Coordinated Regulation of Extracellular Matrix Synthesis by the MicroRNA-29 Family in the Trabecular Meshwork

Primary open-angle glaucoma (POAG) is a chronic, degenerative optic neuropathy that represents one of the leading causes of blindness worldwide.1,2 In the United States, glaucoma is the leading cause of preventable blindness in persons of Latino or African descent.3,4 Despite modern treatment, the estimated risk of blindness from glaucoma over a 15- to 20-year period is between 7% and 24%.5,6 Even in patients with impaired vision, but not blindness, vision loss can have profound consequences on daily functioning. For example, the extent of visual field loss from glaucoma correlates to both an increased risk of a motor vehicle accident and a fall with injury.7,8 Recent studies suggest that the number of Americans with open-angle glaucoma will increase by 50% to 4.2 million by the year 2020.9 The progressive optic nerve damage seen in glaucoma results from an elevation of intraocular pressure (IOP) beyond the structural and vascular capabilities of the optic nerve to withstand. IOP is determined by the balance between aqueous inflow and outflow. In POAG, elevated IOP results from impaired aqueous outflow.10 Normally, between 80% and 90% of aqueous outflow occurs through the trabecular meshwork (TM), with the remaining 10% to 20% occurring through the ciliary body face.11,12 The TM is the critical tissue for the regulation of aqueous outflow, and its dysregulation causes elevated IOP.13 The tissue that includes the inner wall of Schlemm's canal and the juxtacanalicular (JCT) region of the TM is the anatomic location of highest outflow resistance.14 The JCT region is an amorphous and dynamic layer composed of endothelial cells and extracellular matrix (ECM). Alterations in the balance between ECM synthesis and breakdown influence aqueous outflow.15–19 Hence, a rigorous understanding of the molecular mechanisms involved in the control of ECM homeostasis is pivotal for the development of new therapies for POAG. The transforming growth factor-beta (TGF-β) family of cytokines functions as an important regulator of wound healing and ECM synthesis.20 One such isoform, TGF-β2, has been shown in several studies to be elevated in the aqueous humor of POAG patients.21–24 Tripathi et al.24 report total aqueous humor TGF-β2 levels (mean ± SD) to be 1.48 ± 0.68 ng/mL in healthy human subjects compared with 2.70 ± 0.76 ng/mL in age-matched POAG patients. In addition, the authors show that aqueous humor levels of intrinsically active TGF-β2 were elevated in POAG patients (0.45 ± 0.28 ng/mL) compared with age-matched healthy subjects (0.20 ± 0.24 ng/mL).24 Subsequent work has since demonstrated that TGF-β2 decreases aqueous humor outflow facility, which, in turn, elevates IOP.20,25,26 One possible mechanism for the decrease in outflow facility may involve the associated TGF-β2–mediated increase in ECM accumulation in the TM.20,25,26 Specifically, TGF-β2 has been reported to stimulate the expression of fibronectin, thrombospondin-1, laminin, collagen I, and collagen IV, key ECM components in the TM.26–29 MicroRNAs are small (∼22 nucleotides), single-stranded RNAs that modulate the posttranscriptional expression of genes.30 These short RNAs are predicted to regulate as many as 30% of human mRNA transcripts, with a given microRNA potentially binding up to 200 gene targets.31 Recent studies have suggested a critical involvement of microRNAs in the development of tissue fibrosis, with the microRNA-29 (miR-29) family serving as an important mediator of these effects.32–34 In nonocular human tissues, the miR-29 family regulates a number of ECM proteins, including SPARC (secreted protein, acidic, and rich in cysteine), laminin, collagen I, and collagen IV.32–37 Together, these data suggest that miR-29 may function as a node in the regulation of ECM homeostasis. The influence of microRNAs, and in particular the miR-29 family, in the development of elevated IOP remains unknown. To this end, we examined the importance of the miR-29 family in the TM, with a specific focus on its involvement in the regulation of ECM production under basal and TGF-β2 stimulatory conditions.