May 2013
Volume 54, Issue 5
Free
Glaucoma  |   May 2013
Overexpression of SPARC in Human Trabecular Meshwork Increases Intraocular Pressure and Alters Extracellular Matrix
Author Affiliations & Notes
  • Dong-Jin Oh
    Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Harvard Medical School, Boston, Massachusetts
  • Min Hyung Kang
    Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Harvard Medical School, Boston, Massachusetts
  • Yen Hoong Ooi
    Department of Pediatrics, New York Downtown Hospital, New York, New York
  • Kyu Ryong Choi
    Department of Ophthalmology, Ewha Womans University School of Medicine and Medical Center, Mokdong Hospital, Seoul, Korea
  • E. Helene Sage
    Hope Heart Program, Benaroya Research Institute at Virginia Mason, Seattle, Washington
  • Douglas J. Rhee
    Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Harvard Medical School, Boston, Massachusetts
  • Correspondence: Douglas J. Rhee, Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, 243 Charles Street, Boston, MA 02114; DougRhee@aol.com
Investigative Ophthalmology & Visual Science May 2013, Vol.54, 3309-3319. doi:10.1167/iovs.12-11362
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      Dong-Jin Oh, Min Hyung Kang, Yen Hoong Ooi, Kyu Ryong Choi, E. Helene Sage, Douglas J. Rhee; Overexpression of SPARC in Human Trabecular Meshwork Increases Intraocular Pressure and Alters Extracellular Matrix. Invest. Ophthalmol. Vis. Sci. 2013;54(5):3309-3319. doi: 10.1167/iovs.12-11362.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose.: Intraocular pressure (IOP) regulation is largely unknown. SPARC-null mice demonstrate a lower IOP resulting from increased outflow. SPARC is a matricellular protein often associated with fibrosis. We hypothesized that SPARC overexpression would alter IOP by affecting extracellular matrix (ECM) synthesis and/or turnover in the trabecular meshwork (TM).

Methods.: An adenoviral vector containing human SPARC was used to increase SPARC expression in human TM endothelial cells and perfused human anterior segments using multiplicities of infection (MOIs) 25 or 50. Total RNA from TM was used for quantitative PCR, while protein from cell lysates and conditioned media were used for immunoblot analyses and zymography. After completion of perfusion, the anterior segments were fixed, sectioned, and examined by light and confocal microscopy.

Results.: SPARC overexpression increased the IOP of perfused human anterior segments. Fibronectin and collagens IV and I protein levels were elevated in both TM cell cultures and within the juxtacanalicular (JCT) region of perfused anterior segments. Collagen VI and laminin protein levels were increased in TM cell cultures but not in perfused anterior segments. The protein levels of pro-MMP-9 decreased while the kinetic inhibitors of metalloproteinases, TIMP-1 and PAI-1 protein levels, increased at MOI 25. At MOI 50, the protein levels of pro-MMP-1, -3, and -9 also decreased while PAI-1 and TIMP-1 and -3 increased. Only MMP-9 activity was decreased on zymography. mRNA levels of the collagens, fibronectin, and laminin were not affected by SPARC overexpression.

Conclusions.: SPARC overexpression increases IOP in perfused cadaveric human anterior segments resulting from a qualitative change the JCT ECM. Selective decrease of MMP-9 activity is likely part of the mechanism. SPARC is a regulatory node for IOP.

Introduction
Primary open-angle glaucoma (POAG) is one of the leading causes of irreversible blindness. 13 Elevated intraocular pressure (IOP) is a causative risk factor for the development and progression of POAG. 46 The elevated IOP results from dysfunctional aqueous drainage through the trabecular meshwork (TM). 7,8 The exact molecular and cellular processes responsible for the normal physiologic regulation of outflow resistance in the TM remain elusive. However, shifting the equilibrium between extracellular matrix (ECM) synthesis and breakdown within the TM strongly influences IOP. 912 Furthermore, alterations of the ECM within the juxtacanalicular (JCT) portion of the TM have been found to be a primary pathophysiologic association with POAG. 13 Along with other investigators, we believe that proteins known to regulate ECM equilibrium in other tissues will influence IOP. 14  
Matricellular proteins are nonstructural, secreted glycoproteins that facilitate cellular control over their surrounding ECM and play a role in cellular adhesion in many human tissues. 15 The matricellular family includes SPARC (secreted protein, acidic and rich in cysteine), thrombospondins (TSPs) 1 and 2, tenascins C and X, hevin, and osteopontin. 15 Matricellular proteins, and in particular SPARC, are associated with increased fibrosis and aberrant tissue remodeling, processes implicated as major contributors to glaucoma pathogenesis. 15 In resting TM tissue and in TM cells undergoing physiologic stress (mechanical stretch), SPARC is one of the most highly transcribed genes, data indicating that SPARC has a prominent physiological function. 1618 In response to transforming growth factor-β2, which is elevated in the aqueous humor of patients with POAG, SPARC is the most highly up-regulated protein. 19,20 Furthermore, we have demonstrated that SPARC-null mice have a 15% to 20% lower IOP, the result of increased aqueous drainage. 21 These findings implicate SPARC as a significant regulator of IOP. 
We hypothesized that SPARC regulates IOP by shifting the balance of ECM synthesis and turnover in the TM. Accordingly, we increased the expression of SPARC in cadaveric human perfused anterior segments and in cultured human TM cells by adenoviral transfection of cloned human SPARC. We measured the IOP, examined the drainage tissue via immunohistochemistry, and assessed the relative expression of various ECM components. In TM cells, the protein and mRNA levels of specific ECM proteins and matrix metalloproteinases (MMPs), which are prominent ECM-degrading enzymes in TM, and their kinetic inhibitors, tissue inhibitors of metalloproteinases (TIMPs), were determined. 
Methods
Adenoviral Constructs and Adenovirus Production
Adenoviral (Av) vectors containing the human SPARC transgene (Av.hSPARC) and control Av vectors were produced by the use of the transpose-Ad adenoviral vector system (Qbiogene, Carlsbad, CA). A fragment of human SPARC cDNA (973 bp) was prepared by PCR of reverse-transcribed human mRNA, with human-specific primers, 5′ and 3′ ends containing Nhe1 and Xba1 restriction sites, which were subsequently cloned into the corresponding restriction site of pCR259 (Qbiogene). Transgene orientation was verified by analytical PCR. The pCR259 plasmid was prepared without SPARC as a control and both contain the CMV promoter and replication-deficient adenoviral genes (Av.Control). The Av plasmid, Av.CMV.hSPARC, was transformed in DH5α bacteria. The amplified DNA was purified (EndoFree Plasmid Maxi Kit; Qiagen, Valencia, CA) and was subsequently transfected into HEK293 packaging cells (QBI-293A) with the CalPhos Mammalian Transfection Kit (Clontech, Mountain View, CA). We incubated the packaging cells at 37°C, 5% CO2 until the cytopathic effect become evident and collected the viral supernate. The Ad5.hSPARC adenovirus was purified and titered using the ViraBind adenovirus purification kit and QuickTiter adenovirus titer immunoassay kit (Cell Biolabs, San Diego, CA). The control Av plasmid, Av.CMV.empty, received the same handling except that the SPARC cDNA was not included in the plasmid. 
Culture of Human TM Cells and SPARC Overexpression
Primary human TM cell cultures were derived from 12 separate donors (age range: 42–64 years; average age: 48.7 years). Maintenance growth medium consisted of Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum (FBS; Invitrogen-Gibco, Grand Island, NY), 1% L-glutamine (2 mM), and 0.5% or 0.1% gentamicin (50 or 10 μg/mL). For each experiment, TM cultures were seeded into six-well plates (Costar, Corning, NY), allowed to grow to confluence at 37°C in a 10% CO2 atmosphere and given an additional 2 to 3 days for differentiation. For all experiments, TM cells from the fourth or fifth passage were used. 
TM cells were incubated in 2% FBS medium with Ad5.control or Ad5.hSPARC for 18 hours, after which 10% FBS medium and an equal volume were added to the 2% medium for an additional 24 hours (Fig. 1). TM cultures were incubated for another 24 hours in serum-free medium, which was later replaced with culture medium containing adenovirus for 24 hours. The serum-free conditioned medium was analyzed by immunoblot assays. Two different multiplicities of infection (MOIs; 25 and 50) of Ad5.control and Ad5.hSPARC were used. MOI is the ratio of infectious units to infectious targets (cells). 22,23 For zymographic analyses, gelatin (0.1%) for MMP-2 and -9 or β-casein (0.1%) for MMP-1 and -3 were mixed into liquid acrylamide during casting of the polyacrylamide gels. Concentrated conditioned medium mixed with 2× Tris-glycine-SDS zymography sample buffer at a 1:10 ratio was loaded onto 10% SDS-PAGE. The samples were subjected to electrophoresis at 130 V in tank buffer (250 mM Tris, 192 mM glycine, and 0.1% SDS). The gels were incubated with 2.5% Triton X-100 (renaturing buffer) with gentle agitation at room temperature, then transferred to enzyme assay buffer overnight at 37°C. The resultant gels were stained with 0.1% Coomassie Brilliant Blue G-250 (Bio-Rad, Hercules, CA) for at least 3 hours and were destained with fixing/destaining solution until clear bands were visible and contrasted well with the blue background. The gels were scanned using the Odyssey Infrared Imaging System (Li-Cor, Lincoln, NE), and relative densities of the bands were analyzed with the Odyssey densitometric software (version 1.2). All the MMPs were identified based on their molecular weights and were confirmed against purified MMP-1, -2, -3, and -9 (Chemicon, Temecula, CA) as positive controls. 
Figure 1
 
Experimental time course of SPARC overexpression in vitro. TM cultures were incubated with 2% FBS medium with either Ad5.control or Ad5.hSPARC for 18 hours. Then 10% FBS medium of equal volume were added to the 2% medium for an additional 24 hours. The TM cultures were next incubated with serum-free medium (which did not contain virus) for 24 hours. The serum-free media were harvested for immunoblot assays. A representative immunoblot of SPARC expression in conditioned media from TM cells with no adenovirus (N), with Ad5.control (C), and with Ad5.hSPARC (S+) infection is shown; equal total protein was loaded into each well.
Figure 1
 
Experimental time course of SPARC overexpression in vitro. TM cultures were incubated with 2% FBS medium with either Ad5.control or Ad5.hSPARC for 18 hours. Then 10% FBS medium of equal volume were added to the 2% medium for an additional 24 hours. The TM cultures were next incubated with serum-free medium (which did not contain virus) for 24 hours. The serum-free media were harvested for immunoblot assays. A representative immunoblot of SPARC expression in conditioned media from TM cells with no adenovirus (N), with Ad5.control (C), and with Ad5.hSPARC (S+) infection is shown; equal total protein was loaded into each well.
Human Perfused Anterior Segment Culture System
All donor pairs of eyes (aged 42, 56, 78, 78, and 80 years) were obtained from National Disease Research Interchange (NDRI, Philadelphia, PA) according to the provisions of the Declaration of Helsinki for research involving human tissue. Eyes were obtained within 24 hours after death. No donors were known to have a history of glaucoma or other ocular disorder. Human perfused anterior segment cultures were prepared by scoring the surface of the eye around ora serrata with a surgical blade, and the full-thickness incision was completed around the eye with scissors. The vitreous, lens, and iris were removed. Ciliary processes were dissected carefully, leaving in place the longitudinal portion of the ciliary muscle. The anterior segments were rinsed thoroughly with culture media and were mounted into custom plexiglass culture chambers. Anterior segments were perfused at a constant flow rate of 2.5 μL/min with DMEM (Invitrogen-Gibco) containing 1% FBS, 1% L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 U/mL), gentamicin (0.17 mg/mL), and amphotericin-B (0.25 μg/mL) under 5% CO2 at 37°C, using microinfusion pumps (Harvard Apparatus, Holliston, MA). IOP was monitored with a pressure transducer (BD Medical, Franklin Lakes, NJ) and were recorded with an automated computerized system (National Instruments, Austin, TX) every second and averaged each hour. Perfused tissue was allowed to equilibrate at 37°C and 5% CO2 until a stable baseline IOP was achieved, typically 2 to 4 days. Pairs of eyes were treated. The pairs were excluded if the eyes failed to achieve a stable baseline IOP (n = 3) or if the postexperimental toluidine blue–stained light microscopy sections showed acellularity (n = 0). Before injection, perfusion pumps were stopped briefly to lower the pressure in the anterior chamber of the eye. Then one eye was injected intracamerally with Ad5.control while the other received Ad5.hSPARC in a volume of 100 μL containing 1 × 108 infectious units (ifu), similar to the methodology described by Ethier et al. 24 The chambers were kept in a 5% CO2, 37°C humidified incubator. The perfusate drained from scored episcleral veins. The perfusion chambers had a lid to protect against evaporation of media. Perfusate was collected using a sterile Pasteur pipette a day before and at 1 and 2 days after injection for immunoblot analysis of SPARC levels. After 5 days, the anterior segments were fixed and evaluated for viability and morphology by light microscopy at the termination of each study. Effects of SPARC overexpression on IOP were expressed as the percentage change in IOP (compared to baseline values) over 96 hours. Values were expressed as mean ± SEM, and a paired two-tailed Student's t-test was applied to determine the significance of difference in IOP between control and experimental groups at selected time intervals. IOP was normalized at time point 0, the time of injection. Two-way ANOVA testing was applied to the mean IOP values after injection. 
Quantitative PCR
Total RNA from TM endothelial cells was isolated using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Briefly, cultured TM cells were lysed with Trizol buffer, and chloroform was added. After centrifugation, total nucleic acid was isolated from the aqueous layer by ethanol precipitation. After digestion of DNA by DNase I, total RNA was isolated and stored at −80°C for no more than 1 month prior to further experiments. 
Complementary DNA (cDNA) was synthesized using the M-MLV RT kit (Promega, Madison, WI) according to the manufacturer's instructions, with minor modifications. Briefly, 250 ng of total RNA was used as a template in 50 μL of reaction mixture containing 250 ng of oligo-dT primer, 1× reaction buffer, 0.2 mM of dATG, dCTG, dGTG, and dATG, 20 U of RNase inhibitor, and 200 U of M-MLV reverse transcriptase. The reaction mixture was incubated at 42°C for 1 hour and used for quantitative PCR (qPCR). 
Quantitative PCR was performed to detect mRNA in cultured TM cells. Specific mRNAs were amplified in SYBR green I master mix (Applied Biosystems, Inc., Foster City, CA) with the ABI StepOnePlus (Applied Biosystems, Inc.). Quantification of the genes of interest was calculated by fold increase to β-actin expression using StepOne software v2.0 (Applied Biosystems, Inc.). All qPCR were performed in triplicate. Primers for human ECM types and β-actin were designed according to the Primer 3 program (http://frodo.wi.mit.edu) with an expected qPCR product of 200 bp (Table 1). Fold changes were normalized to GAPDH, then calculated relative to control (Ad5.control). 
Table 1. 
 
Primer Sequences for Quantitative PCR
Table 1. 
 
Primer Sequences for Quantitative PCR
Forward Reverse
Collagen α1 (I) 5′-CTGGTCCTGATGGCAAAACT-3′ 5′-CTCCAGCCTCTCCATCTTTG-3′
Collagen α2 (IV) 5′-TCTTTCCTCATGCACACTGC-3′ 5′-TTCAGCGTTTCAGACACAGG-3′
Collagen α1 (VI) 5′-CTGGGCGTCAAAGTCTTCTC-3′ 5′-ATTCGAAGGAGCAGCACACT-3′
Collagen α2 (VI) 5′-GAGATCGACCAGGACACCAT-3′ 5′-GGTCTCCCTGTCTTCCCTTC-3′
Collagen α3 (VI) 5′-CTGGGCAGACATACCATGTG-3′ 5′-GCAAGTTCCTTCGTCTTTCG-3′
Fibronectin 5′-ACCAACCTACGGATGACTCG-3′ 5′-GCTCATCATCTGGCCATTTT-3′
Laminin α5 5′-TGACCTTTTCTGGCTCGTCT-3′ 5′-GTTCAGCACAAAGGGCTCTC-3′
Laminin β1 5′-AGGTTGGAGCTGCCTCAGTA-3′ 5′-TGTTTTCACAACGCTTCTGC-3′
Laminin γ1 5′-GCATCTCTCGAGTGGTCCTC-3′ 5′-TGGATAGGAATTGCCCTGAG-3′
β-actin 5′-GGCATCCTCACCCTGAAGTA-3′ 5′-GGGGTGTTGAAGGTCTCAAA-3′
Immunoblot Analyses
Cultured TM cells infected with adenovirus were lysed in 1× radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH 8.0) with protease inhibitors (25 mTIU/mL aprotinin, 0.05 mg/mL phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 mM EGTA, and 1 μg/mL leupeptin to a final volume of 1 mL with RIPA buffer). The conditioned media were collected and concentrated 30-fold with a centrifugal filter unit (Amicon Ultra-4, 10K; Millipore, Milford, MA). Equal amounts of total protein from conditioned media were mixed with 2× reducing buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.01% bromophenol blue) at a 1:10 ratio and were boiled for 3 minutes with reducing reagent (5 mg/mL dithiothreitol) or without it (nonreducing condition). The samples were analyzed by electrophoresis on 10% polyacrylamide gels (PAGE). SDS-PAGE was performed at 100 V in tank buffer with the XCell SureLock Mini-Cell system (Invitrogen). The separated proteins were transferred onto a nitrocellulose membrane with 0.45-μm pore size (Invitrogen) in blotting buffer (250 mM Tris-HCl, 192 mM glycine, and 10% methanol). The membrane was incubated for 1 hour in 0.5× blocking buffer (Rockland, Gilbertsville, PA) at room temperature. The membrane was next incubated with a primary antibody in 0.5× blocking buffer, overnight at 4°C (Table 2). The next day, the membrane was washed three times with TBS/T (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween20) for 10 minutes and incubated with IRDyeTM 800-conjugated IgG (1:10,000; Rockland) for 1 hour (Table 2). The membrane was washed with 1× TBS/T three times at room temperature for 10 minutes and was scanned on the Odyssey Infrared Imaging System (Li-Cor). The band density of proteins was quantified with the Odyssey densitometric software (version 1.2). Deglycosylation of SPARC was performed using our previously described methodology. 18  
Table 2. 
 
Primary and Secondary Antibodies Used for Immunoblot and Immunofluorescence in This Study
Table 2. 
 
Primary and Secondary Antibodies Used for Immunoblot and Immunofluorescence in This Study
Primary Antibody Company Antibody Host Species Dilution Secondary Antibody Company Dilution
Immunoblot
 MMPs
  MMP-1 R&D Systems Mouse 1:1,000 IRDye 800 antimouse IgG Rockland 1:10,000
  MMP-2 R&D Systems Mouse 1:1,000 IRDye 800 antimouse IgG Rockland 1:10,000
  MMP-3 R&D Systems Mouse 1:1,000 IRDye 800 antimouse IgG Rockland 1:10,000
  MMP-9 R&D Systems Mouse 1:500 IRDye 800 antimouse IgG Rockland 1:10,000
 ECM
  Collagen I Rockland Rabbit 1:10,000 IRDye 800 antirabbit IgG Rockland 1:10,000
  Collagen IV Rockland Rabbit 1:5,000 IRDye 800 antirabbit IgG Rockland 1:10,000
  Collagen VI Rockland Rabbit 1:5,000 IRDye 800 antirabbit IgG Rockland 1:10,000
  Fibronectin Sigma-Aldrich Rabbit 1:5,000 IRDye 800 antirabbit IgG Rockland 1:10,000
  Laminin Sigma-Aldrich Mouse 1:5,000 IRDye 800 antimouse IgG Rockland 1:10,000
 TIMPs
  TIMP-1 Chemicon Rabbit 1:500 IRDye 800 antirabbit IgG Rockland 1:10,000
  TIMP-2 Chemicon Rabbit 1:1,000 IRDye 800 antirabbit IgG Rockland 1:10,000
  TIMP-3 Chemicon Rabbit 1:1,000 IRDye 800 antirabbit IgG Rockland 1:10,000
  TIMP-4 Chemicon Rabbit 1:25,000 IRDye 800 antirabbit IgG Rockland 1:10,000
 Others
  PAI-1 Upstate Rabbit 1:1,000 IRDye 800 antirabbit IgG Rockland 1:10,000
  SPARC Haematologic Tech Mouse 1:10,000 IRDye 800 antimouse IgG Rockland 1:10,000
  GAPDH R&D Systems Rabbit 1:2,500 IRDye 800 antirabbit IgG Rockland 1:10,000
Immunofluorescence
 ECM
  Collagen I Rockland Rabbit 1:100 Goat antirabbit 488 Molecular Probes 1:200
  Collagen IV Rockland Rabbit 1:100 Goat antirabbit 488 Molecular Probes 1:200
  Collagen VI Rockland Rabbit 1:100 Goat antirabbit 488 Molecular Probes 1:200
  Fibronectin Sigma-Aldrich Rabbit 1:100 Goat antirabbit 488 Molecular Probes 1:200
  Laminin Sigma-Aldrich Mouse 1:100 Goat antirabbit 488 Molecular Probes 1:200
 Others
  SPARC Haematologic Tech Mouse 1:500 Goat antimouse 594 Molecular Probes 1:200
Immunofluorescent Staining
Paraffin-embedded TM tissue slides were deparaffinized in xylene for 15 minutes and incubated with xylene for a second 15-minute period. The slides were soaked with TBS/T for 5 minutes, hydrated with ethanol dilutions (100%, 95%, and 70%) for 5 minutes each, and rinsed with 1× TBS/T for 5 minutes. After excess liquid was removed, the tissues were made permeable with 0.2% Triton-100 in 1× PBS for 5 minutes, and were subsequently washed with TBS/T for 5 minutes. The tissues were incubated with blocking solution (10% normal goat serum in PBS) for 1 hour at room temperature and were washed with TBS/T for 10 minutes. Primary antibody (Table 1) diluted in blocking buffer was applied to each section at 4°C overnight. Optimal primary antibody concentrations were empirically determined by serial antibody dilution (1:50–1:200). Slides were washed three times with TBS/T for 10 minutes. Secondary antibodies (1:100) (Table 1) were applied to detect target protein, and nuclei were stained with TO-PRO-3 (1:200; Invitrogen). The slides were washed three times with TBS/T for 10 minutes, and a coverslip was applied with Immuno-Mount (Thermo Scientific, Pittsburgh, PA). Representative sections from two quadrants of each eye were analyzed. Labeled tissues were viewed under the Leica TCS SP2 spectral confocal laser scanning microscope (Leica Microsystems, Exton, PA). The secondary antibody-stained section was used to set up the parameters for nuclear (To-PRO-3) target ECM proteins (Alexa Fluor 488) and SPARC (Alexa Fluor 594) as the basal level. After these parameters were set and saved, we used these exact same conditions to analyze the expression of the target proteins in the remaining sections. Using the ImageJ software (National Institutes of Health, Bethesda, MD), we analyzed five areas selected randomly in the labeled tissues for the average pixel intensity in the green or red channels using arbitrary square box (width × height, 0.3 × 0.3). 
Results
Effect of SPARC Overexpression on SPARC Levels in Human TM Cells
SPARC was expressed in the conditioned media by Ad5.hSPARC 14.43 ± 4.67 times at MOI 25 (P = 0.015) and 44.49 ± 14.52 times at MOI 50 (P = 0.004) in comparison to SPARC protein from the media of Ad5.control (Fig. 2A) treated cells. In the cell lysates, SPARC was increased 3.04 ± 1.35 times at MOI 25 (P = 0.021) and 9.45 ± 4.04 times at MOI 50 (P = 0.001; Fig. 2B). SPARC levels in the cell lysates were lower than that of the conditioned media. SPARC appeared only as a glycosylated band from the conditioned media, but as both an unglycosylated and a glycosylated band from the cell lysates (Fig. 2). 
Figure 2
 
SPARC overexpression in vitro induced by Ad5.hSPARC. (A) Representative immunoblots from the conditioned media (A) and cell lysates (B) of cultured TM56 cells infected with Ad5.hSPARC. There was an increase of SPARC with increasing MOI (n = 12).
Figure 2
 
SPARC overexpression in vitro induced by Ad5.hSPARC. (A) Representative immunoblots from the conditioned media (A) and cell lysates (B) of cultured TM56 cells infected with Ad5.hSPARC. There was an increase of SPARC with increasing MOI (n = 12).
SPARC Overexpression in the Human Anterior Segment Culture System
After adenoviral infection of human anterior segments with Ad5.control or Ad5.hSPARC, we first confirmed that SPARC was in the perfusate. SPARC was increased 36.57 ± 2.29 times at 24 hours postinfection and 43.74 ± 4.06 times at 48 hours postinfection in Ad5.hSPARC-infected perfusate, compared to Ad5.control-infected perfusate (n = 5; Fig. 3A). SPARC levels were 13.45 ± 2.48 times higher (n = 5) in the TM tissue isolated from the anterior segments injected with Ad5.hSPARC, in comparison to the anterior segments injected with Ad5.control at the final day of the perfusion (Fig. 3B). Thus, Ad5.hSPARC increased SPARC expression within the TM tissue as well as secretion into the perfusate. 
Figure 3
 
Immunoblot of SPARC from the perfused anterior segment culture system. SPARC levels were 36.57 ± 2.29 times greater (n = 5) at 24 hours postinfection and 43.74 ± 4.06 times (n = 5) greater at 48 hours postinfection in the perfused conditioned media compared with the perfusate sampled at time of adenovirus injection. C for ad5.control and SPARC for Ad5.hSPARC (A1). SPARC levels were shown in the perfused conditioned media 24 hours before, 24 hours after, and 48 hours after injection (A2). SPARC was 13.45 ± 2.48 times greater (n = 5) in the TM tissue isolated at the final day of the perfusion from the anterior segment injected with Ad5.hSPARC, compared with the anterior segment injected with Ad5.control (B). Representative immunoblots were taken from ex vivo Exp 4 TM78.
Figure 3
 
Immunoblot of SPARC from the perfused anterior segment culture system. SPARC levels were 36.57 ± 2.29 times greater (n = 5) at 24 hours postinfection and 43.74 ± 4.06 times (n = 5) greater at 48 hours postinfection in the perfused conditioned media compared with the perfusate sampled at time of adenovirus injection. C for ad5.control and SPARC for Ad5.hSPARC (A1). SPARC levels were shown in the perfused conditioned media 24 hours before, 24 hours after, and 48 hours after injection (A2). SPARC was 13.45 ± 2.48 times greater (n = 5) in the TM tissue isolated at the final day of the perfusion from the anterior segment injected with Ad5.hSPARC, compared with the anterior segment injected with Ad5.control (B). Representative immunoblots were taken from ex vivo Exp 4 TM78.
Effect of SPARC Overexpression on IOP in Human Anterior Segment Culture System.
After intracameral injection of 1 × 108 ifu of adenoviruses, Ad5.control or Ad5.hSPARC, in paired constant-flow human anterior segment cultures (n = 5), the normalized IOP of Ad5.hSPARC increased an average of 24.31% (P < 0.0001) compared to Ad5.control. At baseline, the raw average IOPs for Ad5.control and Ad5.hSPARC were 10.06 ± 2.81 mm Hg and 12.59 ± 2.07 mm Hg (mean ± SEM), respectively, indicating that our scoring of the episcleral veins was appropriate. After 48 hours, the IOP of Ad5.hSPARC was significantly higher than Ad5.control (P = 0.041; Fig. 4A). The average increase of normalized IOPs between 48 and 98 hours was 27.93% (P < 0.0001) over the same time period. TM tissue infected by Ad5.control and Ad5.hSPARC adenovirus was stained with toluidine blue; there was no difference in cell viability of TM tissues (Fig. 4B). 
Figure 4
 
Effect of SPARC overexpression on IOP in the perfused anterior segments. (A) IOPs were measured in paired constant-flow human anterior segments (n = 5), injected intracamerally with 1 × 108 ifu of adenoviruses, Ad5.control (control), or Ad5.hSPARC (SPARC+). From 48 hours onward, the IOP for Ad5.hSPARC was significantly elevated compared to baseline control (*48, 55, 65, 75, 85, 95 hours; P values = 0.0410, 0.0419, 0.0236, 0.0090, 0.0196, 0.0014, respectively; n = 5). Paired t-tests were used for statistical significance. The IOPs were normalized to 1 at the zero hour time point. (B) TM tissue infected with 1 × 108 ifu of Ad5.control (C) and Ad5.hSPARC (S) adenovirus were stained with toluidine blue; there was no apparent difference in the cell viability of TM tissues infected (n = 5). ×200 magnification.
Figure 4
 
Effect of SPARC overexpression on IOP in the perfused anterior segments. (A) IOPs were measured in paired constant-flow human anterior segments (n = 5), injected intracamerally with 1 × 108 ifu of adenoviruses, Ad5.control (control), or Ad5.hSPARC (SPARC+). From 48 hours onward, the IOP for Ad5.hSPARC was significantly elevated compared to baseline control (*48, 55, 65, 75, 85, 95 hours; P values = 0.0410, 0.0419, 0.0236, 0.0090, 0.0196, 0.0014, respectively; n = 5). Paired t-tests were used for statistical significance. The IOPs were normalized to 1 at the zero hour time point. (B) TM tissue infected with 1 × 108 ifu of Ad5.control (C) and Ad5.hSPARC (S) adenovirus were stained with toluidine blue; there was no apparent difference in the cell viability of TM tissues infected (n = 5). ×200 magnification.
Effect of SPARC Overexpression on ECM Proteins in Perfused Human Anterior Segments.
After completion of perfusion, immunofluorescence of the anterior segments revealed that SPARC was increased 85 ± 51% by Ad5.hSPARC infection (Fig. 5, Table 3). In these segments, fibronectin as well as collagen types I and IV were increased compared to Ad5.control (Fig. 5, Table 3). Collagen type VI and laminin were not affected (Fig. 5, Table 3). 
Figure 5
 
Representative immunolabeling of ECM proteins after infection with Ad5.control and Ad5.hSPARC (1 × 108 ifu). Collagen I (A), collagen IV (B), and fibronectin (FN) (D) were increased, but collagen VI (C) and laminin (LN) (E) were not changed. Collagen I was increased throughout the TM with some increase in the JCT region. Collagen IV was detected more prominently in the JCT region as well as under the outer wall of SC. Fibronectin was increased throughout the TM, but prominently within the JCT region. The secondary only staining (F) was shown as a negative control. Scale bar: 30 μm; C, control, S+, SPARC+; magnification = ×100.
Figure 5
 
Representative immunolabeling of ECM proteins after infection with Ad5.control and Ad5.hSPARC (1 × 108 ifu). Collagen I (A), collagen IV (B), and fibronectin (FN) (D) were increased, but collagen VI (C) and laminin (LN) (E) were not changed. Collagen I was increased throughout the TM with some increase in the JCT region. Collagen IV was detected more prominently in the JCT region as well as under the outer wall of SC. Fibronectin was increased throughout the TM, but prominently within the JCT region. The secondary only staining (F) was shown as a negative control. Scale bar: 30 μm; C, control, S+, SPARC+; magnification = ×100.
Table 3. 
 
Fluorescence Intensity Measurements of ECM and SPARC Proteins From Immunofluorescent Images Postinjection of Ad5.control and Ad5.hSPARC (1 × 108 ifu)
Table 3. 
 
Fluorescence Intensity Measurements of ECM and SPARC Proteins From Immunofluorescent Images Postinjection of Ad5.control and Ad5.hSPARC (1 × 108 ifu)
ECM, n = 5 % Changes P Value
Collagen I 54.54 ± 14.68 0.00027*
Collagen IV 59.29 ± 7.33 0.00005*
Collagen VI 37.07 ± 28.61 0.07210
Fibronectin 49.39 ± 24.72 0.00077*
Laminin 32.75 ± 22.97 0.09750
SPARC 84.85 ± 51.30 0.00059*
Effect of SPARC overexpression on ECM proteins, MMP, and TIMP levels in human TM cells
Effect of In Vitro SPARC Overexpression on ECM Protein Levels.
Overexpression of SPARC was associated with increases in several ECM components (Table 4). Collagen IV was most affected and increased by 45.46 ± 15.83% at MOI 25 and 58.55 ± 15.67% at MOI 50 (Table 4, Fig. 6). Collagen types I and VI, fibronectin, and laminin were increased as well, but only at MOI 50 (Table 4, Fig. 6). 
Figure 6
 
Representative immunoblots demonstrating relative changes in the levels of selected ECM proteins, MMPs, and TIMPs induced by SPARC overexpression in TM cells. At MOI 25, pro-MMP-9 was decreased, while the inhibitors TIMP-1 and PAI-1 as well as collagen IV were increased. At the higher MOI 50, pro-MMP-1, -3, and -9 were decreased while the inhibitors TIMP-1 and -3; PAI-1; collagen I, IV, and VI; fibronectin; and laminin were increased. At both MOIs 25 and 50, only the activity of MMP-9 was decreased.
Figure 6
 
Representative immunoblots demonstrating relative changes in the levels of selected ECM proteins, MMPs, and TIMPs induced by SPARC overexpression in TM cells. At MOI 25, pro-MMP-9 was decreased, while the inhibitors TIMP-1 and PAI-1 as well as collagen IV were increased. At the higher MOI 50, pro-MMP-1, -3, and -9 were decreased while the inhibitors TIMP-1 and -3; PAI-1; collagen I, IV, and VI; fibronectin; and laminin were increased. At both MOIs 25 and 50, only the activity of MMP-9 was decreased.
Table 4. 
 
Changes in the Protein Levels of Selected ECM Components, MMPs, TIMPs, and MMP Activity Following SPARC Overexpression in TM Cells
Table 4. 
 
Changes in the Protein Levels of Selected ECM Components, MMPs, TIMPs, and MMP Activity Following SPARC Overexpression in TM Cells
% Change in Expression Compared to Control
MOI 25 (n) P Value MOI 50 (n) P Value
MMPs (immunoblot)
 MMP-1 −8.69 ± 2.45 (7) 0.062 −18.26 ± 4.64 (7) 0.008*
 MMP-2 0.27 ± 3.72 (8) 0.943 −16.46 ± 7.29 (8) 0.109
 MMP-3 −5.45 ± 1.76 (6) 0.077 −23.87 ± 4.68 (6) 0.004*
 MMP-9 −18.07 ± 4.33 (7) 0.042* −25.26 ± 7.39 (7) 0.014*
MMPs (zymogram)
 MMP-1 1.64 ± 0.37 (8) 0.103 2.46 ± 0.89 (8) 0.078
 MMP-2 −2.12 ± 3.24 (12) 0.527 −7.19 ± 5.85 (12) 0.245
 MMP-9 −10.24 ± 3.56 (8) 0.023* −18.32 ± 4.20 (8) 0.003*
ECM
 Collagen I 5.19 ± 1.84 (5) 0.098 19.18 ± 2.99 (5) 0.037*
 Collagen IV 45.46 ± 15.83 (7) 0.028* 58.55 ± 15.67 (7) 0.010*
 Collagen VI 5.54 ± 1.98 (7) 0.092 22.49 ± 5.43 (7) 0.002*
 Fibronectin 9.87 ± 2.52 (6) 0.061 25.93 ± 9.65 (6) 0.002*
 Laminin 7.12 ± 2.67 (5) 0.106 17.27 ± 3.25 (5) 0.006*
TIMPs
 TIMP-1 16.46 ± 4.71 (7) 0.013* 18.87 ± 6.95 (7) 0.038*
 TIMP-2 10.11 ± 2.91 (6) 0.065 14.66 ± 3.27 (6) 0.300
 TIMP-3 2.83 ± 1.54 (8) 0.158 17.75 ± 6.88 (8) 0.036*
 TIMP-4 9.09 ± 5.31 (7) 0.062 8.61 ± 9.30 (7) 0.082
Other
 PAI-1 23.28 ± 5.08 (8) 0.003* 36.59 ± 7.92 (8) 0.002*
Effect of SPARC Overexpression In Vitro on MMP and TIMP Levels and Activity.
At MOI 25, pro-MMP-9 was decreased. At MOI 50, pro-MMP-1, -3, and -9 were decreased (Table 4, Fig. 6). Pro-MMP-2 was not affected (Table 4, Fig. 6). Only TIMP-1 was significantly increased by 16.46 ± 4.71% at MOI 25. TIMP-1 and -3 increased at MOI 50 (Table 4, Fig. 5). TIMP-2 and -4 were unchanged (Table 4, Fig. 6). Expression of PAI-1, which also inhibits the activity of MMPs, was significantly increased by 23.28 ± 5.08% at MOI 25 and by 36.59 ± 7.92% at MOI 50 (Table 4, Fig. 6). Despite the changes in enzyme and inhibitor levels, zymography demonstrated that only MMP-9 activity was significantly decreased with the higher MOI; intermediate MMP-1 and active MMP-2 activity were unchanged (Table 4, Fig. 6). 
Effect of SPARC Overexpression on the Relative Transcript Levels of Selected ECM Elements, MMPs, and TIMPs in Human TM Cells
The expression of various ECM mRNA transcripts was assessed by qPCR following Ad5.control or Ad5.hSPARC infection. Only collagen VI α3 expression was decreased by 58.34 ± 04.92% at MOI 25 (P = 0.001); however, there were no significant changes in any other ECM mRNAs (Table 5). 
Table 5. 
 
Changes in the mRNA Levels of Selected ECM Proteins Compared With Controls as a Result of Adenoviral Infection by Ad5.control and Ad5.hSPARC
Table 5. 
 
Changes in the mRNA Levels of Selected ECM Proteins Compared With Controls as a Result of Adenoviral Infection by Ad5.control and Ad5.hSPARC
% Change in Expression Compared to Control
MOI 25, n = 6 MOI 50, n = 6
Collagen I α1 −17.17 ± 11.28 −21.60 ± 15.13
Collagen IV α6 −7.81 ± 9.50 8.56 ± 27.17
Collagen VI α1 −17.92 ± 13.73 −7.24 ± 15.03
Collagen VI α2 −10.37 ± 14.05 23.55 ± 30.01
Collagen VI α3 −58.34 ± 4.92 32.69 ± 78.35
Fibronectin 7.18 ± 10.18 43.53 ± 45.12
Laminin α5 −32.20 ± 10.77 −27.11 ± 11.30
Laminin β1 −12.55 ± 14.69 9.79 ± 21.17
Laminin γ1 −30.51 ± 25.05 17.00 ± 40.82
Discussion
In both perfused human anterior segments and cultured TM endothelial cells, we were able to increase SPARC expression in a dose-dependent manner. Methodologically, enhancing SPARC expression by viral transfection has the benefit of tissue/cell-specific posttranslational processing, which yields different results compared to those elicited by recombinant SPARC. 25 SPARC overexpression in the cell lysates was not as prominent as in the conditioned media, chiefly because SPARC is a secreted protein. In perfused human anterior segments, SPARC overexpression increased IOP without altering TM cell viability, as confirmed by toluidine blue stain. A high level of SPARC overexpression was required in order for the IOP to be significantly elevated. Due to the nature of using paired eyes from a single donor, only one control arm is possible. We selected the virus as the control as we were concerned about the virus itself being cytotoxic. Although we considered including a control protein to test if the elevated IOP is a nonspecific result of protein overload, it is unclear what similarly sized and completely inert protein would be appropriate. We believe that the finding of a qualitative difference in the JCT ECM, which was corroborated in TM cell culture, between experimental and control segments implicate that the elevation of IOP is the result of a functional effect of SPARC. Additionally, the overexpression of SPARC resulting in an elevation of IOP is consistent with our earlier report of a lower IOP in SPARC-null mice. 21  
The structure–function relationship, as demonstrated by immunohistologic examination of these segments, revealed an increase in fibronectin and collagen types IV and I within the JCT region; these ECM components were also elevated in TM endothelial cell cultures. Collagen type VI and laminin were increased in TM cell cultures at high levels of SPARC expression. The JCT region, consisting of Schlemm's canal (SC) inner wall cells, subendothelial ECM containing an incomplete basement membrane, and TM endothelial cells represent the anatomic location of maximal outflow resistance. 2629 The regulation of IOP in the JCT region is a complex system with overlapping redundancy to compensate for the numerous physiologic stressors and perturbations. A few processes that have been shown to influence IOP include the regulation of ECM homeostasis, 912,14,30,31 the actin cytoskeleton and cellular tone of JCT TM and inner wall SC cells, 32 and the number of transcellular pores through inner wall SC cells. 33  
In both corneoscleral and JCT TM, the basement membrane is primarily comprised of collagen IV, fibronectin, and laminin. 3436 An increase of basement membrane components, collagen IV, and fibronectin within the JCT TM is a significant structural finding in corticosteroid-induced glaucoma, in which there is an elevation of IOP from increased TM resistance. 3740 In monolayers of TM endothelial cells, high glucose and dexamethasone each induced collagen type IV and laminin, changes resulting in decreased permeability. 41 In ex vivo porcine eyes, overexpression of a constitutively active mutant form of RhoA GTPase increased IOP through greater contractility of inner wall SC cells and increased JCT collagen type IV, fibronectin, and laminin. 42 The increase of JCT basement membrane material seen in our experiments correlates with compromised TM drainage and is likely a significant contributor to the mechanism of increased IOP caused by SPARC overexpression. 
We observed an increase of collagen type I within the JCT region after overexpression of SPARC. However, in TM cells, collagen type I was only increased by the greater degree of SPARC overexpression (MOI 50). Collagen types IV and VI are the predominant collagens in TM, 36,43 but collagen type I is an important structural element. 43 Transgenic mice with a mutation in the α1 subunit of type I collagen that prevents turnover have an elevated IOP due to the increased accumulation within the TM and sclera. 44  
Although collagen VI and laminin were not statistically elevated in the sections of perfused anterior segments, they were increased and almost reached significance. It is possible that with a large sample size, the 33% to 37% increases could reach statistical significance. Thus, these proteins could be part of the qualitative difference in ECM that increased IOP but were altered at a level below the statistical power of our study to definitively discern. In TM cell culture, the mRNA level of collagen VIα3 (Col6A3) was decreased while the protein level of collagen VI was unchanged. Col6A3 is one of the three α chains of type VI collagen and is larger than the α1 and α2 chains due to an increase in the number of subdomains found in the amino terminal globular domain. 45,46 These domains have been shown to bind ECM proteins that assist with organizing matrix components. 4749 The exact significance of the decrease of Col6A1 is unknown, but it may be a negative feedback loop designed to keep the level of collagen VI unchanged while SPARC is acting by a mechanism, other than a simple increase of translation, which could potentially enhance the level of collagen VI if unchecked. 
The mechanistic pathways by which SPARC mediates these changes are unclear. The mRNA levels of these ECM proteins were not elevated results indicating that the mechanism is not a simple increase in production. SPARC may act as a chaperone, a posttranslational control, to stabilize ECM proteins or mRNA allowing for selective accumulation. 5052 In both TM and uveoscleral pathways, alterations of MMP/TIMP balance correlate with aqueous drainage and IOP. 11,53,54 Despite the dose–response related shift in MMP/inhibitor balance of several MMPs, only MMP-9 activity was decreased on zymography. The selective decrease of MMP enzymatic activity is indicative of a qualitative change in composition of the JCT ECM rather than a simple, quantitative increase of ECM components. This assertion is consistent with our observation in SPARC-null mice, which have a lower IOP and enhanced aqueous drainage, but no observable quantitative changes in the JCT ECM at the light microscopy level. 21 Because the ECM of the JCT region is undergoing constant turnover, we hypothesized that the decrease of MMP-9 activity allows for the selective accumulation via decreased turnover of these ECM products. We only studied MMPs-1, -2, -3, and -9. Previously we showed that TM cells also express MMPs-11, -12, -14, -15, -16, -17, -19, and -24, and it is possible that some of the other MMPs could also be affected by SPARC. 54  
Additionally, we believe that SPARC assists the posttranslational processing of collagen. The absence of SPARC is associated with a decreased capacity to compose organized mature collagen fibers. 55 SPARC plays a critical role in collagen fibril formation, recognizing the GVMGFO motif of the collagen triple helix. 56 In TIMP-2 null mice, there was a greater level of fibrosis that was due to increased SPARC and posttranslational stabilization of collagen fibers rather than increased collagen synthesis. 57  
The effects of SPARC modulation on various ECM proteins seen in our study are consistent with previous studies. In pulmonary fibroblasts, SPARC overexpression increased PAI via activation of Akt. 58 In tumor cells, SPARC overexpression resulted in a decrease in pro-MMP-9. 59 Other groups have examined the effects of SPARC suppression. Tumors grown in SPARC-null mice exhibited reduced deposition of fibrillar collagen types I and III, basement membrane collagen IV, and the collagen-associated proteoglycan decorin. 60 SPARC suppression in liver cells resulted in a decrease in collagen type I. 61 Using targeted siRNA to SPARC, Wei et al. 62 found that fibronectin was decreased. These observations with SPARC suppression are supportive of our findings with overexpression of SPARC. 
ECM deposition and turnover are likely regulated by several mechanistic pathways. We have shown that SPARC regulates IOP through a coordinated response involving increased levels of certain ECM proteins in conjunction with a selective decrease of enzymatic activity, resulting in qualitative changes in the JCT ECM. The results within this report, along with our previous observation that SPARC-null mice have a lower IOP and enhanced aqueous outflow, strongly implicate a regulatory role for SPARC in IOP. Work is ongoing to determine the up- and down-stream signaling pathways for SPARC in TM as well as the mechanism responsible for these qualitative changes in the JCT ECM. 
Acknowledgments
Disclosure: D.-J. Oh, None; M.H. Kang, None; Y.H. Ooi, None; K.R. Choi, None; E.H. Sage, None; D.J. Rhee, None 
Supported by National Eye Institute Grants EY 019654-01 and EY 014104 (MEEI Vision-Core Grant), Research to Prevent Blindness, Massachusetts Lions Eye Research Foundation, and American Glaucoma Society. 
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Figure 1
 
Experimental time course of SPARC overexpression in vitro. TM cultures were incubated with 2% FBS medium with either Ad5.control or Ad5.hSPARC for 18 hours. Then 10% FBS medium of equal volume were added to the 2% medium for an additional 24 hours. The TM cultures were next incubated with serum-free medium (which did not contain virus) for 24 hours. The serum-free media were harvested for immunoblot assays. A representative immunoblot of SPARC expression in conditioned media from TM cells with no adenovirus (N), with Ad5.control (C), and with Ad5.hSPARC (S+) infection is shown; equal total protein was loaded into each well.
Figure 1
 
Experimental time course of SPARC overexpression in vitro. TM cultures were incubated with 2% FBS medium with either Ad5.control or Ad5.hSPARC for 18 hours. Then 10% FBS medium of equal volume were added to the 2% medium for an additional 24 hours. The TM cultures were next incubated with serum-free medium (which did not contain virus) for 24 hours. The serum-free media were harvested for immunoblot assays. A representative immunoblot of SPARC expression in conditioned media from TM cells with no adenovirus (N), with Ad5.control (C), and with Ad5.hSPARC (S+) infection is shown; equal total protein was loaded into each well.
Figure 2
 
SPARC overexpression in vitro induced by Ad5.hSPARC. (A) Representative immunoblots from the conditioned media (A) and cell lysates (B) of cultured TM56 cells infected with Ad5.hSPARC. There was an increase of SPARC with increasing MOI (n = 12).
Figure 2
 
SPARC overexpression in vitro induced by Ad5.hSPARC. (A) Representative immunoblots from the conditioned media (A) and cell lysates (B) of cultured TM56 cells infected with Ad5.hSPARC. There was an increase of SPARC with increasing MOI (n = 12).
Figure 3
 
Immunoblot of SPARC from the perfused anterior segment culture system. SPARC levels were 36.57 ± 2.29 times greater (n = 5) at 24 hours postinfection and 43.74 ± 4.06 times (n = 5) greater at 48 hours postinfection in the perfused conditioned media compared with the perfusate sampled at time of adenovirus injection. C for ad5.control and SPARC for Ad5.hSPARC (A1). SPARC levels were shown in the perfused conditioned media 24 hours before, 24 hours after, and 48 hours after injection (A2). SPARC was 13.45 ± 2.48 times greater (n = 5) in the TM tissue isolated at the final day of the perfusion from the anterior segment injected with Ad5.hSPARC, compared with the anterior segment injected with Ad5.control (B). Representative immunoblots were taken from ex vivo Exp 4 TM78.
Figure 3
 
Immunoblot of SPARC from the perfused anterior segment culture system. SPARC levels were 36.57 ± 2.29 times greater (n = 5) at 24 hours postinfection and 43.74 ± 4.06 times (n = 5) greater at 48 hours postinfection in the perfused conditioned media compared with the perfusate sampled at time of adenovirus injection. C for ad5.control and SPARC for Ad5.hSPARC (A1). SPARC levels were shown in the perfused conditioned media 24 hours before, 24 hours after, and 48 hours after injection (A2). SPARC was 13.45 ± 2.48 times greater (n = 5) in the TM tissue isolated at the final day of the perfusion from the anterior segment injected with Ad5.hSPARC, compared with the anterior segment injected with Ad5.control (B). Representative immunoblots were taken from ex vivo Exp 4 TM78.
Figure 4
 
Effect of SPARC overexpression on IOP in the perfused anterior segments. (A) IOPs were measured in paired constant-flow human anterior segments (n = 5), injected intracamerally with 1 × 108 ifu of adenoviruses, Ad5.control (control), or Ad5.hSPARC (SPARC+). From 48 hours onward, the IOP for Ad5.hSPARC was significantly elevated compared to baseline control (*48, 55, 65, 75, 85, 95 hours; P values = 0.0410, 0.0419, 0.0236, 0.0090, 0.0196, 0.0014, respectively; n = 5). Paired t-tests were used for statistical significance. The IOPs were normalized to 1 at the zero hour time point. (B) TM tissue infected with 1 × 108 ifu of Ad5.control (C) and Ad5.hSPARC (S) adenovirus were stained with toluidine blue; there was no apparent difference in the cell viability of TM tissues infected (n = 5). ×200 magnification.
Figure 4
 
Effect of SPARC overexpression on IOP in the perfused anterior segments. (A) IOPs were measured in paired constant-flow human anterior segments (n = 5), injected intracamerally with 1 × 108 ifu of adenoviruses, Ad5.control (control), or Ad5.hSPARC (SPARC+). From 48 hours onward, the IOP for Ad5.hSPARC was significantly elevated compared to baseline control (*48, 55, 65, 75, 85, 95 hours; P values = 0.0410, 0.0419, 0.0236, 0.0090, 0.0196, 0.0014, respectively; n = 5). Paired t-tests were used for statistical significance. The IOPs were normalized to 1 at the zero hour time point. (B) TM tissue infected with 1 × 108 ifu of Ad5.control (C) and Ad5.hSPARC (S) adenovirus were stained with toluidine blue; there was no apparent difference in the cell viability of TM tissues infected (n = 5). ×200 magnification.
Figure 5
 
Representative immunolabeling of ECM proteins after infection with Ad5.control and Ad5.hSPARC (1 × 108 ifu). Collagen I (A), collagen IV (B), and fibronectin (FN) (D) were increased, but collagen VI (C) and laminin (LN) (E) were not changed. Collagen I was increased throughout the TM with some increase in the JCT region. Collagen IV was detected more prominently in the JCT region as well as under the outer wall of SC. Fibronectin was increased throughout the TM, but prominently within the JCT region. The secondary only staining (F) was shown as a negative control. Scale bar: 30 μm; C, control, S+, SPARC+; magnification = ×100.
Figure 5
 
Representative immunolabeling of ECM proteins after infection with Ad5.control and Ad5.hSPARC (1 × 108 ifu). Collagen I (A), collagen IV (B), and fibronectin (FN) (D) were increased, but collagen VI (C) and laminin (LN) (E) were not changed. Collagen I was increased throughout the TM with some increase in the JCT region. Collagen IV was detected more prominently in the JCT region as well as under the outer wall of SC. Fibronectin was increased throughout the TM, but prominently within the JCT region. The secondary only staining (F) was shown as a negative control. Scale bar: 30 μm; C, control, S+, SPARC+; magnification = ×100.
Figure 6
 
Representative immunoblots demonstrating relative changes in the levels of selected ECM proteins, MMPs, and TIMPs induced by SPARC overexpression in TM cells. At MOI 25, pro-MMP-9 was decreased, while the inhibitors TIMP-1 and PAI-1 as well as collagen IV were increased. At the higher MOI 50, pro-MMP-1, -3, and -9 were decreased while the inhibitors TIMP-1 and -3; PAI-1; collagen I, IV, and VI; fibronectin; and laminin were increased. At both MOIs 25 and 50, only the activity of MMP-9 was decreased.
Figure 6
 
Representative immunoblots demonstrating relative changes in the levels of selected ECM proteins, MMPs, and TIMPs induced by SPARC overexpression in TM cells. At MOI 25, pro-MMP-9 was decreased, while the inhibitors TIMP-1 and PAI-1 as well as collagen IV were increased. At the higher MOI 50, pro-MMP-1, -3, and -9 were decreased while the inhibitors TIMP-1 and -3; PAI-1; collagen I, IV, and VI; fibronectin; and laminin were increased. At both MOIs 25 and 50, only the activity of MMP-9 was decreased.
Table 1. 
 
Primer Sequences for Quantitative PCR
Table 1. 
 
Primer Sequences for Quantitative PCR
Forward Reverse
Collagen α1 (I) 5′-CTGGTCCTGATGGCAAAACT-3′ 5′-CTCCAGCCTCTCCATCTTTG-3′
Collagen α2 (IV) 5′-TCTTTCCTCATGCACACTGC-3′ 5′-TTCAGCGTTTCAGACACAGG-3′
Collagen α1 (VI) 5′-CTGGGCGTCAAAGTCTTCTC-3′ 5′-ATTCGAAGGAGCAGCACACT-3′
Collagen α2 (VI) 5′-GAGATCGACCAGGACACCAT-3′ 5′-GGTCTCCCTGTCTTCCCTTC-3′
Collagen α3 (VI) 5′-CTGGGCAGACATACCATGTG-3′ 5′-GCAAGTTCCTTCGTCTTTCG-3′
Fibronectin 5′-ACCAACCTACGGATGACTCG-3′ 5′-GCTCATCATCTGGCCATTTT-3′
Laminin α5 5′-TGACCTTTTCTGGCTCGTCT-3′ 5′-GTTCAGCACAAAGGGCTCTC-3′
Laminin β1 5′-AGGTTGGAGCTGCCTCAGTA-3′ 5′-TGTTTTCACAACGCTTCTGC-3′
Laminin γ1 5′-GCATCTCTCGAGTGGTCCTC-3′ 5′-TGGATAGGAATTGCCCTGAG-3′
β-actin 5′-GGCATCCTCACCCTGAAGTA-3′ 5′-GGGGTGTTGAAGGTCTCAAA-3′
Table 2. 
 
Primary and Secondary Antibodies Used for Immunoblot and Immunofluorescence in This Study
Table 2. 
 
Primary and Secondary Antibodies Used for Immunoblot and Immunofluorescence in This Study
Primary Antibody Company Antibody Host Species Dilution Secondary Antibody Company Dilution
Immunoblot
 MMPs
  MMP-1 R&D Systems Mouse 1:1,000 IRDye 800 antimouse IgG Rockland 1:10,000
  MMP-2 R&D Systems Mouse 1:1,000 IRDye 800 antimouse IgG Rockland 1:10,000
  MMP-3 R&D Systems Mouse 1:1,000 IRDye 800 antimouse IgG Rockland 1:10,000
  MMP-9 R&D Systems Mouse 1:500 IRDye 800 antimouse IgG Rockland 1:10,000
 ECM
  Collagen I Rockland Rabbit 1:10,000 IRDye 800 antirabbit IgG Rockland 1:10,000
  Collagen IV Rockland Rabbit 1:5,000 IRDye 800 antirabbit IgG Rockland 1:10,000
  Collagen VI Rockland Rabbit 1:5,000 IRDye 800 antirabbit IgG Rockland 1:10,000
  Fibronectin Sigma-Aldrich Rabbit 1:5,000 IRDye 800 antirabbit IgG Rockland 1:10,000
  Laminin Sigma-Aldrich Mouse 1:5,000 IRDye 800 antimouse IgG Rockland 1:10,000
 TIMPs
  TIMP-1 Chemicon Rabbit 1:500 IRDye 800 antirabbit IgG Rockland 1:10,000
  TIMP-2 Chemicon Rabbit 1:1,000 IRDye 800 antirabbit IgG Rockland 1:10,000
  TIMP-3 Chemicon Rabbit 1:1,000 IRDye 800 antirabbit IgG Rockland 1:10,000
  TIMP-4 Chemicon Rabbit 1:25,000 IRDye 800 antirabbit IgG Rockland 1:10,000
 Others
  PAI-1 Upstate Rabbit 1:1,000 IRDye 800 antirabbit IgG Rockland 1:10,000
  SPARC Haematologic Tech Mouse 1:10,000 IRDye 800 antimouse IgG Rockland 1:10,000
  GAPDH R&D Systems Rabbit 1:2,500 IRDye 800 antirabbit IgG Rockland 1:10,000
Immunofluorescence
 ECM
  Collagen I Rockland Rabbit 1:100 Goat antirabbit 488 Molecular Probes 1:200
  Collagen IV Rockland Rabbit 1:100 Goat antirabbit 488 Molecular Probes 1:200
  Collagen VI Rockland Rabbit 1:100 Goat antirabbit 488 Molecular Probes 1:200
  Fibronectin Sigma-Aldrich Rabbit 1:100 Goat antirabbit 488 Molecular Probes 1:200
  Laminin Sigma-Aldrich Mouse 1:100 Goat antirabbit 488 Molecular Probes 1:200
 Others
  SPARC Haematologic Tech Mouse 1:500 Goat antimouse 594 Molecular Probes 1:200
Table 3. 
 
Fluorescence Intensity Measurements of ECM and SPARC Proteins From Immunofluorescent Images Postinjection of Ad5.control and Ad5.hSPARC (1 × 108 ifu)
Table 3. 
 
Fluorescence Intensity Measurements of ECM and SPARC Proteins From Immunofluorescent Images Postinjection of Ad5.control and Ad5.hSPARC (1 × 108 ifu)
ECM, n = 5 % Changes P Value
Collagen I 54.54 ± 14.68 0.00027*
Collagen IV 59.29 ± 7.33 0.00005*
Collagen VI 37.07 ± 28.61 0.07210
Fibronectin 49.39 ± 24.72 0.00077*
Laminin 32.75 ± 22.97 0.09750
SPARC 84.85 ± 51.30 0.00059*
Table 4. 
 
Changes in the Protein Levels of Selected ECM Components, MMPs, TIMPs, and MMP Activity Following SPARC Overexpression in TM Cells
Table 4. 
 
Changes in the Protein Levels of Selected ECM Components, MMPs, TIMPs, and MMP Activity Following SPARC Overexpression in TM Cells
% Change in Expression Compared to Control
MOI 25 (n) P Value MOI 50 (n) P Value
MMPs (immunoblot)
 MMP-1 −8.69 ± 2.45 (7) 0.062 −18.26 ± 4.64 (7) 0.008*
 MMP-2 0.27 ± 3.72 (8) 0.943 −16.46 ± 7.29 (8) 0.109
 MMP-3 −5.45 ± 1.76 (6) 0.077 −23.87 ± 4.68 (6) 0.004*
 MMP-9 −18.07 ± 4.33 (7) 0.042* −25.26 ± 7.39 (7) 0.014*
MMPs (zymogram)
 MMP-1 1.64 ± 0.37 (8) 0.103 2.46 ± 0.89 (8) 0.078
 MMP-2 −2.12 ± 3.24 (12) 0.527 −7.19 ± 5.85 (12) 0.245
 MMP-9 −10.24 ± 3.56 (8) 0.023* −18.32 ± 4.20 (8) 0.003*
ECM
 Collagen I 5.19 ± 1.84 (5) 0.098 19.18 ± 2.99 (5) 0.037*
 Collagen IV 45.46 ± 15.83 (7) 0.028* 58.55 ± 15.67 (7) 0.010*
 Collagen VI 5.54 ± 1.98 (7) 0.092 22.49 ± 5.43 (7) 0.002*
 Fibronectin 9.87 ± 2.52 (6) 0.061 25.93 ± 9.65 (6) 0.002*
 Laminin 7.12 ± 2.67 (5) 0.106 17.27 ± 3.25 (5) 0.006*
TIMPs
 TIMP-1 16.46 ± 4.71 (7) 0.013* 18.87 ± 6.95 (7) 0.038*
 TIMP-2 10.11 ± 2.91 (6) 0.065 14.66 ± 3.27 (6) 0.300
 TIMP-3 2.83 ± 1.54 (8) 0.158 17.75 ± 6.88 (8) 0.036*
 TIMP-4 9.09 ± 5.31 (7) 0.062 8.61 ± 9.30 (7) 0.082
Other
 PAI-1 23.28 ± 5.08 (8) 0.003* 36.59 ± 7.92 (8) 0.002*
Table 5. 
 
Changes in the mRNA Levels of Selected ECM Proteins Compared With Controls as a Result of Adenoviral Infection by Ad5.control and Ad5.hSPARC
Table 5. 
 
Changes in the mRNA Levels of Selected ECM Proteins Compared With Controls as a Result of Adenoviral Infection by Ad5.control and Ad5.hSPARC
% Change in Expression Compared to Control
MOI 25, n = 6 MOI 50, n = 6
Collagen I α1 −17.17 ± 11.28 −21.60 ± 15.13
Collagen IV α6 −7.81 ± 9.50 8.56 ± 27.17
Collagen VI α1 −17.92 ± 13.73 −7.24 ± 15.03
Collagen VI α2 −10.37 ± 14.05 23.55 ± 30.01
Collagen VI α3 −58.34 ± 4.92 32.69 ± 78.35
Fibronectin 7.18 ± 10.18 43.53 ± 45.12
Laminin α5 −32.20 ± 10.77 −27.11 ± 11.30
Laminin β1 −12.55 ± 14.69 9.79 ± 21.17
Laminin γ1 −30.51 ± 25.05 17.00 ± 40.82
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