted into the early region 1 (E1) of Ad5. Expression of the ß-gal gene results in a blue staining pattern when cells are exposed to the chromogen 5-bromo-4-chloro-3-indoyl-ß-D-galactopyranoside (X-Gal, Sigma Chemical Co). Viral stocks (10¹¹ to 10¹² pfu/mL) were prepared by passaging adenovirus in 293 cells.²⁰ Cell and tissue culture media and reagents were obtained from Gibco BRL.

Tissue Isolation and Organ Culture
Male Sprague-Dawley rats (Harlan Sprague Dawley Inc, Indianapolis, Ind) weighing between 275 and 300 g were used. The rats were anesthetized with a single intraperitoneal injection of 80 mg/kg ketamine and 1 mg/kg xylazine and then were decapitated. Thoracic aorta (from aortic arch to diaphragm) and both renal arteries (from aorta to kidney) were carefully removed as previously reported.^{21 22} Both aorta and renal artery were cut into fragments (3 mm) and placed in 60-mm dishes containing 5 mL SFM^{21 22} consisting of Dulbecco's modified Eagle's medium and F-12 nutrient mixture (1:1), glutamine (200 µg/mL), penicillin (100 U/mL), streptomycin (100 µg/mL), insulin (5 µg/mL), and transferrin (5 µg/mL). The vessels were then placed in a humidified incubator with a 95% air/5% CO₂ atmosphere at 37°C for 16 hours and used in either gene transfer or [³H]thymidine uptake experiments. The aorta and renal artery from each rat were placed in one dish; n always represents the number of rats.

In Vitro Gene Transfer
After 16 hours of incubation, vessels in each batch were washed twice in PBS and incubated in 0.3 to 0.5 mL PBS containing 2x10⁹ pfu/mL adenovirus for 1 hour.^{12 20} This adenovirus concentration was selected because our dose-response experiments showed that it causes maximal transduction (Fig 1A). After two washes in PBS, vessels were refed with 5 mL SFM plus 10% FBS and incubated for 2 days. The vessels were then homogenized in 50 to 100 µL buffer (100 mmol/L potassium phosphate, pH 7.8, 1 mmol/L dithiothreitol, 100 µg/mL phenylmethylsulfonyl fluoride, 0.1% Triton X-100).¹⁸ After centrifugation for 10 minutes at 16 000g at 4°C, the supernatant was assayed for luciferase or ß-gal activity.

View larger version (13K): [in this window] [in a new window]   Figure 1. Line graphs show concentration-response curve (A) and time course (B) of adenovirus-mediated gene transfer into cultured rat aorta. A, Each curve was generated using aorta from two rats. Vessels were incubated with 0.3 mL PBS containing different concentrations of Ad5-luc3 (from 2x105 to 2x1010 pfu/mL) and then 1 to 8 hours later harvested for luciferase activity assay. B, Each curve was generated using aorta from three rats. Vessels were incubated with 0.3 mL PBS containing 2x109 pfu/mL of Ad5-luc3 and then harvested for luciferase activity assay at different time points (0 to 9 days) after transfection.

Measurement of Luciferase and ß-Gal Activities
For determination of luciferase activity,^{18 23} 10 µL undiluted or serially diluted supernatant was mixed with 100 µL luciferase assay buffer [20 mmol/L Tricine, 1 mmol/L (MgCO₃)₄Mg(OH)₂·5H₂O, 2.67 mmol/L MgSO₄, 0.1 mmol/L EDTA, 33.3 mmol/L dithiothreitol, 270 µmol/L coenzyme A, 470 µmol/L luciferin, 530 µmol/L ATP]. The light emission over the first 15 seconds of reaction measured by an Autolumat LB 953 luminometer (LKB) was recorded as RLU. Activity of ß-gal was measured in a 300-µL reaction volume consisting of 10 to 40 µL supernatant, 100 mmol/L sodium phosphate, pH 7.3, 1 mmol/L MgCl₂, 50 mmol/L ß-mercaptoethanol, and 0.67 mg/mL o-nitro-prenol-ß-D-galactoside. After 30 minutes to 2 hours of incubation at 37°C, the reaction was stopped by addition of 500 µL of 1 mol/L Na₂CO₃. The optical density of each sample was measured at wavelength of 420 nm with a spectrophotometer (Pharmacia Biotech Inc). Luciferase and ß-gal activities were normalized by protein concentrations that were determined with the use of a modified Bradford dye-binding procedure (Bio-Rad).²⁴

Histological Evaluation of ß-Gal Staining
For evaluation of gene transfer at the histological level^{6 11 12} transfected vessels were fixed for 5 minutes in 2% formaldehyde and 0.2% glutaraldehyde in PBS, pH 7.4. The vessels were then washed in PBS several times and placed into X-Gal solution [5 mmol/L K₄Fe(CN)₆, 5 mmol/L K₃Fe(CN)₆, 1 mmol/L MgCl₂, 1 mg/mL X-Gal in PBS]. After 1 day of staining at room temperature samples were postfixed in the same fixative, cut into 5-µm-thick sections, and counterstained with nuclear fast red. With the use of a computerized image-analysis system²⁵ the nuclei stained with nuclear fast red and ß-gal–stained cells were counted from two different circumscribed areas per section, eight sections per rat, and a mean value was calculated for each. The percentage of transduced endothelial cells was calculated as the number of ß-gal–stained endothelial cells divided by the total endothelial cells times 100%. Because of the nonuniform distribution of the cell population in the adventitia, the number of transduced adventitial cells was normalized with the length of vessels in the circumscribed area.

[³H]Thymidine Uptake
DNA synthesis was measured by determination of [³H]thymidine incorporation. Five milliliters SFM containing 2 µCi/mL [³H]thymidine (Amersham) was placed in the culture dishes described above. Two hours later the vessels were washed with PBS three times and homogenized in 0.2 mol/L PCA.²⁶ After centrifugation at 1000g for 10 minutes the pellet was washed with 0.2 mol/L PCA three times. The pellet was then resuspended and heated in 0.5 mol/L PCA for 20 minutes at 90°C to solubilize the DNA. After cooling and recentrifugation at 1000g for 10 minutes the supernatant was used for DNA determinations. [³H]Thymidine incorporation into DNA was assessed with a liquid scintillation system (LS1801, Beckman Instruments Inc). Total DNA content was determined by a fluorochrome compound (Hoechst 33258, Sigma) with the use of a fluorescence spectrophotometer (F-4500, Hitachi Co).²⁷ [³H]Thymidine uptake was normalized to micrograms of DNA content.

Statistical Analysis
All values are expressed as mean±SEM. Differences between groups were determined by Student's t test and ANOVA followed by the Tukey-Kramer multiple comparison test. Significance was accepted at the level of P<.05.

	Results

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Fig 1A demonstrates the dose-response curve of Ad5-luc3 transfection into cultured rat thoracic aorta. Pooled aortas of two rats were used to generate each curve. Increased luciferase activity correlated with increasing virus concentration although not in an entirely linear fashion. When virus concentration increased to 2x10⁹ pfu/mL, the response curve of luciferase activity plateaued. Therefore, a dose of 2x10⁹ pfu/mL Ad5-luc3 produced the maximal transfection efficiency and was chosen for transfection of both the thoracic aorta and renal artery.

Fig 1B shows the duration of luciferase activity in the aorta after the transfection of 2x10⁹ pfu/mL Ad5-luc3. The aortas from three rats were pooled and used for generation of one curve. Luciferase activity was detectable 24 hours after transfection, reaching maximal levels at about 2 days. Although luciferase activity was still easily detectable 7 days after transfection, expression levels were one order of magnitude lower than the peak activity. Luciferase activity returned to basal levels by 9 days after the transfection.

Fig 2A shows the levels of luciferase activity in extracts prepared from thoracic aorta and renal artery exposed to identical concentrations (2x10⁹ pfu/mL) of Ad5-luc3 and AdHCMVsp1LacZ. Luciferase activity was undetectable in extracts from both thoracic aorta (n=4) and renal artery (n=4) exposed to AdHCMVsp1LacZ. Luciferase activity in renal artery exposed to Ad5-luc3 (15.9±2.1x10⁶ RLU/mg protein, n=4) was significantly higher than that in thoracic aorta (8.3±2.0x10⁶ RLU/mg protein, n=4, P<.05).

View larger version (21K): [in this window] [in a new window]   Figure 2. Bar graphs show luciferase (A) and ß-galactosidase (B) activities in cultured aorta and renal artery. Vessels were incubated with 0.5 mL PBS containing 2x109 pfu/mL of either Ad5-luc3 (open bars) or AdHCMVsp1LacZ (shaded bars) and then 48 hours later were harvested for luciferase activity and ß-galactosidase activity assays. n=4 rats per group. TA indicates thoracic aorta; RA, renal artery. *P<.01 vs renal artery; +P<.01 vs Ad5-luc3.

Fig 2B shows the levels of ß-gal activity in extracts prepared from thoracic aorta and renal artery exposed to the same concentrations (2x10⁹ pfu/mL) of Ad5-luc3 and AdHCMVsp1LacZ. Activity of ß-gal was nearly undetectable in extracts from both thoracic aorta (n=4) and renal artery (n=4) exposed to Ad5-luc3. Again, ß-gal activity in renal artery exposed to AdHCMVsp1LacZ (17.7±2.8x10^-3 U/mg protein, n=4) was significantly higher than that in thoracic aorta (7.5±2.1x10^-3 U/mg protein, n=4, P<.05).

To determine the type and proportion of cells in the thoracic aorta and renal artery that expressed ß-gal, we stained tissue sections with X-Gal. As shown in Fig 3 dark blue ß-gal staining was noted in both endothelial and adventitial cells but not in the muscular media of thoracic aorta and renal artery exposed to AdHCMVsp1LacZ (Fig 3A and 3B). No ß-gal staining was detected in thoracic aorta and renal artery transfected with Ad5-luc3 (Fig 3C and 3D). Statistical analysis of the results of the ß-gal staining indicated that the percentage of ß-gal–stained endothelial cells in renal artery (36±4%, n=4) was significantly higher than that in thoracic aorta (22±1%, n=4, P<.05). The number of ß-gal–stained adventitial cells per unit length of renal artery (10.6±0.1/mm, n=4) was also modestly but significantly higher than that of thoracic aorta (7.7±0.7/mm, n=4, P<.05).

View larger version (106K): [in this window] [in a new window]   Figure 3. Photomicrographs show adenovirus-mediated gene transfer into cultured vessels. A and B, Thoracic aorta (A) and renal artery (B) exposed to AdHCMVsp1LacZ and stained with X-Gal. C and D, Thoracic aorta (C) and renal artery (D) exposed to Ad5-luc3 and stained with X-Gal serve as negative controls. Bars in C and D indicate 50 µm. Arrows indicate ß-Gal–stained cells.

To assess whether the efficiency of the transfection is a function of the replication status of the cells, we cultured thoracic aorta with either SFM or SFM plus 10% FBS for 16 hours before transfection with Ad5-luc3. Luciferase activity in aorta cultured with SFM plus 10% FBS (6.5±0.1x10⁶ RLU/mg protein, n=4) was significantly higher than that in aorta cultured with SFM (4.1±0.2x10⁶ RLU/mg protein, n=4, P<.05), indicating that higher efficiency of the transfection is correlated with active growth status of the vessel. We therefore examined [³H]thymidine incorporation in both thoracic aorta and renal artery cultured with SFM. We found that [³H]thymidine uptake of renal artery (2798±235 cpm/µg DNA, n=4) was significantly higher than that of thoracic aorta (1769±220 cpm/µg DNA, n=4, P<.05).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Although the characteristics of adenovirus-mediated gene transfer into aorta and carotid artery have been relatively well defined,^{6 11 12} the nature of adenovirus-mediated gene transfer into renal artery is unknown. Therefore, the goal of these experiments was to characterize the ability of the virus to transfect renal arteries by testing the hypothesis that the efficiencies of adenovirus-mediated gene transfer into organ culture of thoracic aorta and renal artery of rats are different. Using adenovirus containing either firefly luciferase gene or Escherichia coli ß-gal gene, we found that adenovirus results in effective gene transfer into aortic and renal arterial endothelial and adventitial cells. However, the transfection efficiency was much higher in renal artery than in thoracic aorta, suggesting that different properties between renal artery and thoracic aorta may exist.

It has been demonstrated that adenovirus has several advantages over other gene-transfer methods.^{28 29} Among these advantages is exceptionally high efficiency, which is one of the most important features of this virus for therapeutic and research purposes.^{6 11} In the present experiments we found that exposure of cultured aorta to increasing concentrations of Ad5-luc3 (10⁴ to 10¹⁰ pfu/mL) resulted in a dose-dependent increase in luciferase expression, with a peak expression of approximately 2x10⁶ to 4x10⁶ RLU luciferase per milligram protein. This result is consistent with the data obtained from in vivo experiments in which 10¹⁰ pfu/mL adenovirus produced maximally transduced cells in balloon-injured rat carotid arteries.¹¹ The percentage of transduced endothelial cells in our cultured aorta and renal artery was approximately 20% to 40%, which is close to the 30% transfection rate in vivo.¹¹ The failure to transfect the smooth muscle media may result from the barrier function of the internal elastic lamina because it has been demonstrated that when carotid arteries are denuded of endothelium, injection of adenovirus results in effective transfection of the smooth muscle cell.^{6 11} This high transfection efficiency in endothelial and adventitial cells suggests that it may be possible to use adenovirus-mediated gene transfer to study the effects of gene products of autocrine and/or paracrine factors that are linked with the pathogenesis or the prevention of vascular- related diseases.

Another important feature of adenovirus is that adenovirus genome does not integrate into host cell DNA, therefore obviating the hazard of insertional mutagenesis when applied in humans.²⁹ However, this feature also makes permanent transfection impossible. It has been reported that expression of recombinant protein lasts for about 2 weeks after injection of adenovirus into the carotid artery.^{11 12} We found that luciferase expression in cultured aorta was detectable 7 days after transfection but was no longer evident at 9 days. The loss of responsiveness may be due to the lack of permanent integration of adenovirus DNA or the difficulty of maintaining live aorta in the culture. Nonetheless, the peak expression of luciferase can be detected as early as 2 days (Fig 1B), allowing the study of a high level of gene expression at known times.

Interestingly, the efficiencies of the transfection, assessed either at the histological level by counting of ß-gal–stained cells in intact vessels or at the biochemical level by measurement of ß-gal or luciferase activity in tissue extracts, were higher in renal artery than thoracic aorta. These results indicate that there is considerable heterogeneity of the efficiency of adenovirus-mediated gene transfer according to either the anatomic location or size of the vessels studied. If this phenomenon holds true in vivo, it may have significant clinical implications. It has been demonstrated that early narrowing of the afferent arteriole contributes to the development of hypertension in spontaneously hypertensive rats.³⁰ Narrowing of the renal artery also causes rapid development of renal hypertension.³¹ It is therefore conceivable that in cases in which the renal artery itself is the target for gene therapy, the ability to deliver gene products by adenovirus transfection may be highly site specific.

The mechanisms whereby adenovirus causes higher transfection efficiency in renal artery is not clear. The fact that the aorta cultured with serum had higher luciferase activity than that cultured without serum indicates that transfection efficiency may be associated with the proliferative state of the vessel. We found that DNA synthesis assessed by [³H]thymidine uptake in renal artery was significantly higher than that in thoracic aorta, indicating that the enhanced turnover rate of renal artery cells may contribute to increased transfection efficiency in renal artery. Furthermore, it is possible that different transfection efficiencies between the renal artery and thoracic aorta are caused by a heterogeneous distribution of adenovirus receptors between these two vessels.³² Further investigation into these areas is needed.

In conclusion, we demonstrate that adenovirus effectively transfers gene into organ cultured aortic and renal arterial endothelial and adventitial cells. The efficiency of the transfection is significantly higher in renal artery than thoracic aorta. Our data demonstrate the feasibility of using the renal artery as a target for the treatment of diseases such as hypertension and of providing a model for investigation of the interplay of vascular-derived factors and their roles in the pathogenesis of vascular-related diseases.

	Selected Abbreviations and Acronyms

 

FBS = fetal bovine serum PBS = phosphate-buffered saline PCA = perchloric acid pfu = plaque forming units RLU = relative light units SFM = serum-free medium

	Acknowledgments

 
This study was supported in part by National Institutes of Health grant HL-52279 (Dr Donna H. Wang). We thank Dr Richard D. Bukoski for his critical review of this manuscript, Dr Frank L. Graham for kindly providing AdHCMVsp1LacZ and Ad5-luc3, and Dr Allan Brasier for his kind supply of the equipment for the measurement of luciferase activity. We wish to express our thanks to Wilma Frye for her expert secretarial skills.

Received June 18, 1995; first decision August 18, 1995; accepted August 18, 1995.

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