Changes in Extracellular Matrix Gene and Protein Expressions in Human Trabecular Meshwork Cells in Response to Mechanical Fluid Flow Stimulation

Abstract

22Purpose 23 To investigate the changes in extracellular matrix (ECM) gene and protein expressions in 24 human trabecular meshwork (HTM) cells in response to mechanical fluid flow 25 stimulation. 26 Methods 27HTM cells were cultured on a glass plate and exposed to shear stress (0, 0.2, and 1.0 28 dyne/cm 2 ) for 12 hours. Changes in gene expressions were evaluated using microarray 29 analysis. The representative genes related to ECM metabolism underwent real-time 30 reverse-transcriptase polymerase chain reaction. Fibronectin (FN) and collagen (COL) IV 31 levels in the supernatant were evaluated using immunoassays. Rho-associated coiled-coil-32 containing protein kinase (ROCK) activity also was investigated. 33 Results 34After stimulation, transforming growth factor 2 mRNA levels were significantly (p < 35 0.01) lower than that of the static control (0 dyne/cm 2 for 12 hours). Matrix 36 metalloproteinase 2 mRNA levels were significantly (p < 0.05) higher than the static 37 control. COL type 1 alpha 2 mRNA, COL type 4 alpha 2 mRNA, and COL type 6 alpha 1 38 mRNA levels were significantly (p < 0.05, < 0.01, and < 0.05, respectively) higher than 3 39 the static control. The mean ± standard deviation FN levels (ng/mL) in the supernatant 40 after stimulation (0, 0.2, 1.0 dyne/cm 2 ) were 193.7 ± 7.6, 51.5 ± 21.8, and 34.9 ± 23.6, 41 respectively (p < 0.01). The FN and COL IV levels and ROCK activity were significantly 42 (p < 0.01 and < 0.05, respectively) lower than the static control. 43 Conclusions 44 Changes in gene and protein expressions related to ECM metabolism occurred in HTM 45 cells after stimulation. Specifically, the suppression of FN and COL IV production might 46 explain the importance of mechanical fluid flow stimulation on maintenance of the normal 47 aqueous humor outflow.48 49 50 Keywords: trabecular meshwork, shear stress, aqueous humor, extracellular matrix, 51 glaucoma, intraocular pressure 52 53 54 55 56 4 57 60 humor drains through the conventional outflow pathway [2].Therefore, normal function of 61 this outflow component is important to the IOP homeostasis and prevention of glaucoma 62 [3]. Increased aqueous outflow resistance in this component is the main cause of glaucoma 63 accompanied by elevated IOP [4, 5]. 64 The trabecular meshwork (TM), juxtacanalicular meshwork, and Schlemm's canal, the 65 collector channels, and the episcleral veins comprise conventional outflow pathway. 66 Among those, extracellular matrix (ECM) in TM tissue, which are composed of collagen 67 (COL) or fibronectin (FN) [6], is critical for the homeostatic maintenance of the normal 68 outflow resistance [7]. Of note, recent studies revealed that ECM turnover is regulated by 69 mechanical stress, at least in part [8-10]. 70 Experimentally, mechanical stretching to TM cells upregulates gelatinase A and 71 membrane type-1 matrix metalloproteinase (MMP) and reciprocally downregulates tissue 72 inhibitors of metalloproteinases (TIMP)-2 [11]. Additionally, mechanical stretching by 73 pulsatile IOP decreases outflow fac...