There is increasing evidence that biomechanical properties of the trabecular meshwork (TM) are altered in glaucoma. Last et al1 reported that the mean elastic modulus (eg, a measure of tissue stiffness) was significantly increased in the glaucomatous human TM when compared with the age-matched control human TM. They suggested that a change in the physical properties of the TM might directly modulate aqueous humor (AH) outflow resistance and intraocular pressure (IOP). In addition, the authors presented a mathematical model to demonstrate that increased stiffness of an elastic porous membrane (eg, the TM) results in a corresponding increase in outflow resistance.
The term “cross-linking” (CXL) is used to describe the formation of chemical bridges between proteins or other molecules. CXL of extracellular matrix (ECM) proteins change the biomechanical properties of the ECM. CXL in connective tissue can occur during aging, as a side effect of diabetes mellitus and in abnormal fibrosis. CXL of ECM proteins prevents proteolytic breakdown and thus results in decreased ECM turnover, excess ECM accumulation, and tissue stiffness. Major families of CXL enzymes include transglutaminases and lysyl oxidases.
TRANSGLUTAMINASE FAMILY OF CXL ENZYMES
The transglutaminase (TGM) family of CXL enzymes contains 8 members of which tissue transglutaminase (TGM2) is the most ubiquitous and extensively studied.2 TGM2 is a calcium-dependent enzyme involved in specific posttranslational modifications by CXL ECM proteins. TGM2 modifies ECM proteins by CXL epsilon-(gamma-glutamyl) lysine or (gamma-glutamyl) polyamine bonds.3 TGM2 protein and/or enzyme activity is upregulated in a variety of diseases resulting in enhanced accumulation of cross-linked ECM proteins.4 Substrates of TGM2 associated with the TM include fibronectin, collagen, laminin, and elastin. TGM2 enzyme CXL activity has been implicated as a causative factor for many fibrotic diseases including pulmonary fibrosis, liver fibrosis, renal fibrosis, and atherosclerosis.
Cultured human TM cells and TM tissues express and secrete TGM2, and treatment with transforming growth factor beta 2 (TGF-β2) further induces TGM2 expression.5,6 Our laboratory further reported elevated TGM2 expression in both isolated glaucomatous TM cells and glaucomatous tissues.6 In addition, we demonstrated increased TGM2 enzymatic activity in glaucomatous TM cells and tissues.6These results suggest that elevated TGF-β2 levels in the AH and TM lead to an increase in TGM2-mediated CXL of ECM proteins in the TM. A review of TGM2 function and relevance in ocular diseases is available.7
LYSYL OXIDASE (LOX) FAMILY OF CXL ENZYMES
LOX is 1 of 5 LOX family members (LOX and LOXL-1-4) (Fig.1). LOX was initially reported to be expressed and secreted only by fibrogenic cells but is now known to be expressed in several other cell types. LOX oxidizes peptidyl lysine and hydroxylysine residues in collagen and lysine residues in elastin to produce peptidyl alpha-aminoadipic-delta-semialdehydes.9 These aldehyde modifications can spontaneously combine with vicinal peptidyl aldehydes or with epsilon-amino groups of peptidyl lysine to form covalent CXLs that stabilize and cause collagen and elastin fibers to be insoluble in the ECM. LOX is synthesized as a 50 kDa preprotein containing 3 domains: the N-terminal signal peptide sequence, the N-terminal propeptide domain, and the C-terminal catalytic domain.10 The signal peptide is cleaved and the propeptide domain is N-glycosylated in the endoplasmic reticulum and Golgi apparatus yielding a proenzyme, which is then secreted from cells as a catalytically inactive protein. The 32 kDa active enzyme (C-terminal domain) is released by proteolytic cleavage of the propeptide by procollagen C-proteinase (bone morphogenetic protein 1; BMP-1)11 (Fig. 1). LOX and LOXL enzymes are associated with several abnormalities related to an imbalance in ECM synthesis and/or degradation including fibrotic disorders of the heart (myocardial fibrosis), vasculature (atherosclerosis), lungs (pulmonary fibrosis), skin (hypertrophic scarring), kidney (diabetic nephropathy), and liver (liver fibrosis).10,11
We have demonstrated that human TM cells express all members of the LOX family. Significantly, with respect to primary open angle glaucoma, both mRNA and protein levels are induced by exogenous TGF-β2.12 In addition, we reported that TGF-β2 induction of LOX utilized both canonical (eg, receptor-regulated SMAD) and noncanonical signaling pathways. These results suggest that complex regulation of CXL enzymes occurs in the TM. We have also authored a review of LOX function and relevance in ocular diseases.13
LOXL-1 AND EXFOLIATION GLAUCOMA
An indication that an alteration in expression or function of CXL enzymes in the TM is related to glaucoma comes from studies of exfoliation syndrome (XFS) and exfoliation glaucoma. The systemic disease XFS presents with significant eye involvement.14,15 Picht et al,16 reported that TGF-β2 was not elevated in the AH of PXG patients. However, TGF-β1 and TGF-β3 levels are elevated in the AH of XFS and exfoliation glaucoma patients.17
Single nucleotide polymorphisms (SNPs) of the LOXL-1 gene are strongly associated risk factors for XFS. Microfibers coated with amorphous material (eg, exfoliation deposits) coat various structures including the TM.15 The LOXL-1 enzyme is necessary for tropoelastin CXL and elastic fiber formation, maintenance, and remodeling.18,19 At specific sites for elastic fiber formation, the inactive precursor form of LOXL-1 (eg, pro-LOXL-1) binds to both fibulin-5 and tropoelastin and targets the formation of elastic microfibrils. At this site, a scaffold is built using fibrillins and microfibril-associated glycoproteins that aid in the alignment of tropoelastin CXL domains. Significantly, following LOXL-1 proform binding to the scaffold, BMP-1 (eg, procollagen C-terminal proteinase) cleaves the proform resulting in LOXL-1 enzymatic activation. As a result of LOXL-1 activation, lysine residues are covalently cross-linked and elastin fibers become highly resistant to degradation or turnover.19 The SNPs associated with XFS are located in exon 1 that codes for an N-terminal domain of the proform of LOXL-1.20 It is predicted that this domain is involved in LOXL-1 activation and binding to the scaffold. However, it is not clear how a polymorphism in this region may affect enzyme function and subsequent exfoliation material production.
BMP-1 (PROCOLLAGEN C-PROTEINASE)
BMP-1 was erroneously included as a member of the BMP subfamily but has no homology to other BMP proteins.21 In actuality, BMP-1 is a zinc protease that converts secreted precursor proproteins into mature, functional proteins. Both LOX and LOXL-1 are substrates for BMP-1 (Fig. 1).22
Because BMP-1 is involved in the activation of ECM protein CXL enzymes, the regulation of BMP-1 expression and biological activity may be important in understanding TM stiffness and AH outflow resistance. We recently reported that TM cells and tissues express BMP-1 mRNA and protein and that BMP-1 protein is induced by TGF-β2.23 Using a LOX activity assay, we also demonstrated that secreted BMP-1 from human TM cells was biologically active and increased secreted LOX enzyme activity. Significantly, we showed that glaucomatous TM cells secreted higher levels of BMP-1 and that BMP-1 secretion in glaucomatous TM cells was further induced by exogenous TGF-β2 treatment compared with normal TM cells.23
PROPOSED MECHANISM OF LOX AND LOXL-1 IN GLAUCOMA
As exogenous TGF-β2 and gremlin increase CXL enzyme expression in cultured human TM cells, we have suggested that in primary open angle glaucoma, elevated levels of TGF-β and gremlin in the TM increases expression and secretion of TGM2, LOX, and BMP-1. The presence of BMP-1 cleaves the precursor proforms of LOX and LOXL-1 to yield enzymatically active molecules. Subsequent covalent CXL of collagen/elastic fibers increases stiffness of the TM that may result in AH outflow resistance and elevated IOP (Fig. 2). It should be noted that the molecular mechanism for activation of TGM2 has not been clearly defined and warrants examination in the human TM cell. As all preliminary data have been obtained by cell culture experiments, future studies using animals and/or in situ models are needed to verify that overexpression of CXL enzymes in the TM leads to increased TM stiffness, increased AH outflow resistance, and elevated IOP.
1. Last JA, Pan T, Ding Y, et al.. Elastic modulus determination of normal and glaucomatous human trabecular meshwork. Invest Ophthamol Vis Sci. 2011; 52:2147–2152.
2. Collighan RJ, Griffin M. Transglutaminase 2 cross-linking of matrix proteins: biological significance and medical applications. Amino Acids. 2008; 36:659–670.
3. Belkin AM. Extracellular TG2: emerging functions and regulation. FEBS J. 2011; 278:4704–4716.
4. Ricotta M, Iannuzzi M, De Vivo G, et al.. Physio-pathological roles of transglutaminase-catalyzed reactions. World. J Biol Chem. 2010; 1:181–187.
5. Welge-Lussen U, May A, Lutjen-Drecoll E. Induction of tissue transglutaminase in the trabecular meshwork by TGF-β1 and TGF-β2. Invest Ophthamol Vis Sci. 2000; 41:2229–2238.
6. Tovar-Vidales T, Roque R, Clark AF, et al.. Tissue transglutaminase expression and activity in normal and glaucomatous human trabecular meshwork cells and tissues. Invest Ophthamol Vis Sci. 2008; 49:622–628.
7. Tovar-Vidales T, Clark F, Wordinger RJ. Focus on molecules: transglutaminase 2. Exp Eye Res. 2011; 93:2–3.
8. Baker HE, Cox TR, Erler JT. The rationale for targeting the LOX family in cancer. Nat Rev Cancer. 2012; 12:540–552.
9. Smith-Mungo L, Kagan HM. Lysyl oxidase: properties, regulation and multiple functions in biology. Matrix Biol. 1998; 16:387–398.
10. Csiszar K. Lysyl oxidases: a novel multifunctional amine oxidase family. Prog Nucleic Acid Res Mol Biol. 2001; 70:1–32.
11. Kagan HM, Li W. Lysyl oxidase: properties, specificity and biological roles inside and outside the cell. J Cell Biochem. 2003; 88:660–672.
12. Sethi A, Mao W, Wordinger RJ, et al.. Transforming growth factor-beta induces extracellular matrix protein cross-linking lysyl oxidase (LOX) genes in human trabecular meshwork cells. Invest Ophthamol Vis Sci. 2011; 52:5240–5250.
13. Sethi A, Wordinger RJ, Clark AF. Focus on molecules: lysyl oxidase. Exp Eye Res. 2012; 104:97–98.
14. Ritch R, Schlotzer-Schrehardt U. Exfoliation syndrome
. Surv Ophthamol. 2001; 45:265–315.
15. Schlotzer-Schrehardt U, Naumann GO. Ocular and systemic pseudoexfoliation syndrome. Am J Ophthamol. 2006; 141:921–937.
16. Picht P, Welge-Lussen U, Grehn F, et al.. Transforming growth factor beta 2 in the aqueous humor in different types of glaucoma
and the relation to filtering bleb development. Graefes Arch Clin Exp Ophthamol. 2001; 239:199–207.
17. Schlotzer-Schrehardt U, Zenkel M, Küchle M, et al.. Role of transforming growth factor-beta1 and its latent form binding protein in pseudoexfoliation syndrome. Exp Eye Res. 2001; 73:765–780.
18. Decitre M, Gleyzal C, Raccurt M, et al.. Lysyl oxidase-like protein localizes to sites of de novo fibrin genesis in fibrosis and in the early stromal reaction of ductal breast carcinomas. Lab Invest. 1998; 78:143–151.
19. Liu X, Zhao Y, Gao J, et al.. Elastic fiber homeostasis requires lysyl oxidase-like 1 protein. Nat Genet. 2004; 36:178–182.
20. Elhawy E, Kamthan G, Dong CQ, et al.. Pseudoexfoliation syndrome, a systemic disorder with ocular manifestations. Hum Genomics. 2012; 6:22–32.
21. Bragdon B, Moseychuk O, Saldanha S, et al.. Bone morphogenetic proteins: a critical review. Cell Signal. 2011; 23:609–620.
22. Hopkins DR, Keles K, Greenspan DS. The bone morphogenetic protein 1/Tolloid-like metalloproteinases. Matrix Biol. 2007; 26:508–523.
23. Tovar-Vidales T, Fitzgerald AM, Clark AF, et al.. Transforming growth factor-β2 induces expression of biologically active bone morphogenetic protein-1 in human trabecular meshwork cells. Invest Ophthamol Vis Sci. 2013; 54:4741–4748.