Changes in Gene Expression by Trabecular Meshwork Cells in Response to Mechanical Stretching Vasavi Vittal, Anastasia Rose, Kate E. Gregory, Mary J. Kelley, and Ted S. Acott PURPOSE. Trabecular meshwork (TM) cells appear to sense to 5% of people exhibit pathologic elevations in IOP with changes in intraocular pressure (IOP) as mechanical stretching. subsequent optic nerve damage, even at advanced ages.1,2 We In response, they make homeostatic corrections in the aqueous have hypothesized that TM cells can adjust outflow resistance humor outflow resistance, partially by increasing extracellular over a timescale of hours to days by modulating trabecular ECM matrix (ECM) turnover initiated by the matrix metalloprotein- turnover and subsequent biosynthetic replacement.3–6 Manip- ases. To understand this homeostatic adjustment process fur- ulation of the trabecular activity of a family of ECM turnover ther, studies were conducted to evaluate changes in TM gene enzymes, the matrix metalloproteinases (MMPs), reversibly expression that occur in response to mechanical stretching. modulates outflow facility.7 Inhibition of the endogenous ECM METHODS. Porcine TM cells were subjected to sustained me- turnover, which is initiated by these MMPs, increases the chanical stretching, and RNA was isolated after 12, 24, or 48 outflow resistance. Therefore, ongoing ECM turnover must be hours. Changes in gene expression were evaluated with mi- necessary for homeostatic maintenance of the IOP. In addition, croarrays containing approximately 8000 cDNAs. Select mRNA laser trabeculoplasty, a common treatment for glaucoma, ap- changes were then compared by quantitative reverse transcrip- pears to owe its efficacy to producing relatively sustained tion–polymerase chain reaction (qRT-PCR). Western immuno- trabecular MMP elevations, particularly within the juxtacanal- blots were used to determine whether some of these changes icular region of the meshwork.8–10 were associated with changes in protein levels. Much of the aqueous humor outflow resistance appears to RESULTS. On the microarrays, 126 genes were significantly up- reside within the deepest portion of the TM.11 Consequently, regulated, and 29 genes were significantly downregulated at this resistance is functionally stretched like a diaphragm across one or more time points, according to very conservative sta- Schlemm’s canal. Alterations in the outflow resistance that are tistical and biological criteria. Of the genes that changed, sev- sufficient to affect IOP change the degree of stretching and eral ECM regulatory genes, cytoskeletal-regulatory genes, sig- distortion of the juxtacanalicular TM. A likely sensing mecha- nal-transduction genes, and stress-response genes were nism in TM cells, indicating that an increase or decrease in notable. These included several proteoglycans and matricellu- outflow resistance is needed, is that of alterations in mechan- lar ECM proteins composed of common repetitive binding domains. The results of analysis of mRNA changes in more than ical tension or stretching. This sensing could be mediated and transduced by integrin–ECM or similar types of interac- 20 selected genes by qRT-PCR supported the findings in the 12,13 microarray analysis. Western immunoblots of several proteins tions. We and others have found that trabecular cells can demonstrated protein level changes associated with changes in sense changes in IOP and mechanical stretching and produce 14–24 the level of mRNA. several distinctive responses. In support of our overall hypothesis of IOP homeostasis, trabecular cells respond to CONCLUSIONS. The expression of a variety of TM genes is signif- icantly affected by mechanical stretching. These include sev- pressure elevations in perfused organ culture or to mechanical eral ECM proteins that contain multiple binding sites and may stretching in cell culture by increasing MMP-2 (gelatinase A) serve organizational roles in the TM. Several proteins that and MMP-14 (membrane-type-1-MMP) activity or levels, while could contribute to the homeostatic modification of aque- dramatically reducing levels of their primary tissue inhibitor, 14 ous humor outflow resistance are also upregulated or down- TIMP-2. Increased trabecular MMP activity results in reduced 7 regulated. (Invest Ophthalmol Vis Sci. 2005;46:2857–2868) outflow resistance and thus should restore IOP to normal DOI:10.1167/iovs.05-0075 levels. Therefore, the components of a self-contained trabecu- lar IOP homeostasis mechanism are present and functional. In he mechanisms that provide normal IOP homeostasis are perfused anterior segment organ culture, increased flow rates Tonly partially understood. A relatively effective homeo- produce initial elevations of IOP, which return to normal over static mechanism must exist, because only approximately 2% several days, even with sustained increases in perfusion rate.14,23 An important component of this putative IOP homeostasis From the Casey Eye Institute, Oregon Health and Science Univer- mechanism is the nature of the changes in the ECM that adjust sity, Portland, Oregon. the outflow resistance. The ECM turnover process is initiated Supported by National Institutes of Health Grants EY003279, by secretion/activation of the MMPs, which partially degrades EY008247, and EY010572 and by grants from the Glaucoma Research select ECM proteins. This phase is followed by the removal of Foundation (San Francisco, CA), Research to Prevent Blindness, and proteolytic ECM fragments, presumably facilitated by TM cell Alcon Labs (Fort Worth, TX). Submitted for publication January 21, 2005; revised April 11, endocytosis. New materials must then be synthesized by TM 2005; accepted April 22, 2005. cells to replace degraded ECM components. To adjust the Disclosure: V. Vittal, None; A. Rose, None; K.E. Gregory, None; outflow resistance, changes in the amount, composition, or M.J. Kelley, None; T.S. Acott, Alcon Labs (F) organization of this ECM are likely to be instituted. This pro- The publication costs of this article were defrayed in part by page cess must occur within the active pathway of aqueous humor charge payment. This article must therefore be marked ”advertise- outflow without allowing excessive structural disorganization. ment“ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Corresponding author: Ted S. Acott, Casey Eye Institute (CERES), To begin unraveling the details of this complex process, mi- Oregon Health and Science University, 3375 SW Terwilliger, Portland, croarray analysis of TM gene expression changes occurring OR 97239-4197; [email protected]. after sustained mechanical stretching were conducted. Investigative Ophthalmology & Visual Science, August 2005, Vol. 46, No. 8 Copyright © Association for Research in Vision and Ophthalmology 2857 Downloaded from iovs.arvojournals.org on 09/29/2021 2858 Vittal et al. IOVS, August 2005, Vol. 46, No. 8 MATERIALS AND METHODS The two types of OHSU SMC microarray chips that were used had duplicate separated spottings of approximately 5700 or 8400 human Porcine eyes were obtained from Carlton Packing (Carlton, OR); pro- cDNA clones per slide. The data presented herein are from the 8400 teinase inhibitor cocktail, phenylmethylsulfonyl fluoride (PMSF), di- chips, but very similar results were obtained earlier with the 5700 thiothreitol (DTT), and horseradish peroxidase-conjugated secondary chips. The libraries used were prepared by the IMAGE consortium32 antibodies from Sigma-Aldrich (St. Louis, MO); a DNA quantitation and distributed in sequence-verified form by Invitrogen (Carlsbad, CA). reagent (PicoGreen) from Molecular Probes (Eugene, OR); fibronectin Amplified PCR products were printed onto microarray slides (Ultra- antibody from Transduction Laboratories-BD Biosciences (San Diego, GAPS; Corning Costar, Corning, NY) using microarray printing pins CA); tenascin C antibody from Santa Cruz Biotechnology (Santa Cruz, (TeleChem 16 CMP-3) and an array printer (Cartesian PixSys 5500 XL; CA); Dulbecco’s modified Eagle’s medium (DMEM), antibiotics, and Genomic Solutions, Irvine, CA). Twenty-three plant genes (Arabidop- antimycotics from Invitrogen-Gibco (Grand Island, NY); fetal bovine sis thaliana) spotted twice per chip, were used for standard reference serum from HyClone (Logan, UT); chemiluminescence detection kits providing both a positive and a negative control. In addition, 176 spots (Super Signal) from Pierce (Rockford, IL); Falcon cell culture inserts were left blank on each chip to provide another type of negative (PET track-etched 3-␮m pore membranes in six-well format) from BD control. Scanned images were analyzed by computer (ImaGene; Bio- Biosciences (Franklin Lakes, NJ); and RNA extraction kits (RNeasy) Discovery, Marina del Rey, CA) to determine relative fluorescence from Ambion (Austin, TX). intensity for each label; to identify spot locations, shapes, sizes, and anomalies; and to determine spot-specific background intensity for TM Cell Culture, Mechanical Stretching, each label. At each of the three time points (12, 24, and 48 hours), Treatments, and Extractions three totally independent experiments were conducted. Each sample 5,25–27 from each experiment was analyzed on two separate identical micro- Porcine TM cells were cultured as previously described. By chips. On each chip, each of the 8400 genes was spotted twice in passages 3 to 5, cells were plated at a density of approximately 90% separate regions. Consequently, at each of the three time points, there confluence onto cell culture insert membranes in six-well