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(TSP1)–Null and TSP2-Null Mice Exhibit Lower Intraocular Pressures

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(TSP1)–Null and TSP2-Null Mice Exhibit Lower Intraocular Pressures Glaucoma Thrombospondin-1 (TSP1)–Null and TSP2-Null Mice Exhibit Lower Intraocular Pressures Ramez I. Haddadin,1 Dong-Jin Oh,1 Min Hyung Kang,1 Guadalupe Villarreal Jr,1 Ja-Heon Kang,2 Rui Jin,3 Haiyan Gong,3 and Douglas J. Rhee1 PURPOSE. Thrombospondin-1 (TSP1) and TSP2 are matricellular alteration in the extracellular matrix may contribute to this proteins that have been shown to regulate cytoskeleton, cell finding. (Invest Ophthalmol Vis Sci. 2012;53:6708–6717) DOI: adhesion, and extracellular matrix remodeling. Both TSP1 and 10.1167/iovs.11-9013 TSP2 are found in the trabecular meshwork (TM). In cadaver eyes with primary open-angle glaucoma (POAG), TSP1 is increased in one third of patients. We hypothesized that TSP1 laucoma is the leading cause of irreversible blindness 1 and TSP2 participate in the regulation of intraocular pressure Gworldwide. Elevated intraocular pressure (IOP) is a major (IOP). risk factor for glaucoma. The relatively elevated IOP of open- angle glaucoma is caused by impaired aqueous drainage METHODS. IOPs of TSP1-null, TSP2-null mice, and their through the trabecular meshwork (TM) (i.e., conventional corresponding wild-type (WT) mice were measured using a pathway).2 Although not all of the physiologic processes commercial rebound tonometer. Fluorophotometric measure- responsible for the regulation of TM and ciliary body (CB) ments assessed aqueous turnover. Central corneal thickness drainage are known, extracellular matrix (ECM) turnover is at (CCT) was measured by optical coherence tomography. least one contributory factor.3–5 The mechanisms regulating Iridocorneal angles were examined using light microscopy the deposition and turnover of the ECM are not fully (LM), immunofluorescence (IF), and transmission electron understood. microscopy (TEM). Matricellular proteins are a group of extracellular proteins that modulate cellular interactions with ECM during embryo- RESULTS. Average IOPs of TSP1-null and TSP2-null mice were genesis and in adult tissues that continue to undergo 10% and 7% less than that of the corresponding WT mice, remodeling.6 The family includes SPARC (secreted protein, respectively. CCTs were 6.5% less in TSP1-null mice (P < acidic and rich in cysteine), thrombospondin-1 (TSP1) and 0.05) and 1.1% less in TSP2-null mice (P > 0.05). TSP2, tenascin C and X, SC1/hevin, and osteopontin. A number Fluorophotometric measurements suggest that aqueous of these matricellular proteins are widely expressed in ocular turnover rates in TSP1-null and TSP2-null mice are greater tissues including cornea, lens, retina, vitreous, aqueous, and than those of WT mice. LM of the TSP1-null and TSP2-null TM, playing specific roles in each tissue.7–15 iridocorneal angles reveals morphology, which is indistin- TSP1 and TSP2 are matricellular proteins that have been guishable from that of their corresponding WTs. IF revealed shown to regulate cytoskeleton, cell adhesion, and ECM possible concurrent underexpression of TSP2 in TSP1-null remodeling.7 Both TSP1 and TSP2 are found in the TM; TSP1 mice and of TSP1 in TSP2-null mice. TEM revealed larger is expressed throughout the TM, with a predominance in the collagen fibril diameters in TSP1-null and TSP2-null mice juxtacanalicular connective tissue (JCT) region, whereas TSP2 compared with WTs. is more concentrated in the uveal meshwork.14 TSP1 has been implicated in the pathogenesis of glaucoma because its CONCLUSIONS. TSP1-null and TSP2-null mice have lower IOPs than their WT counterparts. The rate of aqueous turnover expression is increased in one third of patients with primary open-angle glaucoma (POAG).16 Consistent with this correla- suggests that the mechanism is enhanced outflow facility. An tion are the findings that TSP1 expression is increased in situations of fibrosis, wound healing, and other circumstances in which there is significant matrix production.14,17,18 From the 1Department of Ophthalmology, Massachusetts Eye The creation of mice deficient in various matricellular and Ear Infirmary, Harvard Medical School, Boston, Massachusetts; proteins has also helped investigate their potential role in IOP 2 the Department of Ophthalmology, Kyung Hee University Hospital regulation.6 We have previously shown that the prototypical at Gangdong, College of Medicine, Kyung Hee University, Seoul, 3 matricellular protein, SPARC, is highly expressed throughout Korea; and the Department of Ophthalmology, Boston University 12 School of Medicine, Boston, Massachusetts. the TM and particularly within the JCT region. SPARC is one 15 Supported in part by National Eye Institute Grants EY of the most highly expressed genes by TM cells at rest and 019654-01 and EY 014104 (Massachusetts Eye and Ear Infirmary following mechanical stretch.20 SPARC-null mice exhibit lower Core Grant), Fight for Sight (RIH), Research to Prevent Blindness, IOPs compared with that of their wild-type counterparts, Massachusetts Lions Eye Research Foundation, and American which is the result of enhanced aqueous outflow and not the Glaucoma Society. result of an artifact of central corneal thickness (CCT).19 Submitted for publication November 3, 2011; revised July 31 The TSP1-null mouse phenotype includes reduced dermal and August 16, 2012; accepted August 16, 2012. matrix, thoracic kyphosis, hyperplasia of various epithelial Disclosure: ,None; ,None; , R.I. Haddadin D.-J. Oh M.H. Kang cells, and pulmonary inflammation. Culture of TSP1-null cell None; G. Villarreal Jr,None;J.-H. Kang,None;R. Jin,None;H. 21 Gong,None;D.J. Rhee,None isolates revealed one eighth the amount of active TGF-b1. Corresponding author: Douglas J. Rhee, Department of Oph- The TSP2-null mouse phenotype includes fragile skin, lax thalmology, Massachusetts Eye and Ear Infirmary, 243 Charles Street; tendons, abnormal collagen fibrils, accelerated skin wound Boston, MA 02114; [email protected]. healing, increased bone density, and a bleeding diasthesis.22–25 Investigative Ophthalmology & Visual Science, September 2012, Vol. 53, No. 10 6708 Copyright 2012 The Association for Research in Vision and Ophthalmology, Inc. Downloaded from jov.arvojournals.org on 10/02/2021 IOVS, September 2012, Vol. 53, No. 10 TSP1-Null and TSP2-Null Mice Have Lower IOPs 6709 Many of these findings, however, are limited to specific tissues, Medical Supply). IOP measurements were taken between 11:00 AM and the effects of TSP1 and TSP2 in the TM have yet to be and 3:00 PM. determined. Given our findings in SPARC-null mice, we hypothesized that TSP1 and TSP2 participate in the regulation Measurement of Central Corneal Thickness of IOP. We tested our hypothesis by comparing the IOP, CCT, and aqueous fluorescein dye clearance of TSP1-null, TSP2-null, In humans, Goldmann applanation tonometry may be affected by and their corresponding wild-type (WT) mice. We subsequent- central corneal thickness (CCT). To assess whether this confounder is ly investigated any differences evident by light microscopy contributing to any observed IOP difference, CCTs of WT and TSP-null (LM), immunofluorescence (IF), and transmission electron mice were measured using optical coherence tomography (OCT). As microscopy (TEM). we have previously reported, mice were imaged with a Stratus OCT (Carl Zeiss Meditec, Inc., Dublin, CA) under anesthesia, and the process of locating the central corneal reflex was repeated three times MATERIALS AND METHODS to produce three different images.19 CCT was obtained using the internal software (version 4.0.7; Carl Zeiss Meditec, Inc.) by measuring Animal Husbandry the peak-to-peak amplitude distance. All experiments were performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Assessment of Aqueous Humor Turnover TSP1-null and TSP2-null mice were previously generated,25,26 and each To investigate the mechanism of any IOP difference observed between strain was compared with its own background strain. TSP1-null mice WT and TSP-null mice, we noninvasively measured aqueous humor were backcrossed onto a C57BL/6 background for at least eight turnover using a previously published fluorophotometric tech- generations; and the WT strain used was C57BL/6. TSP2-null mice nique.19,31 In brief, all measurements were made between 11:00 AM were created from a C57BL/6 3 129/SvJ background, which was the and 3:00 PM to reduce potential variability related to diurnal variation WT strain used. All four strains were obtained from The Jackson of aqueous inflow or outflow. After anesthetizing each mouse with the Laboratory (Bar Harbor, ME). Each strain of mice was bred same anesthesia mixture used for IOP measurement, 10 lL of 0.02% independently and genotyped to confirm homozygosity. Briefly, DNA benzalkonium chloride (BAC) in saline was applied to the right eye to was isolated using a commercial kit including proteinase, and DNA permeabilize the cornea to fluorescein.32 After 5 minutes, the BAC purification reagents (Qiagen, Germantown, MD). PCR was performed solution was blotted at the lid margin without contacting the corneal with 40 cycles at the following temperatures: denaturation at 958Cfor epithelium and 10 lL of 0.02% fluorescein in saline was applied to the 30 seconds, annealing at 608C for 30 seconds, and polymerization at eye for 5 minutes. The eye and lids were then carefully washed with 728C for 1 minute, with an additional 5-minute extension for 600 lL of saline. The microscope was focused to a depth intermediate polymerization at the end of the last cycle. The amplified DNA to the iris and cornea, and images were captured in 10-minute intervals products were analyzed on 1.5% of agarose gel. The primer sequences thereafter for 1 hour (AxioCam ICC 1 camera and Stemi SV11 used for genotyping of TSP1-null and TSP2-null mice were obtained microscope, equipped with a GFP filter; Carl Zeiss Meditec, Inc.), online from The Jackson Laboratory (www.jax.org). Electron micros- using acquisition software (AxioVision, Release 4.6.3; Carl Zeiss copy and immunofluorescence were performed at a later date using Meditec, Inc.).
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