Molecular Biology Reports

, Volume 40, Issue 11, pp 6091–6096

Adenovirus conducted connective tissue growth factor on extracellular matrix in trabecular meshwork and its role on aqueous humor outflow facility

Authors

  • Ying Su
    • Department of OphthalmologyFirst Clinical College of Harbin Medical University
    • Department of Ophthalmology, Department of Anatomy and NeurobiologyBoston University School of Medicine
  • Jingli Cheng
    • Department of OphthalmologyFirst Clinical College of Harbin Medical University
  • Hongtao Liu
    • Department of OphthalmologyFirst Clinical College of Harbin Medical University
    • Department of OphthalmologyFirst Clinical College of Harbin Medical University
  • Shiguang Zhao
    • Department of NeurosurgeryFirst Clinical College of Harbin Medical University
Article

DOI: 10.1007/s11033-013-2720-2

Cite this article as:
Su, Y., Cheng, J., Liu, H. et al. Mol Biol Rep (2013) 40: 6091. doi:10.1007/s11033-013-2720-2

Abstract

Deposition of extracellular matrix (ECM) in trabecular meshwork, such as fibronectin, collagen IV, elastin. leads to increased resistance of trabecular meshwork in primary open angle glaucoma (POAG). Connective tissue growth factor (CTGF) is known to regulate the ECM deposits. In this study, we detect the effect of adenovirus conducted CTGF (Adv-CTGF) transfection on either the expression of ECM components or aqueous humor outflow facility. Adv-CTGF was used to transfect rat trabecular meshwork cells in vivo and in vitro. Aqueous humor outflow facility was test by microbeads perfusion. Protein expression of CTGF, fibronectin, and collagen IV was determined using Western blot. In the Adv-CTGF group, the outflow facility displayed a significant decrease from baseline. It appears as though the transfection with Adv-CTGF significantly affects the aqueous humor outflow pattern. A negative correlation between IOP and PEFL indicated that a decrease in the area of bead deposition corresponded to an overall decrease of outflow, leading to an elevated IOP. Adv-CTGF can enhance the expression of CTGF, fibronectin and collagen IV. CTGF is the novel target for treatment of POAG. It is necessary to further study to test inhibition of CTGF expression for treatment of POAG.

Keywords

GlaucomaTrabecular meshworkConnective tissue growth factorMicrobeadFacility

Introduction

Glaucoma is the leading cause of blindness worldwide [1, 2]. Elevated intraocular pressure is confirmed to be major factor of glaucoma [35]. Increased resistance of aqueous humor outflow pathway, especially in trabecular meshwork was the major reason of primary open angle glaucoma [68]. Deposition of extracellular matrix in trabecular meshwork, such as fibronectin, collagen IV, elastin, etc. leads to increased resistance of trabecular meshwork [9, 10].

The synthesis and turnover of ECM is regulated by physiological factors, TGF-β, cytokines, connective tissue growth factor (CTGF) [11, 12]. The human CTGF gene is located on chromosome 6q23.1 and consists of five exons and four introns [1315]. The CTGF promoter contains Smad binding elements (SBE) and a unique TGF-β response element, suggesting it is a TGF-β1 responsive gene [16, 17]. CTGF is a secreted protein which contains four distinct structural modules [18, 19].

TGF-β is a multifunctional cytokine and it has been reported to play various roles in wound healing, chemotaxis, mitogenesis, apoptosis, migration, differentiation, extracellular matrix synthesis and immunomodulation [20, 21]. Increased levels of endogenous TGF-β subsequently induce ECM proteins (such as fibronectin and collagen IV) via CTGF dependent and independent pathways [22].

In the present study, we generated rat model which overexpress CTGF in the eye. We provide evidence for the first time that overexpression of CTGF can decrease the aqueous humor outflow which leads to high intraocular pressure in rat.

Materials and methods

Construction of Adv-CTGF

To enhance CTGF expression in the cells, full length of CTGF cDNA was subcloned into an adenoviral shuttle vector. Vector was generated with PTE (Imgenex, San Diego, CA, USA). Recombinant adenovirus carrying CTGF (Adv-CTGF) was constructed by homologous recombination in human embryonic kidney (HEK) 293 cells. The adenovirus with no insert (Adv-PTE) was used as control.

Transfection rTM with Adv-CTGF

This study and all the procedures were approved by the Ethics Committee of the University of Harbin Medical University. For in vivo studies, rats were examined by direct ophthalmoscopy (YZ6E, Suzhou company, Suzhou, China) to confirm a normal appearance, free of any signs of ocular disease. At day 0, rats were anesthetized using ketamine 60 mg/kg and xylazine 80 mg/kg, intraperitoneal injection. Eyes given intracameral injection were pretreated with 1–2 drops 1 % Benoxil topically anesthetized. Then adenovirus of a specified titer in a volume of 5 μL was injected into anterior chamber of the rat eye. Ocular injections were administered using a Hamilton (Reno, NV) glass microsyringe fitted with 35-gauge needle, while the uninjected eye served as a control. Each injection was made over the course of approximately 50 s [23].

Intraocular pressure examination

A rebound tonometer (TonoLab; Colonial Medical Supply, Franconia, NH) was used to measure the IOP of the right eye after the topically anesthesia. The IOP was calculated and recorded for the rat. The rat was positioned to allow the probe to contact the central cornea perpendicularly [24].

Rat microbead injection

Rat will be stabilized on a mounting stage under magnification of a surgery microscope. A 35G needle (NF35BL-2, World Precision Instruments, Sarasota, FL, USA) is loaded with 1 μL of 1:100 solution of fluorescent tracer (FluoSpheres carboxylate-modified 20-nm microspheres; Invitrogen, Eugene, OR, USA) as well as 2 μL of Karnovsky’s fixative (KII solution including 2 % paraformaldehyde and 2.5 % glutaraldehyde in phosphate buffer, pH 7.3) separated by a small air bubble. The needle is then inserted into the OD anterior chamber centrally in order to allow for uniform distribution of the tracer throughout the eye. The 1 μL of tracer then is delivered at 5 nL/s through a 10 μL Hamilton microsyringe (Nanofil; World Precision Instruments, Sarasota, FL) by a microprocessor-based microsyringe pump controller (Micro4; World Precision Instruments, Sarasota, FL). After 30 min, half of the original dose of anesthesia administered was given in order to maintain the mouse is at the appropriate level of unconsciousness. After 45 min of tracer injection, 5 μL of KII solution will be injected into the eye, and KII solution is also simultaneously applied to the exterior of the eye multiple times using a plastic dropper. The rat will be sacrificed by anesthetic overdose (4× the original amount administered). The needle is then removed, and both injected eye and uninjected eye (control) are enucleated. Eyes will be enucleated using a lateral canthotomy procedure in order to reduce trauma to the eyeball. After overnight fixation with KII solution, eyes will be transferred into PBS containing 50 % KII solution at 4 °C to store for further processing [24, 25].

Confocal microscope scan of section of rat eye

The vitreous body and lens of each eye were carefully removed. Frontal Sections were washed with PBS for 3 times after incubated with 0.19 Trinton X-100, then with 3 successive 5-min washes in PBS, counterstained in 4.5 μL To-pro3 (Molecular Probes, Inc., Eugene, OR, USA.) in 3 mL PBS for 30 min to visualize cell nuclei. After 3 successive 5-min washes in PBS, the sections were examined under confocal microscopy (Carl Zeiss 510, Axiovert 100 M Laser Scanning Microscope, Heidelberg, Germany). Images were taken along the inner wall of Schlemm’s canal (SC). The total length of the inner wall (TL) and the tracer-decorated length of the inner wall (L) were measured in frontal sections of each eye. A minimum of 16 images per eye were measured and the average percent effective filtration length (PEFL = FL/TL) in each eye was calculated [26].

Light microscope (LM) examination

Frontal section were fixed in Karnovsky’s solution (10 % paraformaldehyde and 50 % glutaraldehyde in 0.1 M cacodylate buffer) for 24 h. Then was fixed in a mixture of 4 % OsO4 and 0.8 % potassium ferrocyanide in 0.1 M cacodylate buffer for 2 h at 4 °C. Eyes were then dehydrated in a graded series of ethanol and embedded in Epon (Serva, Heidelberg, Germany). Frontal sections (1 μm) were collected on glass slides and stained with methylene blue/azure II for 1 min at room temperature [27].

TM cell culture and transfection with Adv-CTGF

The lens, cornea, retina, iris and ciliary body were extracted first. Then TM cells between Descemet membrane and scleral spur were dissected using fine forceps and placed in a 35 mm2 culture dish where cells were adhered to the plastic. The cell culture medium, Dulbecco’s Modified Eagle’s Medium (DMEM) (low glucose) supplemented with 10 % fetal bovine serum (FBS), l-glutamine (0.292 mg/mL), penicillin (100 units/mL), streptomycin (0.1 mg/mL), and amphotericin B (4 mg/mL) (HyClone Labs, Logan, UT, USA), was changed every 2 days. TM cells between passages 5 and 8 were selected for experiment. Cells were serum starved for 24 h prior to treatment with transfection with Adv-CTGF for 24 h [11, 12].

TM cells were transfected with plasmid containing Adv-CTGF, Adv-PTE, were served as experimental, vehicle control respectively. Transfection was performed in 60 mm plates using 3 μg (1 μg/μL) vector in 10 μL of Metafectene Pro reagent (Biontex, Martinstried, Germany). After 48 h of transfection, cells was screened for expression of TM cells by Western blot analysis.

Western blot analysis

Conditioned medium was collected from TM cells after treatment with Adv-CTGF in serum-free medium containing 0.5 mg/mL BSA (HyClone Labs, Logan, UT, USA). Protein concentration was measured using absorbance spectroscopy. Protein was separated on a 10 % SDS–polyacrylamide gel and transferred to nitrocellulose membranes. After blocking with 5 % nonfat milk, membranes were incubated with primary antibodies against CTGF, fibronectin, and collagen IV (Santa cruz biotechnology Inc, Santa Cruz, CA, USA) overnight at 4 °C, followed by incubation with secondary antibodies. The membrane was then assayed using the enhanced chemiluminescent kit (ECL, Thermo Scientific, Rockford, IL) and scanned with ChemiDoc™Doc XRS+ system (Bio-Rad, Hercules, CA, USA). The density of each band was obtained using Quantity One 4.6.2 basic software (Bio-Rad). Values were expressed as fold change relative to control and normalized to a loading control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz biotechnology Inc, Santa Cruz, CA, USA) [15].

Statistical analysis

The data were analyzed by the two-tailed Student’s t test using SPSS 10.0 (IBM Inc., Beijing, China) and p < 0.05 was considered significance.

Result

Adv-CTGF on IOP

We confirmed that transfection with Adv-CTGF increase IOP in rat, with a 35.2 % increase compared to control mice. The mean IOP was 12.5 ± 22.1 and 25.6 ± 3.5 mm Hg for control and Adv-CTGF rat, respectively (p < 0.001, N = 15 pairs, Fig. 1).
https://static-content.springer.com/image/art%3A10.1007%2Fs11033-013-2720-2/MediaObjects/11033_2013_2720_Fig1_HTML.gif
Fig. 1

Transfection with Adv-CTGF on IOP We confirmed that transfection with Adv-CTGF increase IOP in rat, with a 35.2 % increase compared to control rat. The mean IOP was 12.5 ± 22.1 mm Hg and 25.6 ± 3.5 mm Hg for control and Adv-CTGF rat, respectively (p < 0.001, N = 15 pairs)

Adv-CTGF on aqueous humor outflow facility

Changes in outflow facility among Adv-CTGF and control groups are shown in Figure. In the control group, outflow facility increased 25.2 ± 9.5 % from baseline. In the Adv-CTGF group, the outflow facility displayed a significant decrease of 52.5 ± 12.3 % from baseline (p < 0.01, Fig. 2).
https://static-content.springer.com/image/art%3A10.1007%2Fs11033-013-2720-2/MediaObjects/11033_2013_2720_Fig2_HTML.gif
Fig. 2

Transfection with Adv-CTGF on aqueous humor outflow facility In the Adv-CTGF group, the outflow facility displayed a significant decrease of 52.5 ± 12.3 % from baseline compared with control group (p < 0.01)

Adv-CTGF on PEFL

Distribution of tracer in the trabecular meshwork was found by confocal microscopy in control eyes. In contrast, there isn’t tracer found in the trabecular meshwork in Adv-CTGF eyes. PEFL in Adv-CTGF group (1.5 ± 2.1 %) was less than that of control eyes (95.3 ± 15.2 %, p < 0.001) Thus, it appears as though the transfection with Adv-CTGF significantly affects the aqueous humor outflow pattern. A negative correlation between IOP and PEFL was observed (Fig. 3). PEFL decreased as IOP increased. This suggests that a decrease in the area of bead deposition corresponded to an overall decrease of outflow, leading to an elevated IOP.
https://static-content.springer.com/image/art%3A10.1007%2Fs11033-013-2720-2/MediaObjects/11033_2013_2720_Fig3_HTML.gif
Fig. 3

Transfection with Adv-CTGF on PEFL PEFL in Adv-CTGF group (1.5 ± 2.1 %) was less than that of control eyes (95.3 ± 15.2 %, p < 0.001). A negative correlation between IOP and PEFL was observed

LM examination

Light microscopy analysis revealed that the inner wall of SC appeared mostly compact with the underlying JCT matrix in Adv-CTGF eyes than in control eyes (Fig. 4).
https://static-content.springer.com/image/art%3A10.1007%2Fs11033-013-2720-2/MediaObjects/11033_2013_2720_Fig4_HTML.jpg
Fig. 4

LM examination light microscopy analysis revealed that the inner wall of SC appeared mostly compact with the underlying JCT matrix in Adv-CTGF eyes than in control eyes

Upregulation of CTGF expression by transfection with Adv-CTGF

Expression of CTGF was detected in TM cells transfected with Adv-CTGF compared with control group (p < 0.01) (Fig. 5)
https://static-content.springer.com/image/art%3A10.1007%2Fs11033-013-2720-2/MediaObjects/11033_2013_2720_Fig5_HTML.gif
Fig. 5

Upregultion of CTGF expression by Adv-CTGF Expression of CTGF was detected in TM cells transfected with Adv-CTGF compared with control group (p < 0.01)

Transfection with Adv-CTGF on expression of fibronectin and collagen IV in vitro

Fibronectin and collagen IV protein was expressed in the TM cells transfected with Adv-CTGF, control group (p < 0.01). Downregulation of fibronectin and collagen IV was detected in TM cells transfected with Adv-CTGF (Fig. 6).
https://static-content.springer.com/image/art%3A10.1007%2Fs11033-013-2720-2/MediaObjects/11033_2013_2720_Fig6_HTML.gif
Fig. 6

Transfection with Adv-CTGF on expression of fibronectin and collagen IV in vitro Downregulation of fibronectin and collagen IV was detected in TM cells transfected with Adv-CTGF compared with control group (p < 0.01)

Discussion

Iyer et al. [28] found that CTGF influences ECM synthesis, including fibronectin, laminin, actin cytoskeletal dynamics and contractile properties in TM cells and that the expression of CTGF is closely regulated by Rho GTPase.

Junglas et al. [29] found that CTGF induces TM fibronectin and α-SMA in animals, whereas actin stress fibers and contractility are both induced in cultured TM cells. Depletion of CTGF by RNA interference leads to a marked attenuation of the actin cytoskeleton. Rho kinase inhibitors cause a reversible decline in the IOP of CTGF-overexpressing mice to levels seen in control littermates [30]. Research demonstrated that CTGF is related to pseudoexfoliation glaucoma [31, 32].

In our study, transfection with Adv-CTGF increase IOP in rat, with a 35.2 % increase compared to control rat which CTGF can leads to high IOP. The result is in agreement with previous study [28, 29]. CTGF is an effective factor to increase IOP because it can change the structure of JCT. In our study, structure of TM transfected with Adv-CTGF was compact than the control group. Additionally, CTGF can enhance the expression of collagen IV and fibronection which is similar to previous study [2830].

These are the first data to demonstrate transfection with AH outflow facility of Adv-CTGF group is less than that of control group which indicate transfection with Adv-CTGF can establish the model of POAG.

We confirmed that it appears as though the transfection with Adv-CTGF significantly affects the aqueous humor outflow pattern because there is negative correlation between IOP and PEFL. PEFL decreased as IOP increased. This suggests that a decrease in the area of bead deposition corresponded to an overall decrease of outflow, leading to an elevated IOP.

In summary, the present work demonstrates that transfection with Adv-CTGF can increase the IOP in mice which is the cause of decrease of AH outflow facility. The above result indicates it is the novel target for treatment of POAG. It is necessary to further study to test if inhibition of CTGF expression can be effective for treatment of POAG.

Acknowledgments

This work was supported by the grant of nature science science foundation of China (81100659), the grant of nature science foundation of Heilongjiang province of China (No. D2007–80), Scientific foundation of education ministry of China (20092307120003), and Postdoctral foundation of China (20080430137, 200902418). Dr. Feng Wang and Dr. Shiguang Zhao work equally to this work and can be regarded as co-corresponding author.

Conflict of interest

The authors declare that there is no conflict of interest.

Copyright information

© Springer Science+Business Media Dordrecht 2013