|Molecular Vision 2006;
Received 7 June 2006 | Accepted 4 September 2006 | Published 30 September 2006
Regulation of connective tissue growth factor expression in the aqueous humor outflow pathway
Saumil M. Chudgar,1
David L. Epstein,1
P. Vasantha Rao1,2
1Department of Ophthalmology and 2Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, NC
Correspondence to: P. V. Rao, Ph.D., Department of Ophthalmology, Duke University School of Medicine, Erwin Road, Durham, NC, 27710; Phone: (919) 681-5883; FAX: (919) 684-8983; email: firstname.lastname@example.org
Purpose: Connective Tissue Growth Factor (CTGF) is an inducible secretory protein known to regulate proliferation and extracellular matrix production in various cell types. We hypothesize that CTGF plays a critical role in the physiological regulation of aqueous humor outflow through the trabecular meshwork (TM) by influencing extracellular matrix synthesis and organization.
Methods: To determine the expression of CTGF in tissues of the aqueous outflow pathway, cells obtained from human TM and Schlemm's Canal (SC) were analyzed by PCR and western blot analysis. To understand the regulation of CTGF expression in TM cells, TM cells were either treated with various physiologic factors or subjected to cyclical stretch prior to analysis of CTGF expression by RT-PCR and western blot analysis. To study the effect of increased intraocular pressure on CTGF production, we perfused porcine eyes at high pressure (50 mm Hg) for 5 h, followed by analysis of CTGF expression by RT-PCR and western blotting.
Results: Treatment of human TM cells treated with either serum or transforming growth factor-beta 1 led to a robust stimulation, compared to thrombin, lysophosphatidic acid (LPA), and dexamethasone, which elicited a relatively moderate induction of CTGF expression. Both high pressure perfusion and mechanical stretch were associated with increases in the levels of CTGF at the protein and transcript levels.
Conclusions: This study demonstrates that CTGF expression in TM cells is modulated by several physiological agonists and by increased ocular pressure and mechanical stretch. These results suggest that the regulation of CTGF expression within tissues of the outflow pathway may play a role in the homeostasis of intraocular pressure, possibly by modulation of ECM production in these tissues.
Primary open-angle glaucoma (POAG) is an ocular disease characterized by increased resistance to aqueous humor outflow through the trabecular meshwork (TM) and Schlemm's canal (SC) [1,2]. It is thought that impairment of trabecular meshwork function is a major cause for the elevated intraocular pressure, which eventually leads to the death of retinal ganglion cells associated with POAG [1,3,4]. Thus, understanding the mechanisms that regulate aqueous humor outflow facility may prove of great importance as we try to understand the etiology of glaucoma and increased intraocular pressure. The extracellular matrix (ECM) of the trabecular meshwork and regulation of ECM production by TM cells play an important role in the homeostasis of intraocular pressure via influencing aqueous humor outflow [5-7]. Increased production of ECM by the TM could be one cause of decreased aqueous humor outflow, a condition that can lead to increased intraocular pressure [2,5-8]. Identifying the factors that influence the synthesis and degradation of the ECM within the aqueous outflow pathway is therefore pivotal for understanding the mechanistic bases which may underlie the pathophysiology of glaucoma.
Connective Tissue Growth Factor (CTGF) is a 38 kDa secretory protein that is a member of the CCN family (CYR61, CTGF, and NOV) [9,10]. It is thought to mediate many different functions, including acting as a mitogen in fibroblast cell cultures and enhancing the production of extracellular matrix components (Type I collagen, fibronectin) in various cell types [9,10]. CTGF is also known to play a role in angiogenesis, fibrosis, and wound healing [9-11]. Various fibrotic conditions such as renal fibrosis, scleroderma, and idiopathic pulmonary fibrosis have been associated with increased production of CTGF [9-14]. In addition, a 50 fold increase of CTGF mRNA expression has been reported in atherosclerotic plaques, as compared to the normal arterial wall .
Several studies in the literature have looked at the effects of various physiological factors on CTGF expression in different cell types. Transforming growth factor-beta (TGF-β), for instance, is known to share several of the functions of CTGF, including fibroblast proliferation and extracellular matrix synthesis [9,16,17]. CTGF is actually thought to be a downstream mediator of TGF-β, and production of CTGF is known to be stimulated by TGF-β [2,16,17]. Other factors are also known to stimulate CTGF expression. CTGF is increased in fibroblasts treated with dexamethasone , thrombin , endothelin-1 , and in renal mesangial cells treated with lysophosphatidic acid (LPA) . Additionally, mechanical strain/shear stress has been shown to increase CTGF mRNA expression in both fibroblasts  and rat mesangial cells . Also, Rho GTPase activity and actin cytoskeletal integrity have been demonstrated to influence the expression of CTGF in different cell types [21,24-27].
The role and regulation of CTGF in the aqueous humor outflow pathway has not been studied in detail [28-30]. By determining what effect factors implicated in the pathophysiology of glaucoma has on CTGF production, we may be able to better understand the role of CTGF in aqueous humor outflow. In this study, we hypothesize that CTGF plays an important role in the regulation of aqueous humor outflow facility via influencing extracellular matrix production in the trabecular meshwork. To explore this hypothesis, we studied several physiological factors that affect the production of CTGF in the outflow pathway tissues and considered the potential impact that CTGF may have on intraocular pressure.
The goat polyclonal antibody to recombinant human CTGF was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). cDNA libraries of human TM and SC cells and TM tissues were provided by Pedro Gonzalez, Duke University Eye Center and the details on these libraries have been described earlier . TGF-β1, dexamethasone, LPA, anti-β-actin monoclonal antibody was purchased from Sigma-Aldrich (St. Louis, MO). Human thrombin was from Calbiochem (La Jolla, CA).
Primary cell cultures of human trabecular meshwork and Schlemm's canal were maintained as described previously [32,33] in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37 °C with 5% CO2. For western blot analysis, cells were grown to confluence in the presence of serum. To investigate CTGF regulation by different physiological mediators, TM cells were initially grown to confluence and then serum starved for 24 h before treatment with human TGF-β1 (20 ng/ml), serum (10% fetal bovine), human thrombin (5 U/ml), LPA (20 μM) and dexamethasone (5 μM) for 4 h. Both TM and SC cells were isolated from postmortem human donor eyes as described previously  and passaged through 3 to 5 times.
TM cells from porcine eyes were harvested and grown in the medium as described above for human TM. For the stretch experiments described below, cells were maintained in cell culture medium containing serum. Passages 3 and 4 were used in each experiment.
Porcine TM tissue lysates were prepared by chopping the collected TM tissue finely and then grinding it with a plastic pestle. The remaining steps for processing were the same as that described in the following section for cells. TM and SC cell and tissue lysates were prepared by probe sonication in 20 mM Tris buffer (pH 7.4) containing 1 mM sodium orthovanadate, 0.2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 0.1 M NaCl, 50 mM NaF, aprotinin (25 μg/ml) and leupeptin (25 μg/ml). Centrifugation at 13,000 rpm for 15 min at 4 °C was performed, and the soluble fractions were collected. Total protein concentration for the lysates was determined by the Bradford method . Equal amounts (60 μg) of protein from each sample were separated on 12.5% SDS-polyacrylamide gels followed by electrophoretic transfer of resolved proteins to a nitrocellulose membrane using a Bio-Rad transfer apparatus. The membranes were probed for CTGF using the polyclonal anti-CTGF antibody (1:1000 dilution), followed by incubation with a perioxidase-linked secondary antibody (1:4000 dilution). Detection of immunoreactivity was done by enhanced chemiluminescence (ECL) as described by the manufacturer (Amersham Biosciences, Piscataway, NJ). Equal loading of protein was confirmed wherever indicated by probing for actin as an internal control, and a negative control lacking primary antibody was carried out. When quantification was necessary, blots were scanned and relative concentrations were determined by densitometric analysis using the NIH Scion Image software. A mean of the readings from different trials was taken, and the paired t-test was used to determine statistical significance. Values were expressed as mean±SEM.
In order to determine the extracellular CTGF secreted by HTM cells, confluent cultures of HTM cells were serum starved for 48 h and stimulated with TGF-β1 for 4 h. This serum-free medium was collected and concentrated using an Amicon Ultra 10K filter device (Millipore Corp, Bedford, MA). The protein content of the concentrated medium was determined by the Bradford method , and an equal amount of protein (10 μg) from both control and TGF-β1 treated was analyzed for the CTGF by immunoblotting, per the aforedescribed method.
PCR and RT-PCR
To confirm CTGF expression in human TM tissue and human TM and SC cells, cDNA libraries were amplified by PCR using human CTGF-specific oligonucleotide primers. The following set of forward and reverse primers was used for the human TM tissue and cell PCR reactions: GTG GAG TAT GTA CCG ACG GCC and AAG CTG TCC AGT CTA ATC GAC AGG. PCR reactions were carried out for 30 cycles using the Advantage cDNA PCR kit (BD Biosciences, Palo Alto, CA) and the GeneAmp 9700 PE Applied Biosystem thermal cycler (PE Applied Biosystems, Foster City, CA). PCR products were separated on a 1.5% agarose gel and stained with ethidium bromide for ultraviolet visualization. Product was sequenced to confirm the CTGF specificity.
To determine the relative expression of CTGF after treatments, TM tissue from the perfused eyes and cell samples were collected, and RNA was extracted using the Qiagen RNeasy Mini kit (Qiagen, Valencia, CA). DNase I treatment (Invitrogen, Carlsbad, CA) was performed to remove any genomic DNA contamination. RNA concentration was determined using RiboGreen (Molecular Probes, Eugene, OR), and RT-PCR was performed on equal amounts (1 μg) of RNA from the samples using the Advantage RT-for-PCR kit (BD Biosciences, Palo Alto, CA). PCR amplifications (cycle numbers) were performed within the linear range of DNA product formation.
Porcine TM CTGF was amplified from cDNA libraries using oligonucleotide primers specific for porcine CTGF (CTT TGG AGG AAC GGT GTA CCG and CAA ACG TGT CTT CCA GTC GGT AA) as already described. Internal controls glyceraldehyde 3-phosphate dehydrogenase (G3PDH; CCG AGC TGA GCA TAG ACA TT and TCC ACC ACC CTG TTG CTG TA, 452 bp product) and β-actin were used to confirm equal amounts of DNA in PCR reactions. PCR amplifications (cycle numbers) were performed within the linear range of DNA product formation. The products were run on a 1.5% agarose gel with ethidium bromide, and images under ultraviolet illumination were captured using Polaroid film. The images were subjected to densiometric analysis to determine quantitative differences in CTGF expression in control versus experimental samples. A mean of the densiometric readings from different trials was taken, and the paired t-test was used to determine statistical significance. Values were presented as mean±SEM.
Real-time quantitative RT-PCR
Real-time quantification of CTGF mRNA in TGF-β1-treated HTM cells was performed using the iCycler iQ detection system (Bio-Rad, Hercules, CA). mRNAs of different samples were normalized to an endogenous housekeeping gene, β-actin. The following human CTGF and β-actin specific oligonucleotide primers were used, respectively, in the real-time PCR analysis: CTGF; CTG GTC CAG ACC ACA GAG TGG AG/CTT CCA GGT CAG CTT CGC AAG G (146 bp) and β-actin; AAG GAG AAG CTG TGC TAC GTC G/C ATG ATG GAG TTG AAG GTA GTT TCG T (212 bp). Briefly, the PCR master mix (iQ supermix, Bio-Rad) consisted of 1 μl of template cDNA in 20 μl reaction, 2x PCR master mix, 10 nM fluorescein calibration dye (Bio-Rad), 1 μl of a 1:1500 dilution of 10,000x nucleic acid dye (SYBR Green 1; Molecular Probes), and 500 nM of each gene specific oligonucleotide pair. Duplicate PCR reactions were carried out using the following amplification protocol: 95 °C for 2 min followed by 50 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 15 s. The increase in fluorescence was measured in real time during the extension step, and melt curves were obtained immediately after amplification by increasing temperature in 0.4 °C increments from 65 °C for 85 cycles of 10 s each and analyzed using Bio-Rad iCycler software. The fold difference in CTGF expression between control and TGF-β-treated samples normalized to the housekeeping gene (β-actin) was calculated by the comparative threshold (CT) method as described by the manufacturer (Prism 7700 Sequence Detection System; Applied Biosystem, Inc.).
High pressure perfusion
Fresh paired enucleated porcine eyes were obtained from a local abattoir and fitted with a Grant corneal fitting using a technique described previously . The eyes were perfused at room temperature with an isotonic solution consisting of Dulbecco's PBS with glucose (5.5 mM) adjusted to pH 7.4. Both eyes were initially maintained at a pressure of 15 mm Hg (height of buffer column=20.4 cm) for 30 min to allow for anterior chamber filling and equilibration. Then, the experimental eye was raised to a pressure of 50 mm Hg (height of buffer column=68.0 cm) for 5 h, while the control eye was maintained at 15 mm Hg. TM was harvested after 5 h, and analysis for CTGF expression was conducted by western blot and RT-PCR analyses.
Cultured porcine TM cells were plated onto BioFlex six-well plates that contained a collagen-coated surface (Flexcell International, Hillsborough, NC). After the cells reached approximately an 80% confluence, they were exposed to mechanical stretch using the FlexCell FX-4000 system (Flexcell International). A cyclic stretch of 1 cycle/s was applied to the experimental plates for 16 h at 37 °C, giving a total stretch of 15% from control. Control plates were loaded into the system but did not undergo stretch. After stretch treatment, the cells were scraped and harvested for Western Blot and RT-PCR analysis.
Expression of connective tissue growth factor in the aqueous humor outflow pathway
The initial part of our studies involved determining the expression of CTGF in tissues of the outflow pathway, using PCR and western blotting techniques. Initially, CTGF transcripts were detected in human TM and SC cells and in human TM tissue by PCR amplification of representative cDNA libraries. PCR assays carried out with oligonucleotide primers specific to human CTGF yielded the expected 740 base pair DNA product. Expression was demonstrated in all three samples (Figure 1A). Sequencing of the product confirmed that it was specific to CTGF mRNA.
After establishing the presence of CTGF mRNA in cells of the outflow pathway, we performed an analysis of CTGF protein. Immunoblot analysis of lysates from human SC and TM cells confirmed the presence of two closely migrated immunopositive bands (native and glycosylated) at the range of 40 kDa, which corresponds closely to the reported molecular weight (38 kDa) of CTGF (Figure 1B) [9,35]. A specificity control wherein cell lysates were treated with secondary antibody alone showed no immunopositive reaction (data not shown). Immunoblot analysis of cell culture medium obtained from the serum-starved (24 h) confluent cultures of HTM cells also demonstrated a specific immunopositive protein bands for CTGF (data not shown). Although we found a single, specific immunopositive band closely corresponding to the molecular weight of CTGF, the blots developed with culture medium showed a diffused band as compared to the immunoblots developed with cell lysates perhaps due to glycosylation of the secreted CTGF protein.
Regulation of connective tissue growth factor expression in human trabecular meshwork cells
To investigate the regulation of CTGF expression, we treated serum-starved human TM cells with different factors including TGF-β1 (20 ng/ml), dexamethasone (5μM), LPA (20 μM), thrombin (5 U/ml), and serum (10%) for 4 h. Total RNA extracted from these samples was used for semiquantitative estimation of CTGF by RT-PCR analysis using β-actin as an internal control. Due to short supply of human SC cells, we have not carried out these and further experiments with SC cells in this study.
Figure 2 illustrates representative data for CTGF expression patterns in human TM cells treated with the aforedescribed different agonists. Densitometric analysis of RT-PCR-amplified CTGF signal from two independent experiments showed a robust induction of CTGF expression by serum (15 fold), TGF-β1 (7 fold), thrombin (4.4 fold), dexamethasone (4 fold), and LPA (3 fold) when compared to serum starved control cells (Figure 2C).
Since TGF-β1 elicited a strong induction of CTGF expression in HTM cells, we evaluated the kinetics and dose-dependent response of this factor. After treating serum-starved (24 h) TM cell cultures with TGF-β1 (20 ng/ml) for given periods of time (0, 0.5, 1, 4, 8, and 24 h), cells were analyzed by RT-PCR amplification to determine expression of CTGF. A time-dependent increase was noted, with a steady rise occurring between the time of addition to 4 h; maximal expression was seen after 8 h of TGF-β1 addition (Figure 3). A dose-response effect of TGF-β1 on CTGF expression after treatment for 4 h revealed a strong induction with 0.5 ng/ml (70% over the untreated sample) and with 5 ng/ml reaching maximum of 90% induction over the control (Figure 4). The induction with 20 ng/ml was found to be similar to that of 5 ng/ml (data not shown). Figure 5A,B show quantitative changes in CTGF mRNA in TGF-β1-treated HTM cells by real-time RT-PCR analysis. Serum-starved HTM cells treated with 20 ng/ml TGF-β for 4 h (n=5) demonstrated a 3.89 fold increase in CTGF expression compared to untreated HTM cells.
In addition to the expression status of CTGF, we have also tested the effects of TGF-β1 on the protein levels of CTGF in serum-starved HTM cells treated with 5 ng/ml for 4 h. Two independent samples showed a marginal but consistent increasing trend based on the immunoblotting analysis of equal amounts of total cell lysate (Figure 6). Also, the secreted levels of CTGF were found to have only a marginal increase in TGF-β-treated cells compared to untreated cells (data not shown).
Effects of increased IOP on CTGF expression in TM tissue
Porcine eyes were perfused at either 15 mm Hg (control) or 50 mm Hg (high pressure) for 5 h to determine the impact of increased intraocular pressure on CTGF expression in TM tissue. TM tissue was first analyzed by western blotting to determine changes in CTGF protein levels. Densitometric analyses of blots from a mean of four trials show a statistically significant upregulation (of approximately 50%) of CTGF protein expression in the eyes perfused at high pressure compared to controls (n=4, p<0.014; Figure 7).
TM tissue obtained from the eyes perfused at high pressure (50 mm Hg) was also analyzed by RT-PCR to determine the relative levels of CTGF mRNA expression. Confirmation that equal amounts of cDNA were used in the analyses was obtained using G3PDH as an internal control. Relative expression of CTGF was determined by densitometric analysis of Polaroid pictures of agarose gels taken under ultraviolet fluorescent light. A mean of four trials demonstrated a statistically significant increase (about 20%) in CTGF expression in eyes subjected to high pressure perfusion as compared to control eyes (n=4, p<0.009; Figure 8).
Effects of mechanical stretch on CTGF expression in TM cells
To determine the effects of mechanical stretch on CTGF expression, porcine TM cells were subjected to cyclic mechanical stretch for a period of 16 h and then harvested for analysis. Mean (n=4) values obtained from densitometric scans of western blot analysis of CTGF protein levels showed a small, but statistically significant (p<0.009), upregulation in CTGF production of 13% in cells that were stretched, as compared to control cells (Figure 9).
RT-PCR analysis of cDNA libraries prepared from the control and stretched cells was also carried out to determine relative CTGF mRNA expression using methods described in the methods section. A mean increase of 18% was seen in cells that underwent stretch. However, these results were not statistically significant using the paired t-test (n=4, p<0.09; Figure 10).
The results from this study document the expression of CTGF in cells of the aqueous humor outflow pathway, including the TM and SC and in TM tissue. Additionally, CTGF expression was found to be upregulated in TM cells by a number of physiological agonists including TGF-β1, thrombin, serum, dexamethasone, and LPA. Importantly, CTGF expression was found to be upregulated in TM tissue at both the transcript and protein levels in response to intraocular pressure in intact eyes and to mechanical stretch in TM cells. This study establishes the presence of CTGF in the outflow pathway and begins to elucidate the regulation of CTGF expression in aqueous outflow pathway tissues by physiological agonists and also as a potential role for this growth factor in the effects of elevated intraocular pressure.
These data support the hypothesis that CTGF plays a potentially pivotal role in aqueous humor outflow pathway. CTGF has previously been shown to play a role in extracellular matrix production and in fibrosis [9,10] in other cell types, and it is conceivable that a similar mechanism may account for the increased resistance to aqueous humor outflow under certain pathophysiological conditions involving increased levels of CTGF in ocular tissues. For instance, situations where there is a chronic increase of intraocular pressure and mechanical strain, such as those seen in many cases of POAG, could elicit increases in CTGF production, which could in turn potentially modulate aqueous outflow facility by affecting the production and organization of ECM in the outflow pathway [9,10,28,29,36,37].
The choice of physiological agonists examined in this study were based on published literature supporting either a direct association of one or more of these agonists with the glaucomatous condition, or with situations involving decreased aqueous humor outflow [8,31,38-40]. For example, it is well documented that the levels of TGF-β2 increase in the aqueous humor of glaucomatous eyes when compared to nonglaucomatous eyes [38,41,42]. Given that CTGF is thought to be a downstream mediator of TGF-β action , increased levels of TGF-β could potentially stimulate CTGF expression and thereby impair aqueous outflow facility in glaucomatous eyes. Importantly, Ho et al.  recently documented that aqueous humor obtained from the pseudoexfoliation patients with glaucoma contains significantly higher levels of CTGF. In this study, we observed a definite induction of CTGF expression in HTM cells treated with TGF-β1 (Figure 5). Although we confirmed a 4 fold increase in CTGF expression based on quantitative PCR analysis (Figure 5), under similar conditions, the levels of CTGF protein were increased only marginally (Figure 6). Additionally, the levels of secreted CTGF were found to be only marginally different between TGF-β treated and control HTM cells. However, in this study, we have not examined the long-term effects of TGF-β on TM cell CTGF protein levels (both intracellular and secreted), and this response may be different from what we found with 4 h exposure (Figure 6).
Interestingly, in this study we also detected increased expression of CTGF in TM cells stimulated with agonists of G-protein coupled receptors, such as thrombin and LPA. Both thrombin and LPA have been documented to decrease aqueous humor outflow facility in porcine eyes; this effect is thought to be mediated by activation of the Rho/Rho kinase pathway [31,39]. Intriguingly, expression of CTGF is known to be regulated by the Rho/Rho kinase pathway, and treatment with a Rho/Rho kinase inhibitor and statins has been reported to inhibit LPA and TGF-β-mediated induction of CTGF in other cell types [21,24,44]. Similar to thrombin and LPA, dexamethasone, a corticosteroid also caused an increased expression of CTGF in HTM cells (Figure 2). Increased intraocular pressure is one of the significant complications associated with the use of corticosteroids in clinical ophthalmology.
In addition to considering the effect of physiologic factors, we also studied mechanical forces on the TM and their effects on CTGF. Both experimental models utilized in this study, one which increased intraocular pressure in a whole eye perfusion model, and the other which directly stretched TM cells in culture, simulate increased mechanical stress on outflow pathway tissues. CTGF was found to be upregulated in both situations (Figure 6 and Figure 8), which is consistent with other data derived from human and porcine TM cells [28,29]. Increased intraocular pressure and increased mechanical stress on the TM are often noted in cases of POAG [1-4,28,45]. Our studies found that both kinds of stress upregulate the expression of CTGF, further implicating CTGF in the pathophysiology of glaucoma.
The level of upregulation of CTGF protein was much greater in the high pressure whole eye perfusion model (50%), compared to the mechanical stretching of TM cells (13%). This difference may be attributed to either the strength of the mechanical stress utilized or the experimental model. Whole eyes perfused at 50 mm Hg pressure might have responded to much stronger mechanical stress than the cells under 15% stretch. The former model might provide a more realistic picture of the situation encountered in POAG. The high-pressure perfusion and stretching studies were based on analysis at a single time-point. Thus, further studies involving long-term perfusion over several days or different levels of stretch and pressure and time intervals should provide important insights into the influence of mechanical stress on CTGF expression.
The idea that increased mechanical stretch can be sensed by the TM, and result in morphological and cellular and molecular alterations that might impact outflow facility, has been established in previous studies [46-50]. Our data show that CTGF might be another factor produced in response to this stress and lead us to propose that the effects of this growth factor may be mediated by its effects on the production of increased extracellular matrix deposition within tissues of the aqueous outflow pathway. Previous studies have demonstrated an increase in matrix metalloproteinase (MMP) production in TM cells in response to stretch [28,46,51], speculating that increased MMP activity may decrease resistance to outflow by degrading the ECM in the TM . We propose, on the other hand, that mechanical stress-induced increases in CTGF might induce a wound healing response in the TM, as it does in other tissues, and actually increase the synthesis of ECM. This could be one mechanism that causes decreased outflow and increased intraocular pressure. Elucidation of the mechanisms underlying this hypothesis may be a topic of future study in this area. Additionally, as reported recently, increased production of CTGF might lead to wound healing and failure of glaucoma bleb following glaucoma filtration surgery .
Further studies are required to determine the specific role played by CTGF in the pathophysiology of increased intraocular pressure. Specifically, evaluating whether ECM production is increased in the TM exposed to CTGF would serve to confirm the mechanistic basis by which CTGF potentially modulates aqueous outflow facility. Interestingly, Fuchshofer et al.  have reported that increased expression of ECM components in astrocytes of optic nerve head are associated with increased levels of CTGF under TGF-β treatment. Thus, measuring changes in intraocular pressure after perfusion of recombinant CTGF into an anterior chamber perfusion model might give an accurate picture of the exact role on CTGF in influencing outflow facility.
In summary, our studies have demonstrated the presence of CTGF in the aqueous humor outflow pathway of the eye and provide data on the effects of TGF-β1, dexamethasone, thrombin, agonists of G-protein coupled receptors, mechanical stretch, and increased intraocular pressure on CTGF expression in these tissues. Regulation of CTGF might thus be critical for homeostasis of aqueous outflow.
We thank Dr. Pedro Gonzalez for providing the cDNA libraries of TM and SC cells and Jessica Ebright for help in Real-Time PCR analysis. This work was supported by the National Eye Institute/NIH R01 grants EY-013573 and EY-12201 (P.V.R.).
1. Epstein DL. Open angle glaucoma. Why not a cure? Arch Ophthalmol 1987; 105:1187-8.
2. Lutjen-Drecoll E. Functional morphology of the trabecular meshwork in primate eyes. Prog Retin Eye Res 1999; 18:91-119.
3. Quigley HA. Open-angle glaucoma. N Engl J Med 1993; 328:1097-106.
4. Weinreb RN, Khaw PT. Primary open-angle glaucoma. Lancet 2004; 363:1711-20.
5. Acott TS. Trabecular extracellular matrix regulation. In: Drance SM, Van Buskirk EM, Neufeld AH, editors. Pharmacology of glaucoma. Baltimore: Williams & Wilkins; 1992. p. 125-57.
6. Alexander JP, Acott TS. Involvement of the Erk-MAP kinase pathway in TNFalpha regulation of trabecular matrix metalloproteinases and TIMPs. Invest Ophthalmol Vis Sci 2003; 44:164-9.
7. Yue BY. The extracellular matrix and its modulation in the trabecular meshwork. Surv Ophthalmol 1996; 40:379-90.
8. Gabelt BT, Kaufman PL. Changes in aqueous humor dynamics with age and glaucoma. Prog Retin Eye Res 2005; 24:612-37.
9. Ihn H. Pathogenesis of fibrosis: role of TGF-beta and CTGF. Curr Opin Rheumatol 2002; 14:681-5.
10. Leask A, Holmes A, Abraham DJ. Connective tissue growth factor: a new and important player in the pathogenesis of fibrosis. Curr Rheumatol Rep 2002; 4:136-42.
11. Moussad EE, Brigstock DR. Connective tissue growth factor: what's in a name? Mol Genet Metab 2000; 71:276-92.
12. Kikuchi K, Kadono T, Ihn H, Sato S, Igarashi A, Nakagawa H, Tamaki K, Takehara K. Growth regulation in scleroderma fibroblasts: increased response to transforming growth factor-beta 1. J Invest Dermatol 1995; 105:128-32.
13. Crean JK, Lappin D, Godson C, Brady HR. Connective tissue growth factor: an attractive therapeutic target in fibrotic renal disease. Expert Opin Ther Targets 2001; 5:519-530.
14. Sato S, Nagaoka T, Hasegawa M, Tamatani T, Nakanishi T, Takigawa M, Takehara K. Serum levels of connective tissue growth factor are elevated in patients with systemic sclerosis: association with extent of skin sclerosis and severity of pulmonary fibrosis. J Rheumatol 2000; 27:149-54.
15. Oemar BS, Werner A, Garnier JM, Do DD, Godoy N, Nauck M, Marz W, Rupp J, Pech M, Luscher TF. Human connective tissue growth factor is expressed in advanced atherosclerotic lesions. Circulation 1997; 95:831-9.
16. Grotendorst GR. Connective tissue growth factor: a mediator of TGF-beta action on fibroblasts. Cytokine Growth Factor Rev 1997; 8:171-9.
17. Leask A, Abraham DJ. TGF-beta signaling and the fibrotic response. FASEB J 2004; 18:816-27.
18. Dammeier J, Beer HD, Brauchle M, Werner S. Dexamethasone is a novel potent inducer of connective tissue growth factor expression. Implications for glucocorticoid therapy. J Biol Chem 1998; 273:18185-90.
19. Chambers RC, Leoni P, Blanc-Brude OP, Wembridge DE, Laurent GJ. Thrombin is a potent inducer of connective tissue growth factor production via proteolytic activation of protease-activated receptor-1. J Biol Chem 2000; 275:35584-91.
20. Kemp TJ, Aggeli IK, Sugden PH, Clerk A. Phenylephrine and endothelin-1 upregulate connective tissue growth factor in neonatal rat cardiac myocytes. J Mol Cell Cardiol 2004; 37:603-6.
21. Hahn A, Heusinger-Ribeiro J, Lanz T, Zenkel S, Goppelt-Struebe M. Induction of connective tissue growth factor by activation of heptahelical receptors. Modulation by Rho proteins and the actin cytoskeleton. J Biol Chem 2000; 275:37429-35.
22. Schild C, Trueb B. Mechanical stress is required for high-level expression of connective tissue growth factor. Exp Cell Res 2002; 274:83-91.
23. Riser BL, Denichilo M, Cortes P, Baker C, Grondin JM, Yee J, Narins RG. Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis. J Am Soc Nephrol 2000; 11:25-38.
24. Eberlein M, Heusinger-Ribeiro J, Goppelt-Struebe M. Rho-dependent inhibition of the induction of connective tissue growth factor (CTGF) by HMG CoA reductase inhibitors (statins). Br J Pharmacol 2001; 133:1172-80.
25. Heusinger-Ribeiro J, Eberlein M, Wahab NA, Goppelt-Struebe M. Expression of connective tissue growth factor in human renal fibroblasts: regulatory roles of RhoA and cAMP. J Am Soc Nephrol 2001; 12:1853-61.
26. Ott C, Iwanciw D, Graness A, Giehl K, Goppelt-Struebe M. Modulation of the expression of connective tissue growth factor by alterations of the cytoskeleton. J Biol Chem 2003; 278:44305-11.
27. Chowdhury I, Chaqour B. Regulation of connective tissue growth factor (CTGF/CCN2) gene transcription and mRNA stability in smooth muscle cells. Involvement of RhoA GTPase and p38 MAP kinase and sensitivity to actin dynamics. Eur J Biochem 2004; 271:4436-50.
28. Vittal V, Rose A, Gregory KE, Kelley MJ, Acott TS. Changes in gene expression by trabecular meshwork cells in response to mechanical stretching. Invest Ophthalmol Vis Sci 2005; 46:2857-68.
29. Liton PB, Liu X, Stamer WD, Challa P, Epstein DL, Gonzalez P. Specific targeting of gene expression to a subset of human trabecular meshwork cells using the chitinase 3-like 1 promoter. Invest Ophthalmol Vis Sci 2005; 46:183-90.
30. Liang Y, Li C, Guzman VM, Evinger AJ 3rd, Protzman CE, Krauss AH, Woodward DF. Comparison of prostaglandin F2alpha, bimatoprost (prostamide), and butaprost (EP2 agonist) on Cyr61 and connective tissue growth factor gene expression. J Biol Chem 2003; 278:27267-77.
31. Mettu PS, Deng PF, Misra UK, Gawdi G, Epstein DL, Rao PV. Role of lysophospholipid growth factors in the modulation of aqueous humor outflow facility. Invest Ophthalmol Vis Sci 2004; 45:2263-71.
32. Stamer WD, Roberts BC, Howell DN, Epstein DL. Isolation, culture, and characterization of endothelial cells from Schlemm's canal. Invest Ophthalmol Vis Sci 1998; 39:1804-12.
33. Rao PV, Deng PF, Kumar J, Epstein DL. Modulation of aqueous humor outflow facility by the Rho kinase-specific inhibitor Y-27632. Invest Ophthalmol Vis Sci 2001; 42:1029-37. Erratum in: Invest Ophthalmol Vis Sci 2001; 42:1690.
34. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248-54.
35. Yang DH, Kim HS, Wilson EM, Rosenfeld RG, Oh Y. Identification of glycosylated 38-kDa connective tissue growth factor (IGFBP-related protein 2) and proteolytic fragments in human biological fluids, and up-regulation of IGFBP-rP2 expression by TGF-beta in Hs578T human breast cancer cells. J Clin Endocrinol Metab 1998; 83:2593-6.
36. Fuchshofer R, Birke M, Welge-Lussen U, Kook D, Lutjen-Drecoll E. Transforming growth factor-beta 2 modulated extracellular matrix component expression in cultured human optic nerve head astrocytes. Invest Ophthalmol Vis Sci 2005; 46:568-78.
37. Rachfal AW, Brigstock DR. Connective tissue growth factor (CTGF/CCN2) in hepatic fibrosis. Hepatol Res 2003; 26:1-9.
38. Tripathi RC, Li J, Chan WF, Tripathi BJ. Aqueous humor in glaucomatous eyes contains an increased level of TGF-beta 2. Exp Eye Res 1994; 59:723-7.
39. Rao PV, Deng P, Sasaki Y, Epstein DL. Regulation of myosin light chain phosphorylation in the trabecular meshwork: role in aqueous humour outflow facility. Exp Eye Res 2005; 80:197-206.
40. Gottanka J, Chan D, Eichhorn M, Lutjen-Drecoll E, Ethier CR. Effects of TGF-beta2 in perfused human eyes. Invest Ophthalmol Vis Sci 2004; 45:153-8.
41. Picht G, Welge-Luessen U, Grehn F, Lutjen-Drecoll E. Transforming growth factor beta 2 levels in the aqueous humor in different types of glaucoma and the relation to filtering bleb development. Graefes Arch Clin Exp Ophthalmol 2001; 239:199-207.
42. Ochiai Y, Ochiai H. Higher concentration of transforming growth factor-beta in aqueous humor of glaucomatous eyes and diabetic eyes. Jpn J Ophthalmol 2002; 46:249-53.
43. Ho SL, Dogar GF, Wang J, Crean J, Wu QD, Oliver N, Weitz S, Murray A, Cleary PE, O'Brien C. Elevated aqueous humour tissue inhibitor of matrix metalloproteinase-1 and connective tissue growth factor in pseudoexfoliation syndrome. Br J Ophthalmol 2005; 89:169-73.
44. Watts KL, Spiteri MA. Connective tissue growth factor expression and induction by transforming growth factor-beta is abrogated by simvastatin via a Rho signaling mechanism. Am J Physiol Lung Cell Mol Physiol 2004; 287:L1323-32.
45. Borras T. Gene expression in the trabecular meshwork and the influence of intraocular pressure. Prog Retin Eye Res 2003; 22:435-63.
46. Bradley JM, Kelley MJ, Zhu X, Anderssohn AM, Alexander JP, Acott TS. Effects of mechanical stretching on trabecular matrix metalloproteinases. Invest Ophthalmol Vis Sci 2001; 42:1505-13.
47. WuDunn D. The effect of mechanical strain on matrix metalloproteinase production by bovine trabecular meshwork cells. Curr Eye Res 2001; 22:394-7.
48. Stamer WD, Roberts BC, Epstein DL. Hydraulic pressure stimulates adenosine 3',5'-cyclic monophosphate accumulation in endothelial cells from Schlemm's canal. Invest Ophthalmol Vis Sci 1999; 40:1983-8.
49. Mitton KP, Tumminia SJ, Arora J, Zelenka P, Epstein DL, Russell P. Transient loss of alphaB-crystallin: an early cellular response to mechanical stretch. Biochem Biophys Res Commun 1997; 235:69-73.
50. Tumminia SJ, Mitton KP, Arora J, Zelenka P, Epstein DL, Russell P. Mechanical stretch alters the actin cytoskeletal network and signal transduction in human trabecular meshwork cells. Invest Ophthalmol Vis Sci 1998; 39:1361-71.
51. Bradley JM, Kelley MJ, Rose A, Acott TS. Signaling pathways used in trabecular matrix metalloproteinase response to mechanical stretch. Invest Ophthalmol Vis Sci 2003; 44:5174-81.
52. Bradley JM, Vranka J, Colvis CM, Conger DM, Alexander JP, Fisk AS, Samples JR, Acott TS. Effect of matrix metalloproteinases activity on outflow in perfused human organ culture. Invest Ophthalmol Vis Sci 1998; 39:2649-58.
53. Esson DW, Neelakantan A, Iyer SA, Blalock TD, Balasubramanian L, Grotendorst GR, Schultz GS, Sherwood MB. Expression of connective tissue growth factor after glaucoma filtration surgery in a rabbit model. Invest Ophthalmol Vis Sci 2004; 45:485-91.