1Department of Biochemistry and Nutrition, Medical School, Université Libre de Bruxelles and 2Division of Nephrology, Université catholique de Louvain, Brussels, Belgium
Correspondence and offprint requests to: C. Delporte, Department of Biochemistry and Nutrition CP 611, Blg G/E, Medical School, Université Libre de Bruxelles, Route de Lennik 808, B-1070 Brussels, Belgium. Email: cdelport{at}ulb.ac.be
Abstract
Background. Initial studies of adenovirus-mediated gene transfer to the peritoneum have shown transgene expression in the mesothelium from the parietal peritoneum. Using a replication-deficient adenovirus encoding ß-galactosidase (AdßGal), we investigated the expression efficiency and the distribution of the transgene to different areas of both visceral and parietal peritoneum and to extra-peritoneal tissues.
Methods. Male Wistar rats received an intraperitoneal injection of 15 ml of 0.9% NaCl alone or containing 1 x 109 or 3 x 109 p.f.u. of AdßGal. Evaluations of the histology of the peritoneum, the transgene expression and the safety of adenovirus-mediated gene transfer, using measurement of both ßGal activity and staining, were performed 1, 3 and 5 days post-injection.
Results. At 1 day post-injection of x 109 p.f.u. of AdßGal, significant ßGal activity and staining were detected in the omentum and mesenteric peritoneum. ßGal staining was observed in endothelial cells, mesothelial cells and adipocytes. Focal mononuclear infiltrates restricted to the submesothelial area of the visceral peritoneum were also observed. No expression was detected in the mesocolon and parietal peritoneum, where the mesothelium was damaged. Significant ßGal activity and staining were observed in lymph nodes, lungs, liver, heart and kidneys, in the absence of inflammatory changes.
Conclusions. Intraperitoneal delivery of adenoviral vectors allows highly efficient transgene expression in mesothelial cells, but also in endothelial cells and adipocytes of the visceral peritoneum. Adverse focal mononuclear infiltrates, as well as spreading of the adenoviral vector from the abdominal cavity to the systemic circulation, were observed in parallel. Transgene expression in endothelial cells is potentially important since the latter play a key role in the alterations of the peritoneal membrane associated with long-term peritoneal dialysis. However, these data emphasize the need for less immunogenic adenoviral vectors, ideally containing an endothelial cell-specific promoter, to overcome immune response-related problems and spreading to extra-peritoneal tissues.
Keywords: adenovirus; endothelium; gene transfer; mesothelium; peritoneum
Introduction
Gene transfer in the peritoneal cavity holds considerable promise for the investigation of mediators or pathways operating in the peritoneal membrane (PM) and, perhaps, participation in the management of a variety of conditions such as peritoneal dialysis (PD), adhesions or intra-abdominal malignancies [1]. In the past few years, in vivo gene transfer to the peritoneal cavity using adenoviral vectors has been used in rat and mouse models. Intraperitoneal (i.p.) administration of such vectors resulted in transgene expression restricted to the peritoneal mesothelium [24], and in the transduction of multiple tissues in the developing murine fetus [5].
The endothelium lining peritoneal capillaries represents the functional barrier during PD [6] and an obvious target for gene transfer in the PM. On the other hand, the PM is characterized by a large, highly vascularized surface area (1 m2) and an abundant lymphatic drainage [7]. These characteristics raise the feasibility of endothelial targeting and the issue of safety and dissemination of adenoviral vectors administered i.p. Using two separate methods, we investigated in detail the expression and distribution of the transgene to different areas of both visceral and peritoneal peritoneum as well as to extra-peritoneal tissues.
Materials and methods
Recombinant adenovirus
Adenovirus encoding ß-galactosidase (AdßGal) was kindly provided by Dr R. Gerard. Culture and purification of recombinant adenovirus were performed as previously described [8].
In vivo gene delivery in rat peritoneum
Male Wistar rats (400 g) were obtained from Harlan CBP (Zeist, The Netherlands). All experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the local Ethics Committee for animal studies. The rats received a single 15 ml i.p. injection of either 0.9% NaCl (control group; n = 6) or 1 x 109 (n = 6) or 3 x 109 p.f.u. (n = 7) of AdßGal diluted in 0.9% NaCl. One day before the i.p. injection, and daily until sacrifice, the animals received an intramuscular injection of 4 mg/kg dexamethasone (Aacidexam, NV Organon, Oss, The Netherlands). Animals were put under light anaesthesis with sevoflurane (Abbott NV, Ottignies, Belgium) when the i.p. injection was administrated. No mortality resulting from that protocol was recorded. Rats were sacrificed at day 1 or 5 post-i.p. injection.
Histological evaluation of peritoneum
Samples from the omentum, mesenteric and mesocolon regions of the visceral peritoneum and from the anterior and posterior abdominal cavity of the parietal peritoneum were obtained 1, 3 and 5 days post-injection, then processed for paraffin embedding and sectioned. The integrity of the mesothelium was carefully assessed by observation of cilitated cells or immunostaining using a monoclonal anti-Pan cytokeratin (Sigma, St Louis, MO).
Evaluation of transgene expression
At 1, 3 and 5 days post-injection, samples from the different regions of the visceral and parietal peritoneum were removed and fixed for 2 h in a solution of 4% formaldehyde in phosphate-buffered saline (PBS) pH 7.4, washed in PBS pH 7.4 before being incubated at 37°C for 24 h in a solution of 5 mM K4Fe (CN)6, 5 mM K3Fe(CN)6, 2 mM MgCl2 and 0.025% of Igepal in PBS pH 7.4 containing 500 µg/ml of X-Gal substrate, and finally rinsed in PBS pH 7.4. After macroscopic analysis, peritoneum samples were post-fixed, embedded in paraffin, sectioned, and counter-stained with haematoxylineosin. Cells positive for ßGal staining displayed a blue nuclear/perinuclear staining. To assess the percentage of positive transduced cells, the total and stained cells were counted in six separate fields of each section.
Safety of in vivo adenovirus-mediated gene transfer in peritoneum
Homogenates of rat tissue samples from the duodenum, stomach, small intestine, colon, pancreas, liver, lung, spleen, kidney and heart were centrifuged at 1000 g for 10 min at 4°C, then the supernatants were centrifuged further at 17 000 g for 20 min at 4°C. The final supernatants were kept for ßGal assay (Invitrogen, San Diego, CA). ßGal activity was expressed in ßGal units per mg of protein.
The concentrations of total proteins, GOT, GPT, lipase, amylase and urea from blood samples were determined by standard automated analysis [9].
Results
Morphological evaluation of peritoneum
Detailed histological examination of the parietal (anterior, posterior left and right abdominal wall) and visceral (omentum, mesenteric and mesocolon regions) peritoneum was performed at 1, 3 and 5 days post-injection (Figure 1). None of the injected rats and visceral peritoneum showed massive infiltrate and oedema suggestive of acute peritonitis or focal areas of vascular proliferation.
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Evaluation of transgene expression: determination of ßGal activity and staining
Higher ßGal activity was observed in animals injected with 3 x 109 p.f.u. compared with 1 x 109 p.f.u. of AdßGal. At 1 day post-injection of 3 x 109 p.f.u. AdßGal, the omentum and mesenteric peritoneum exhibited significant ßGal activity, while no significant activity was detected in the mesocolon and parietal peritoneum (Figure 2). In extra-peritoneal tissues, ßGal activity was considerably increased in liver, lung and kidney, and slightly increased in heart (Figure 2). At 5 days post-injection of 3 x 109 p.f.u. AdßGal, the ßGal activity in peritoneum samples and other extra-peritoneal tissues was considerably lower (data not shown).
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Extra-peritoneal transgene expression
Lymphatic nodes from the peritoneal cavity of rats injected with AdßGal showed massive ßGal staining of the ganglionic cells (>90% of cells), contrasting with the negligible endogenous ßGal staining in control rats (Figure 4A and B). A significant ßGal staining was also observed in the spleen (strong reaction in the capsule), liver, lungs, kidney and heart (Figure 4CG). In the lungs, low and dispersed ßGal staining was observed in alveolar walls (Figure 4C). In the kidneys, ßGal staining was observed mainly in the proximal tubule cells of nephrons located in the outer cortex (Figure 4D). In the liver, ßGal staining was intense in the capsule, and included numerous hepatocytes in the hilar region (Figure 4E and F). Isolated positive cardiomyocytes were also detected in the heart (Figure 4G). No inflammatory infiltrates were observed in the lungs, kidneys, liver and heart.
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In vivo gene therapy offers possibilities to improve the understanding or treatment of structural damage induced in the PM [2,3,10,11]. Several studies have reported the feasibility of in vivo adenovirus-mediated gene transfer into rat peritoneal cavity [14]. Thus far, the transgene expression has been restricted to the mesothelium [24]. In view of the vascularization and lymphatic drainage of the PM, the aim of this study was to evaluate the potential to induce transgene expression in the endothelium lining peritoneal capillaries, as well as adverse effects and other safety concerns that might arise from the i.p. administration of adenoviral vectors.
The experimental procedure used to deliver the adenoviral vector to the PM may influence the nature of the cells transduced and the efficiency of the process. Previous studies reported that a single i.p. injection of recombinant adenovirus (15 x 109 p.f.u.) in a small volume (50 µl to 1 ml) induced transgene expression in mesothelial cells at and around the site of injection and into the muscular abdominal wall [2,3,12]. Our data show that delivery of similar doses of adenovirus in a higher volume (15 ml) ensured a better distribution and a higher transgene expression to a large peritoneal membrane area. Transgene expression was found in endothelial cells, adipocytes and mesothelial cells of the omentum, as well as in the mesenteric and mesocolon regions of the visceral peritoneum. In fact, 70% of cells within the omentum, the most superficial region of the peritoneum, were transfected at day 1 post-injection of AdßGal. The similar transgene expression using two different doses of adenoviruses suggests that the volume of infusate, rather than the dose of adenovirus, has a strong impact on the transgene distribution. The observation that transgene expression in the visceral peritoneum was much higher at day 1 than at day 5 post-injection confirmed the intrinsic property of adenoviral vector to induce transient gene transfer.
Our data show that the transgene expression in the visceral peritoneum involves mesothelial cells, as reported by others [3,12], but also adipocytes and endothelial cells. The endothelium lining peritoneal capillaries represents the major functional barrier to solute exchange during PD [6,7]. In that respect, transgene expression in endothelial cells could offer important prospects, since endothelial cell proliferation, and modifications in essential components such as endothelial nitric oxide synthase or the water channel aquaporin-1, are involved in the pathophysiology of the alterations of the PM associated with long-term PD [13]. In contrast to previous studies [3,11], no transgene expression could be observed in the mesothelial cells lining the parietal peritoneum. The loss of mesothelial cells in this part of the peritoneal membrane is probably due to the protocol of large volume infusion rather than related to tissue dissection and processing (the mesothelial cells are well preserved in the visceral peritoneum).
The use of adenoviruses has been associated with the development of a dose-dependent inflammation at the site of delivery. This acute inflammation can usually be suppressed by dexamethasone administration [14]. The administration of high-dose dexamethasone in our study prevented most of the inflammatory response after injection of AdßGal. Discrete and very limited cellular infiltrates were observed in the visceral peritoneum (Figure 1C and D), whereas no infiltrate could be documented in the parietal peritoneum. Previous studies have shown that the cytotoxicity (due to cell arrest in G2/M phase) and the immune response observed after adenovirus delivery could be partially due to the adenovirus and the transgene [15].
The safety of i.p. delivery of adenoviral vectors was assessed using ßGal enzymatic activity and staining. We observed increased ßGal activity and positive staining in heart, kidney, liver and lung, in the absence of inflammatory infiltrate. These data confirm that liver and lung are the preferential targets for recombinant adenovirus following systemic or i.p. injection [5,12,16]. Since the peritoneum has abundant lymphatic drainage [2,5] and as the intraperitoneal fluid volume has a direct impact on lymphatic clearance [17], delivery in a large volume of fluid could explain the spreading of the vector to extra-peritoneal tissues. This assertion was supported by the analysis of visceral peritoneal ganglia, showing the presence of the transgene in 90% of the ganglionic cells compared with minimal endogenous ßGal activity in control rats. Furthermore, lymphatic drainage was also reported to account for adenoviral spreading in other adenoviral-mediated gene delivery models [18]. Serum chemistry analysis from rats injected with AdßGal was similar to control animals, in agreement with a previous study [16].
Despite spreading of the adenoviral vector to extra-peritoneal tissues, transgene expression in endothelial cells was associated with the use of a large volume of infusate. New strategies will be required to increase transgene expression in peritoneal endothelial cells, for instance by using new less immunogenic adenoviral vectors containing an endothelial cell-specific promoter.
Acknowledgments
We thank Professor R. T. Krediet and Dr M. Zweers for fruitful discussions. The expert technical assistance of Mrs Y. Cnops, Mrs M. Stiévenart and L. Wenderickx is greatly appreciated. This work was supported by grant 3.4502.99 from the Fund for Medical Scientific Research (Belgium), the Belgian agencies FNRS and FRSM, the ARC 00/05-260, and grants from the Société de Néphrologie and Baxter Belgium.
Conflict of interest statement. None declared.
References
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