Nanotechnology and adeno-associated virus-based decorin gene therapy ameliorates peritoneal fibrosis

Abstract

Peritoneal dialysis (PD) is a life-sustaining therapy for end-stage renal disease (ESRD), used by 10–15% of the dialysis population worldwide. Peritoneal fibrosis (PF) is a known complication of long-term PD and frequently follows episodes of peritonitis, rendering the peritoneal membrane inadequate for dialysis. Transforming growth factor (TGF)-β is an inducer of fibrosis in several tissues and organs, and its overexpression has been correlated with PF. Animal models of peritonitis have shown an increase in expression of TGF-β in the peritoneal tissue. Decorin, a proteoglycan and component of the extracellular matrix, inactivates TGF-β, consequently reducing fibrosis in many tissues. Recently, gold nanoparticles (GNP) have been used for drug delivery in a variety of settings. In the present study, we tested the possibility that GNP-delivered decorin gene therapy ameliorates zymosan-mediated PF. We created a PF model using zymosan-induced peritonitis. Rats were treated with no decorin, GNP-decorin, or adeno-associated virus-decorin (AAV-decorin) and compared with controls. Tissue samples were then stained for Masson's trichrome, enface silver, and hematoxylin and eosin, and immunohistochemistry was carried out with antibodies to TGF-β1, α-smooth muscle actin (α-SMA), and VEGF. Animals which were treated with GNP-decorin and AAV-decorin gene therapy had significant reductions in PF compared with untreated animals. Compared with untreated animals, the treated animals had better preserved peritoneal mesothelial cell size, a significant decrease in peritoneal thickness, and decreased α-SMA. Quantitative PCR measurements showed a significant decrease in the peritoneal tissue levels of α-SMA, TGF-β, and VEGF in treated vs. untreated animals. This study shows that both GNP-delivered and AAV-mediated decorin gene therapies significantly decrease PF in vivo in a rodent model. This approach has important clinical translational potential in providing a therapeutic strategy to prevent PF in PD patients.

peritoneal fibrosis (PF) is a recognized complication of long-term peritoneal dialysis (PD), leading to failure of therapy. A major obstacle to performing long-term PD is the deleterious structural and functional alteration in peritoneal membrane due to long-term exposure to PD solution and peritonitis-related injury. Due to the loss of membrane function, most PD patients are unable to sustain PD and are forced to switch to the more expensive hemodialysis (HD), which inflates health care costs. A single episode of peritonitis may not be severe enough to cause significant PF. However, multiple episodes of peritonitis leave residual fibrosis, ultimately causing significant PF and failure of therapy (11, 36). Profibrotic cytokines, such as transforming growth factor (TGF)-β, are upregulated after peritonitis episodes and have been shown to play a central role in tissue fibrosis, leading to progressive loss of peritoneal mesothelial cells (25). TGF-β is a potent profibrotic factor, and its overexpression has been reported to increase production of extracellular matrix and mesenchymal-associated molecules such as Snail, fibronectin, collagen I, and α-smooth muscle actin (α-SMA) (1, 18, 22). In experimental animal models, TGF-β delivery into the peritoneal cavity caused epithelial-to-mesenchymal transition (EMT) and fibrosis (23, 33, 40). Also, increased TGF-β following injury has been reported to cause fibrosis in various other organs, including the kidneys, lungs, cornea, and liver (9, 20, 39). Interventions targeting TGF-β inhibition in the peritoneum has resulted in significant resolution of PF in several animal models (8, 10, 13, 19, 34).

Gene therapy is a promising approach to treating PF, as demonstrated a decade ago using an adenovirus vector (21). Nonetheless, the progress in this area was limited due to the lack of safe and potent gene transfer vectors. Recent advancements in virology have led to the development of a potent, safe, and nonpathogenic adeno-associated virus (AAV). Similarly, innovations in nanotechnology have rendered numerous efficient and safe nanoparticles for gene therapy. These newer generation gene therapy vectors have significantly improved the translational application of gene therapy for PF. Our recent studies demonstrated that decorin, a small leucine-rich proteoglycan, delivered into keratocytes of the cornea via hybrid gold nanoparticles (GNP) or AAV, significantly inhibits myofibroblast formation, fibrosis, and angiogenesis in vivo in rabbit corneal fibrosis and angiogenesis disease models (26, 27). In this study, we tested the potential of nanoparticle- and AAV-mediated decorin gene therapy in the treatment of PF in vivo using a zymosan-induced rat model of PF.

MATERIALS AND METHODS

Animals.

All animal studies were approved and carried out according to the University of Missouri-Columbia and Harry S. Truman Memorial Veterans' Hospital Animal Care and Usage Committee guidelines. Twenty-eight Sprague-Dawley rats (200–250 mg) were used and divided into five groups: group 1: naive controls (n = 4); group 2: zymosan-induced peritonitis (n = 6); group 3: zymosan-induced peritonitis+decorin gene delivery by GNP (n = 7); group 4: zymosan-induced peritonitis+decorin gene delivery via AAV (n = 7); and group 5: zymosan+naked vectors group (n = 4).

Treatment protocol.

Peritonitis in the rats was induced by injecting two doses of zymosan (15 mg/kg body wt ip) 48 h apart. In our experiments, we induced peritonitis in rats with zymosan as demonstrated in the literature (24). Briefly, abdominal skin was disinfected with Amuchin, a chlorine antiseptic solution, and each animal received 25 ml of commercial peritoneal dialysis solution into the peritoneal cavity through the lower right portion of the abdomen via a 35-ml syringe with a 22-gauge needle. The injection was the initiation point of the peritoneal equilibration test (PET). Two hours after instillation, blood and dialysate samples were taken for analysis. Following the conclusion of the PET, animals were given buprenorphine (0.05 mg/kg) and zymosan (15 mg/kg body wt ip). A second dose of zymosan was given in similar fashion after 48 h. For the AAV animals (group 4), the AAV-decorin (5 × 1,010 genomic copies/ml) injection was given 5 days before the first dose of zymosan, and the GNP animals (group 3) received a GNP-decorin transfection mixture (100 μl/rat) 24 h before the first dose of zymosan (25, 30). The animals were euthanized 14 days after the last dose of zymosan. Histological studies were done using silver, trichrome, and hematoxylin and eosin staining and immunofluorescence for TGF-β1, α-SMA, and VEGF.

PET.

Before the induction of peritonitis, a baseline PET was carried out. At time 0, 25 ml of a 2.5% glucose commercial dialysis solution (Baxter Healthcare, Chicago, IL) was injected into the peritoneal cavity under light isoflurane anesthesia. A dose (0.01–0.05 mg/kg) of the analgesic buprenorphine was also given. At 55 min, the animal was anesthetized with isoflurane and 2 ml of blood was removed via cardiocentesis, and the animal was allowed to recover. The blood specimen stood for 20 min and then was centrifuged at 2,000 g for 15 min, and serum was removed and refrigerated. At 115 min, the animal was again anesthetized, and a 1.5- to 2-ml sample of dialysate was removed from the abdominal cavity at the 120-min mark. The animal was then allowed to recover. The serum and dialysate specimens were analyzed on a Synchron CX5-Delta (Beckman-Coulter, Brea, CA). The dialysate/plasma ratios (D/P) for urea and creatinine were calculated. A second PET was carried at the end of the study period identical to the first in methodology for comparison.

Histology.

Tissue samples from the lower anterior abdominal wall, liver, and mesentery were collected after euthanasia. Sections from both sides of the midline were taken and fixed in a sufficient amount of 4% phosphate-buffered formalin. The tissue samples were then processed, embedded in paraffin, and 5-μm sections were prepared. The trichrome, silver, and hematoxylin and eosin staining and immunofluorescence were carried out with antibodies specific to TGF-β1, α-SMA, and VEGF.

Quantitative real-time PCR.

Peritoneal tissues were homogenized in RLT buffer using TissueLyser (Qiagen, Valencia, CA) for 2 min at 20 Hz. Total RNA was extracted using an RNeasy kit (Qiagen) and reverse-transcribed to cDNA following the vendor's instructions (Promega, Madison, WI).

Statistics.

Analysis was carried out using SigmaPlot v 11.2 software (Systat Software, San Jose, CA). Student's t-test was used except where the data failed the tests for normality and equal variance; then, the Mann-Whitney test was used. The statistical comparison between the groups was performed using ANOVA with Bonferroni-Dunn adjustments for repeated measures. Values are presented as means ± SD, and P values <0.05 were considered significant.

RESULTS

Zymosan significantly altered peritoneal membrane histology.

Zymosan infusion led to inflammation of the submesothelial zone with significantly increased fibrinogen content of mesenteric tissue, leading to chronic fibrotic changes in test animals. As expected, our study showed that zymosan significantly altered the structure of the parietal peritoneum compared with untreated animals.

Animals treated with decorin gene transfer showed better preservation of peritoneal membranes.

We compared changes in the peritoneal membrane of three groups of zymosan-treated rats [one treated with zymosan only, a second treated with intraperitoneal AAV-decorin (AAV-dcn), and the third treated with GNP-decorin (GNP-dcn)] with the untreated naive group of rats. Animals that received decorin gene therapy showed better preservation of peritoneal membranes and significantly less PF in immunofluorescence staining compared with the non-decorin-delivered group. We also compared the histology of negative control animals (naive and null vector) with three groups of treated animals; zymosan treated only, zymosan+AAV-dcn, and zymosan+GNP-dcn. Animals of both the AAV-dcn and GNP-dcn therapy groups showed a remarkable decrease in PF on gross appearance compared with the group treated with zymosan only. Similarly, tissues of both decorin treated groups (AAV-dcn and GNP-dcn) showed better retention of cell morphology by silver staining compared with the zymosan only group (Fig. 1). The cell size was better preserved in both the decorin-delivered animals compared with the untreated animals: naive 656.8 vs. zymosan 216.6 vs. GNP-treated 272.2 vs. AAV-treated 292.8 μm² (P < 0.001) (Fig. 1). Hemtoxylin and eosin staining images showed a decrease in parietal peritoneum thickness in decorin-treated groups compared with the zymosan only group (Fig. 2A). Both GNP- and AAV-mediated decorin gene therapy significantly (P < 0.001) decreased PF compared with the zymosan only group (GNP-dcn 43.3 ± 3.9 and AAV-dcn 42.4 ± 5.4%) (Fig. 2B). A marked reduction in fibrosis marker protein α-SMA was detected in the abdominal wall (Fig. 3A) in both decorin-delivered groups compared with nondelivered groups. Quantification of α-SMA+vehicle cells/×200 magnification field in the tissue sections obtained from parietal peritoneum showed significant reduction (P < 0.001) of α-SMA+vehicle cells in the GNP (58 ± 4.3%)- and AAV (60.8 ± 3.8%)-mediated decorin gene therapy groups compared with the non-decorin-delivered (zymosan only) group (Fig. 3B).

Fig. 1.

Representative image using silver stain showing normal and abnormal membrane histology. AAV, adeno-associated virus; GNP, gold nanoparticles; NT, no treatment.

Fig. 2.

A: representative hematoxylin and eosin images showing abdominal wall parietal peritoneum thickness in treated and untreated animals. B: measurements of effects of GNP- and AAV-mediated decorin (dcn) gene therapy on parietal peritoneum thickness. *P < 0.001. *#P = not significant (NS).

Fig. 3.

A: immunohistochemistry showing levels of α-smooth muscle actin (SMA) in treated vs. untreated animals. B: quantification of α-SMA-positive cells/×200 column in the tissue sections obtained from abdominal wall parietal peritoneum. *P < 0.001. ΦP = NS.

Animals treated with decorin gene transfer showed decreased levels of markers of fibrosis by RT-PCR.

Quantitative real-time PCR analysis detected differential changes in α-SMA, TGF-β, and VEGF expression in parietal peritoneum of Sprague-Dawley rats with/without decorin gene therapy (Fig. 4). Untreated animals had a 6.1-fold increase in α-SMA (left), a 8.4-fold increase in TGF-β (middle), and a 6.2-fold increase in VEGF levels (right). AAV-dcn and GNP-dcn gene delivery inhibited zymosan-induced α-SMA, TGF-β, and VEGF expression significantly (*P < 0.001 vs. control and *#P < 0.01 vs. dcn gene therapy via AAV and GNP) [Fig 4].

Fig. 4.

Quantitative real-time PCR showing differential change in α-SMA, transforming growth factor (TGF)-β, and VEGF expression in parietal peritoneum of treated vs. untreated animals. *P < 0.001 vs. control and *#P < 0.01 vs. dcn gene therapy via AAV and GNP.

Decorin gene transfer effects on PET.

In a comparison of pre- and post-PET results, there was a statistically significant increase in D/P urea and decrease in the dialysate concentration ratio at the beginning and ending of PET (D/Do) for glucose in the decorin-untreated animals. In both decorin-treated group of rats, there was an increase in the D/P urea and decrease in the D/Do glucose as well. However, changes in the D/P urea and D/Do glucose between three groups did not show statistical difference. The D/P creatinine also increased in all three groups but did not reach statistical significance (Fig. 5).

Fig. 5.

Peritoneal equilibration test (PET) test data showing changes pre- and post zymosan exposure in zymosan only, GNP-dcn, and AAV-dcn groups. D/P, dialysate/plasma ratio; D/Do, dialysate concentration ratio at the beginning and ending of PET.

Both AAV and GNP delivered the decorin plasmid efficiently to the peritoneum.

We measured the delivered transgene gene copy numbers by real-time PCR quantification in rat parietal peritoneum tissue. The AAV and GNP vectors delivered 2.03 × 10⁵ and 1.8 × 10⁵/μg DNA, respectively, of decorin plasmid copies in abdominal wall tissue (Fig. 6).

Fig. 6.

Real-time PCR quantification of delivered transgene gene copy number in Sprague-Dawley rat parietal peritoneum tissue.

DISCUSSION

We have presented here the first report to the best of our knowledge of using nanoparticles in drug delivery for prevention of PF. The present study was designed to investigate the protective effects of decorin gene therapy using GNP or AAV as a delivery vehicle in zymosan-induced PF in rats. Our study showed nanoparticles efficiently delivered the decorin plasmid in the peritoneum. Decorin gene therapy delivered via nanoparticles decreased PF in a rat model of PF and decreased expression of fibrosis markers, i.e., α-SMA, TGF-β, and VEGF. At present, no specific treatment which can prevent PF in PD patients is available. Although many interventions have been tried to prevent PF in patients undergoing PD, none has been clinically successful. Small observational studies have shown that patients receiving renin-angiotensin system blockade tended to have slightly more favorable peritoneal transport characteristics (16) but had no survival advantage. In animal models, drugs such as angiotensin-converting enzyme inhibitors and angiotensin receptor blockers have been tried and shown to reduce PF, although human data have been equivocal (35). Nevertheless, these drug therapies do not likely offer any additional advantage from a clinical perspective because most PD patients are already on these medications for coexisting cardiovascular problems but still develop PF during the course of PD therapy. Upregulation of cyclooxygenase (COX)-2 during EMT has been reported in an in vitro setting, and celecoxib treatment resulted in a reduction of fibrosis and blunted ultrafiltration failure (2). In a rat model, oral administration of celecoxib drastically reduced prostaglandin E₂ levels, angiogenesis, and lymphangiogenesis, and preserved ultrafiltration failure induced by administration of classic PD fluids (6). However, in view of the adverse cardiovascular effects associated with COX-2 inhibition and the adverse renal effects of NSAIDs in general, its use in patients on PD is very limited. More recently, use of TGF-β-blocking peptides has shown to ameliorate PF and angiogenesis in an animal model of PD (19). Decorin is a 100-kDa proteoglycan found in the interstitial ECM and has been shown to decrease fibrosis in many tissues (8, 13). Decorin belongs to the family of small leucine-rich proteoglycans composed of a 40-kDa core protein and a single chondroitin sulfate side chain. The core protein is capable of binding TGF-β with high affinity, which together forms an inactive TGF-β -decorin complex (19). Decorin is reported to reduce collagen accumulation via inhibition of TGF-β in vitro and in vivo (8, 13, 21). It has been shown that decorin has multiple direct effects as a signaling molecule through multiple signaling pathways principally affecting growth, differentiation, or programmed cell death in a cell/tissue-specific manner (31). Numerous studies have reported decorin's role in the modulation of TGF-β, affecting cell cycle machinery, tissue organization, and development (12). Recent research has demonstrated that decorin suppresses endothelial migration as well as formation of vascular tubes, suggesting its role in angiogenesis (4, 15). Decorin has been effective in treating lung fibrosis by direct application and in renal fibrosis through systemic gene therapy (8, 13). A role for decorin has been established in healing and has led to its use as a therapeutic agent to facilitate wound healing in various model systems, including topical gene therapy in corneal fibrosis and angiogenesis as well as cutaneous wound healing and angiogenesis (14, 28). An interesting finding in this study was the decreased mRNA levels of TGF-β (Fig. 4), pointing to its decreased transcription, which is in alignment with other published reports (30, 42). TGF-β has been shown to increase its own transcription levels through an autocrine loop in scar tissue (30). Therefore, decorin, by binding and inactivating TGF-β, can block this autocrine loop, which possibly explains the noted effect of decorin gene therapy on TGF-β mRNA transcription. A similar decrease in TGF-β mRNA expression has been shown by recombinant decorin treatment in keloid fibroblasts (42), which supports the results of our study. Additionally, decorin gene therapy can also cause a decrease in TGF-β expression by decreasing the myofibroblast population, because transdifferentiation of mesothelial cells to myofibroblasts in peritoneal fibrosis is mainly responsible for the noted increase in TGF-β expression.

After being injected into the peritoneal cavity, the GNP-decorin complex enters the peritoneal mesothelial cells by receptor-mediated endocytosis (3). After entering mesothelial cells, GNPs mainly reside inside vesicles that evolve to form late endosomes and endolysosomes (vesicle size and number of particles per vesicle increase with time). Due to the “proton-sponge effect,” cationic charges on polyethylenimine (PEI) attract chloride ions and cause endocytic vesicle swelling and rupturing, and releases plasmid into the cytoplasm, providing high decorin levels in the peritoneum (38). This mechanism leads to attenuation of TGF-β action, prevents PF, and preserves peritoneal membrane structure and function. The small size, efficient binding, and releasing properties make GNPs an ideal gene therapy vector. As a delivery system, PEI-GNP has multiple advantages: 1) efficient complexing with nucleic acids, 2) high gene transfection efficiency and excellent cellular uptake, 3) controlled gene expression through modification of the nitrogen/phosphate ratio, 4) protection of therapeutic genes from nuclease degradation, and 5) long-term stability at room temperature. The gene therapies have been found to be generally safe and are not injurious to the applied tissue. Adenovirus given by an intraperitoneal route did not result in any peritoneal fibrosis or increase in markers of fibrosis (20, 21, 29). Studies involving GNP have found it to be noncytotoxic, noninflammatory, and safe in the human cornea in vitro and the rabbit cornea in vivo, and GNP has been used for gene transfer in the rabbit cornea to prevent corneal fibrosis (26, 37).

There are some limitations of the study. The PF model using zymosan-induced peritonitis has a more vigorous effect, whereas the PF in PD patients is mainly secondary to intermittent infections and chronic glucose and glucose degradation product exposure (5). The results of the physiological PET were negative, which could be due to the different behavior of the peritoneal membrane in rats compared with humans. Also, besides TGF-β there are other mediators of membrane injury, including inflammatory cytokines such as interleukin-6, which also determine solute transport characteristics at the start of peritoneal dialysis and can alter membrane function and contribute to PF (7, 17).

In conclusion, our study results show that rats treated with both GNP-delivered and adenovirus-mediated decorin had less PF than untreated animals. There are several theoretical and therapeutic benefits of using GNP over an adenovirus. This has significant translational potential since decorin is a naturally occurring inhibitor of TGF-β and will likely be very suitable for clinical use than other gene-based therapies.

GRANTS

This work was primarily supported by the Paul Teschan Research Fund and a research grant from the Dialysis Clinic, Inc. (K. Chaudhary and R. R. Mohan), and partially by the Ruth M. Kraeuchi Missouri Endowment Fund (R. R. Mohan), Grant RO1EY17924 (R. R. Mohan) from the National Eye Institute, and Merit Grant 1l01BX000357-01 (R. R. Mohan) from the US Veterans Health Administration.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: K.C., H.M., R.K., and R.R.M. provided conception and design of research; K.C., H.M., A.T., S.G., and R.R.M. analyzed data; K.C., H.M., A.T., S.G., R.K., and R.R.M. interpreted results of experiments; K.C., H.M., R.K., and R.R.M. drafted manuscript; K.C., R.K., and R.R.M. edited and revised manuscript; K.C., H.M., A.T., R.K., and R.R.M. approved final version of manuscript; H.M., A.T., and S.G. performed experiments; H.M., A.T., and R.R.M. prepared figures.