Stromelysin Gene Transfer into Cultured Human Trabecular Cells and Rat Trabecular Meshwork In Vivo

Abstract

purpose. To determine whether stromelysin gene can be introduced into and expressed in the cultured human trabecular cells as well as in the rat eye in vivo through means of a recombinant replication-deficient adenovirus.

methods. Stromelysin cDNA was obtained by reverse transcription-polymerase chain reaction with mRNA extracted from the cultured human trabecular cells after induction with interleukin 1α. Adenovirus vector that contains stromelysin cDNA was constructed by cotransfection of pJM17 and pΔA.CMV-str into the 293 cells. The expression of stromelysin in the cultured human trabecular cells was assayed by Western blot and zymography. The expression of stromelysin in the trabecular meshwork of the rat eyes was detected by in situ hybridization and immunohistochemistry.

results. The constructed adenovirus vector contained stromelysin cDNA, but no E1 region. Western blot and zymogram revealed that the stromelysin could be expressed and that it possessed enzymatic activity in cultured human trabecular cells. In situ hybridization and immunostaining of the stromelysin showed that the complete form of stromelysin was expressed in the trabecular meshwork, the iris, and the uveoscleral outflow pathway of the rat eye.

conclusions. Stromelysin, a functional gene, can be transferred in vivo into rat eyes and in vitro into cultured human trabecular cells using a replication-deficient adenovirus vector. This shows the possibility of gene therapy in glaucoma.

Primary open-angle glaucoma is thought to occur because of the increased resistance of aqueous humor outflow through the trabecular meshwork. ¹ Trabecular extracellular matrix (ECM), particularly the highly charged glycosaminoglycans, is a likely site of the normal aqueous humor outflow resistance. ^{2 3 4} ECM turnover appears to be achieved by members of the matrix metalloproteinases (MMPs) family. ^{5 6} Stromelysin, which is a member of the MMP family, has broad substrate specificity and degrades the globular domains of proteoglycan core proteins, laminin, fibronectin, type IV collagen, and a variety of other proteins. ^{7 8} It has been found that trabecular cells secrete stromelysin. ^{5 6} Although stromelysin has broad specificity in all cell biology, it may have more specificity in the trabecular meshwork; therefore, an increase in stromelysin should degrade trabecular proteoglycans, the putative outflow resistance source, and allow their uptake and further degradation by the trabecular cells. ^{5 6 7 8 9} If diminished trabecular ECM turnover is responsible for the reduction in aqueous humor outflow in glaucoma, an increase in stromelysin in the trabecular meshwork should ameliorate the outflow resistance. 

It has been reported that argon laser trabeculoplasty increases the outflow facility by biosynthetic and biodegradative effect in the trabecular meshwork and increases stromelysin in the trabecular meshwork. ⁹ It has also been reported that intracamerally given interleukin (IL) 1α, a known stimulator of the expression of trabecular MMPs, significantly increased the outflow facility in rat eyes. ¹⁰ Hence, stromelysin could be a good candidate gene for gene therapy in glaucoma. 

Recently, successful in vivo gene transfer into the mouse and rabbit trabecular meshwork was reported with the use of an adenoviral vector containing lacZ reporter gene. ^{11 12}  

The present study was designed to determine whether the stromelysin gene, a candidate therapeutic gene for the glaucoma, could be introduced and expressed in the human trabecular cells as well as in the rat eyes in vivo through the recombinant replication-deficient adenovirus vector. If it is possible, gene therapy could be applied as a new modality in the treatment of glaucoma. 

Materials and Methods

Cell Culture

Trabecular meshwork cell culture was initiated from human eyes provided by the Samsung Medical Center Eye Bank. Trabecular meshwork explants were isolated by blunt dissection from the eyes enucleated within 24 hours postmortem, and cells were obtained after digestion of the ECM with collagenase (type XI; Sigma Chemical Co., St. Louis, MO) for 2 hours with vigorous shaking at room temperature. ¹³ After washing briefly, the cells were plated on a 35-mm dish containing 2 ml DMEM supplemented with 20% FBS. Once confluent, the cells were subcultured by trypsinization at a split ratio of 1:4 and used for the following experiments within six passages. The 293 cells were grown in EMEM containing 10% FBS and used for viral propagation within 40 passages. 

Adenoviral Vector Construction

The replication-deficient adenovirus expressing stromelysin was constructed as described by Graham and Prevec. ¹⁴ The cDNA for human stromelysin was obtained by reverse transcription-polymerase chain reaction (RT-PCR) using total RNA extracted from interleukin lα–stimulated human trabecular meshwork cells. The primers used were designed from available sequences (GenBank accession No. X05232) and contained restriction sites (BamHI and EcoRI at each 5′ end) to facilitate the cloning. The primers used were 5′-GGATCCGAAATGAAGAGTCTTCCAATC [forward primer, −3 to 17 nucleotides (nt) human stromelysin cDNA] and 5′-GAATTCCTTTCAACAATTAAGCCAGCT (reverse primer, 1437 to 1417 nt human stromelysin cDNA). Each primer contains six extra nt in its 5′ end corresponding to BamHI and EcoRI restriction sites, to facilitate the cloning. Human stromelysin cDNA nt numbering is according to the translation initiation site (GenBank accession No. X05232). The 1452-bp PCR product was directionally cloned into the pΔA.CMV vector, ¹⁵ which contains the first 16 map units of the adenovirus genome, with the region between map unit 1.3 and map unit 9.4 deleted and replaced by the CMV promoter and a multiple cloning site. The constructed plasmid, along with pJM17, the second plasmid consisting of a complete adenovirus-5 genome with a pBR322 insert in the E1 region, which exceeds packaging capacity and allows plasmid replication but not the formation of infectious viruses, ¹⁶ was cotransfected into the 293 cells using lipofectAMINE (Gibco BRL, Rockville, MD) following the protocol recommended by the manufacturer. After 10 days, a single plaque was isolated, propagated in the 293 cells, and then the resultant recombinant adenovirus Ad.CMV-str was purified by cesium chloride gradient ultracentrifugation. The virus with the desired gene construction was identified by PCR, and the absence of E1 containing wild-type adenovirus contamination was also confirmed by PCR, using primers specific for the adenovirus E1 region as described by Sullivan et al. ¹⁷ Viral titer was 4 × 10¹⁰ plaque-forming unit (pfu)/ml, as determined by the 50% tissue culture infection dose (TCID₅₀) method. 

Zymography and Western Blotting

Trabecular meshwork cells cultured in 12-well plate were washed with serum-free culture media and infected with 100 μl of the viral suspension in PBS at a MOI (multiplicity of infection) of 10, 20, and 40 pfu/cell. After 2 hours of infection, the cells were washed and cultured with fresh culture media containing 10% FBS for 24 hours. Because the serum used for cell culture was known to show matrix metalloproteinase activity including stromelysin, the cells infected were washed three times with PBS, replaced with 0.5 ml of serum-free media per well, and then further cultured until harvested. 

Zymographic analysis was performed 5 days after the infection to detect stromelysin activity according to previously described methods. ^{5 6} Briefly, 20 μl of culture media was mixed with 2-mercaptoethanol–free sample buffer and loaded into 10% polyacrylamide gels containing 0.1% casein (Sigma Chemical Co.). After electrophoresis, the gels were incubated twice for 30 minutes each with 2.5% Triton X-100 to remove SDS and then with proteinase reaction buffer, which was composed of 50 mM Tris (pH 8.0), 150 mM NaCl, 10 mM CaCl₂, and l μM ZnCl₂ at 37°C for 48 hours. The gels were then stained with Coomassie blue, destained, and photographed. 

For Western blotting, acrylamide gel electrophoresis was carried out as described above, and the resolved proteins were transferred electrically to nitrocellulose membranes, blocked with 5% skim milk and 0.1% Tween 20 in PBS, and incubated with polyclonal rabbit anti-human stromelysin antibody (Biogenesis Inc., Sandown, NH) diluted 1:1000 in PBS/Tween 20. The membrane was then developed by peroxidase-conjugated secondary antibody diluted 1:5000 in PBS/Tween 20 using the chemiluminescence method (ECL; Amersham Pharmacia Biotech Inc., Piscataway, NJ) according to the manufacturer’s guide. 

In Vivo Delivery

Animals.

Five normal albino rats (Sprague–Dawley) of both sexes, weighing 250 to 300 g, were used. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg/kg). 

In each rat, one eye was injected with adenoviral vector carrying stromelysin cDNA, and the other eye was injected with the null viral vector, which contains no foreign DNA. A 30-gauge needle was inserted into the anterior chamber through the peripheral cornea, and 5 μl of viral suspension (2 × 10⁸ pfu) was injected with microsyringe. All injections were monitored by direct visualization through the ophthalmic surgical microscope. Animals were killed 5 days after the injection, and the eyes were enucleated. Eyes were immediately fixed for 18 hours at 4°C in PBS containing 4% paraformaldehyde, embedded in paraffin, and then sectioned at 8 to 16μ m. 

All procedures were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

In Situ Hybridization.

Detailed protocols were as described by Komminoth. ¹⁸ Briefly, the slides were deparaffinized and rehydrated, followed by permeabilization with 10 mg/ml of proteinase K. The slides were then incubated with prehybridization solution composed of 4× SSC and 40% deionized formamide. Hybridization was carried out at 42°C overnight in a humid chamber with a solution containing 40% deionized formamide, 10% dextran sulfate, 1× Denhardt’s solution, 4× SSC, 10 mM DTT, 1 mg/ml yeast t-RNA, 1 mg/ml sheared salmon sperm DNA, and 10 ng RNA probe in a volume of 30 μl. For preparation of riboprobe, the full-length human stromelysin cDNA was first cloned into the BamHI and EcoRI sites of pSPT-18 vector, and the circular plasmid was linearized by KpnI cut. An approximately 600-bp antisense probe was transcribed with T7 polymerase in the presence of DIG (digoxigenin)–UTP and purified by phenol extraction and ethanol precipitation. Posthybridization was carried out by serial washing with 2× SSC, 1× SSC, 1× SSC, and finally with 0.1× SSC. The slides were then blocked with 5% skim milk and incubated with alkaline phosphatase–conjugated anti-DIG antibody (Boehringer Mannheim, Mannheim, Germany) diluted 1:100 in 100 mM Tris (pH 7.5) and 150 mM NaCl for 2 hours. After washing the slides several times, binding of the antibody was visualized by NBT/BCIP color detection method. 

Immunohistochemistry

The sections were deparaffinized, rehydrated, and then enzymatically digested for 6 minutes at room temperature by incubation with 0.025% protease (type XXIV; Sigma Chemical Co.) in PBS. After washing briefly, the slides were treated for 5 minutes with 3% H₂O₂ to reduce the endogenous peroxidase activity and subsequently blocked for 30 minutes with 5% bovine serum albumin (BSA) in PBS. The slides were washed in PBS and then incubated for 2 hours with polyclonal rabbit anti-human antibody (Biogenesis Inc.) diluted 1:50 in PBS containing 1% BSA. Detection of the primary antibody used the avidin-biotin complex method using biotinylated anti-rabbit immunoglobulin and horseradish peroxide–conjugated streptavidine (DAKO Corp., Carpinteria, CA) according to the manufacturer’s protocol. The peroxidase reaction was induced by incubating the slides in diaminobenzidine chromogen solution (DAKO Corp.) for 10 minutes at room temperature. 

Results

The full sequence of stromelysin cDNA obtained by RT- PCR was authentic, and correctly inserted into the pΔA.CMV (Fig. 1) , as proven by DNA sequencing and restriction enzyme digestions (data not shown). The adenoviral vector produced was identified to be replication-deficient recombinant by confirmation that the vector contained stromelysin cDNA, but not the E1 gene (Fig. 2) . 

Zymography and Western Immunoblots

The efficiency of adenovirus-mediated stromelysin gene expression in vitro was ascertained in human TM cells infected with the virus Ad.CMV-str. On zymographic analysis, major bands of 57 kDa due to the protease activity of stromelysin were clearly observed, and the activity was shown to increase dose-responsively as the MOI increased. By contrast, faint bands were detected in null virus-infected media and uninfected control media (Fig. 3A) . The activity above was certified to be specific to stromelysin by Western blotting in which sharp band with an apparent MW of 57 kDa was seen only in the media from cells infected with Ad. CMV-str, but not in media from uninfected cells and null virus-infected cells (Fig. 3B) . 

In Situ Hybridization of Stromelysin mRNA and Immunohistochemistry

The localization of stromelysin mRNA was constrained primarily to the trabecular meshwork, the ciliary body, and the uveoscleral outflow pathway (Figs. 4A 4B) . Immunohistochemical staining demonstrated a similar dramatic stromelysin immunostaining in the same area (Figs. 5A 5B) . In controls, however, neither immunostaining nor stromelysin mRNA was observed in the outflow pathways (Figs. 4C and 5C) . 

Discussion

The eye has certain distinct advantages as a target for virus-mediated gene therapy including easy accessibility, well-defined anatomy, and translucent media allowing excellent visual localization of the transfer process. Gene therapy for glaucoma has even more advantages. First, intracamerally introduced vectors can easily access into the cells in the trabecular or uveoscleral outflow pathways because they flow out of the anterior chamber. Second, the surface to volume ratio of the trabecular meshwork is so large that the vectors have a high chance of being introduced into the cells. 

The present study demonstrated that intracamerally introduced adenoviral vector successfully transferred a stromelysin gene into the cells of the rat aqueous outflow tract, including the trabecular meshwork and uveoscleral pathway. The transduced stromelysin gene activity could be rapidly lost in the anterior chamber secondary to immune mediated clearance. However, this seems unlikely given the lack of an immune cell infiltrate and the knowledge that the anterior chamber, like the subretinal and vitreous spaces, possesses enhanced immune privilege properties because of immune deviation and immunomodulating factors. ¹⁹ However, further studies should test the possibility, as others have suggested, ¹⁹ that conventional immunity can emerge and overcome the deviant form of antigen-specific systemic immunity of the anterior chamber. 

Western immunoblot analysis demonstrated that the vector expresses stromelysin by the distinctive gel migration pattern, and zymography revealed that the expressed stromelysin possesses enzymatic activities. In situ hybridization and immunohistochemistry showed that cells in the outflow pathway expressed the stromelysin, and this was the complete form. The location of expression was mostly in the trabecular meshwork and uveoscleral outflow pathway. Particularly, the density of expression in the uveoscleral outflow pathway was significant. This can be explained by the significance of aqueous humor outflow through the uveoscleral outflow pathway in the animal eye compared with that of human eye. ²⁰ Immunohistochemistry demonstrated that a recombinant adenovirus could mediate the transfer and expression of stromelysin gene to the cells in the outflow pathway. 

Adenovirus DNA reportedly remains episomal in infected cells, with the loss of transgene expression because of the loss of transferred DNA or its inactivation without DNA loss. ¹⁸ Thus, for practical gene therapy applications, vectors need to be generated to increase the stability of transgene expression. In comparison with retrovirus or replication-deficient herpes simplex virus 1, replication-deficient adenovirus is less cytotoxic and can be prepared and delivered to tissues in vivo at high titers, resulting in high levels of gene transfer without severe tissue damage. ^{21 22} To control the expression of the gene by the adenovirus vector, the virus should be replication deficient, and this could be obtained by removing the E1 region of the virus. However, recombination competent virus (RCV) can be produced in the process of cotransfection with 293 cells. To eliminate the RCV, we harvested only the plaques containing no E1 region, as confirmed by PCR. 

The appropriate choice of regulatory sequences should allow exogenous genes to be expressed as desired in the relevant cells. The ability to introduce stable exogenous genes into the adult mammalian trabecular meshwork could ultimately pave the way for gene therapy as a treatment for glaucoma. Recombinant adenovirus containing the cDNA for stromelysin may be effective in increasing outflow facility. This raises the possibility that in vivo introduction of the stromelysin gene into the trabecular meshwork could potentially be a viable approach to the treatment of glaucoma. 

In conclusion, the results presented here demonstrate that recombinant replication-deficient adenoviruses can deliver the stromelysin gene successfully into human trabecular cells as well as into the rat eyes in vivo. The next step would be a physiologic investigation, which will introduce our vectors into animal eyes and measure the changes in intraocular pressure and the outflow facility. 

 Supported by a grant from the Korea Science and Engineering Foundation, SRC project endowed to the MTRC.

 Submitted for publication November 9, 2000; revised April 19 and June 12, 2001; accepted June 22, 2001.

 Commercial relationships policy: N.

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked“ advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Map of pΔA.CMV-str.

Figure 2.

 

Analysis of recombinant adenoviral vector carrying human stromelysin cDNA by PCR. The virus rescued from 293 cells cotransfected with pJM17 and pΔA.CMV containing cDNA for stromelysin was propagated and purified. Its genomic conformations were then analyzed by PCR using primer pairs of stromelysin (lane 1) and El (lane 2). The presence of 1452 bp and the absence of El product indicate that the viral vector produced was a replication-deficient recombinant adenovirus containing the stromelysin gene.

Figure 3.

 

Viral expression of stromelysin in cultured human trabecular meshwork cells. Trabecular cells plated onto a 12-well plate were infected with viral vector Ad.CMV-str at MOIs of 0, 2, 5, 10, 20, and 40 (lanes 1 to 6), respectively. Proteolytic activities of the stromelysin were detected 5 days after infection by zymography using casein as a substrate (A). Stromelysin expression was assayed 5 days after infection by Western blot using rabbit anti-human stromelysin antibody (B). Major bands of 57 kDa due to the expression of stromelysin were clearly observed, and the expression was shown to increase dose-responsively as the MOI increased. Lane 7 is a negative control infected with null virus.

Figure 4.

 

Adenovirus-mediated expression of stromelysin in the rat eyes by in situ hybridization. Paraffin sections of the rat eyes obtained 5 days after infection with recombinant Ad.CMV-str (A and B) and with null virus (C) were stained using DIG-labeled antisense riboprobe. The nuclei were counterstained with hematoxylin. Positive reaction is indicated by red-stained cytoplasm. Magnification, (A) ×100; (B) ×400; (C) ×100.

Figure 5.

 

Detection of stromelysin expression in the rat eyes by immunohistochemistry. Paraffin sections of the rat eyes obtained 5 days after injection with recombinant adenovirus Ad.CMV-str (A and B) and with null virus (C) were stained with anti-human stromelysin antibody. The nuclei were counterstained with hematoxylin. Positive reaction is indicated by brown-stained cytoplasm. Magnification, (A)× 100; (B) ×400; (C) ×100.