Abstract
purpose. Lysyl oxidase (LOX) cross-links the side chain of collagen and elastin and thereby contributes to extracellular matrix (ECM) integrity. ECM remodeling is seen in various ocular diseases. Until now, there have been no reports on the LOX enzyme’s activity in ocular tissues. The purpose of this study was to estimate LOX activity and expression in human donor ocular tissues and to measure the specific activity of LOX in the vitreous of proliferative diabetic retinopathy (PDR) and rhegmatogenous retinal detachment (RRD).
method. Human donor eyeballs obtained from an eye bank were used to study tissue distribution of LOX. Human vitreous specimens were obtained during vitreoretinal surgery from PDR (n = 16) and RRD (n = 10). LOX activity was estimated by N-acetyl-3,7-dihydroxyphenoxazine assay, immunohistochemistry, and real-time polymerase chain reaction (RT-PCR). Matrix metalloprotease (MMP)-2 and -9 were quantified in the vitreous by sandwich enzyme-linked immunosorbent assay (ELISA).
results. The specific activity of LOX in ocular tissues was on the order of vitreous, iris ciliary body, lens, choroid RPE, and retina, which were comparable by mRNA expression and immunolocalization. The vitreous level of LOX activity decreased significantly in PDR and RRD, with an increase in total MMP-2 and -9 levels compared with normal donor vitreous.
conclusions. LOX activity showed a statistically significant decrease in the vitreous of PDR and RRD relative to control specimens. This effect can contribute to the inadequate collagen cross-linking that causes the ECM changes that occur in these diseases.
The extracellular matrix (ECM) represents a heterogeneous group of macromolecules, including collagen, noncollagenous glycoproteins, elastic fibers, and proteoglycans. The ECM is under constant remodeling by simultaneous degradation and synthesis of matrix components with different turnover rates. Various disease processes, such as inflammatory reactions and neovascularization, occur in the ECM.
1 The structural integrity of ECM depends on the collagen and elastin cross-links. During the formation of intermolecular cross-links, collagen fibers become increasingly insoluble and refractory to the action of enzymes and show a progressive increase in tensile strength,
2 which is essential for normal connective tissue function and wound healing.
Lysyl oxidase (LOX; EC 1.4.3.13) is a copper-dependent amine oxidase that initiates the covalent cross-linking of collagen and elastin in ECM. It is secreted as a glycosylated proenzyme with a molecular weight (Mw) of 50,000, which is proteolytically processed by procollagen C proteinase (bone morphogenic protein-1) into a mature, biologically active Mw 32,000 form.
3 4 Isoforms of LOX, called LOX-like proteins (LOXL)—namely, LOXL, -2, -3, and -4—have been identified, which are fully functional but genetically distinct.
5 Each member of the LOX protein family is characterized by a highly conserved amino acid sequence at their C-terminal end that includes the copper-binding site, residues for carbonyl cofactor formation, and the cytokine receptor–like domain. The conserved C-terminal domains contribute to amine oxidase activity, and the unique N-terminal domains may determine individual functions. Both LOX and LOXL catalyze the oxidative deamination of lysine residues in collagen and elastin.
6 β-Aminopropionitrile (BAPN) is a potent irreversible inhibitor of LOX that binds covalently to the active site of LOX, with an inhibitory constant (
K i) of 3 to 5 μM.
7 BAPN specifically inhibits all LOX isoenzymes except LOXL2.
8 BAPN has been studied for its therapeutic implications as a LOX inhibitor in liver fibrosis
9 and lung fibrosis
10 in animal models.
LOX and LOXL are colocalized in the skin, aorta, heart, lung, liver, cartilage, kidney, stomach, small intestine, colon, retina, ovary, testis, and brain in mouse tissues.
11 There are very few reports showing amine oxidase activity in bovine retina and sclera,
12 including a report on the presence of human retina–specific amine oxidase in the retinal ganglion cell layer.
13 However, very little information is available on LOX in human ocular tissues, in both normal and pathologic conditions.
In vitreoretinal diseases such as proliferative diabetic retinopathy (PDR) and rhegmatogenous retinal detachment (RRD), there is extensive ECM remodeling.
14 PDR is a common complication of diabetes mellitus characterized by preretinal neovascularization and development of epiretinal fibrovascular traction and retinal detachment.
15 RRD is a complex wound-healing pathobiology of proliferative vitreoretinopathy (PVR).
16 Wound healing in PVR in general, involves inflammation, extracellular matrix deposition and tissue remodeling. The matrix metalloproteinases (MMPs), a ubiquitous family of enzymes, are known to play a role in the degradation of the ECM.
17 MMP 2 (72-kDa gelatinase) is constitutively found in normal human vitreous, where it is complexed with TIMP-2
18 and MMP 9 (92-kDa gelatinase) is constitutively expressed in the retinal ganglion cell layer.
19 These MMPs degrade denatured collagen (gelatin) and type IV collagen. Although many MMPs have been reported in retinal disease,
14 characteristic changes in the levels of MMP-2 and -9 activity attributing to ECM remodeling have been reported in both PDR
20 21 22 and RRD.
23 24 However, there are no reports on the LOX activity in the vitreous during ECM remodeling.
Hence, in the present study, we determined the ocular tissue distribution of LOX in human donor eye balls. We estimated LOX activity in the vitreous of PDR and RRD, in light of the changes seen in the levels of MMP-2 and -9.
All experiments involving human subjects adhered to the tenets of the Declaration of Helsinki. Human donor eye balls obtained from the CU Shah Eye Bank (Sankara Nethralaya, India), were used after light microscopic examination and cornea removal (age 65 ± 9, years; seven male; three female), for determining specific activity of LOX (n = 10), real-time gene expression, and immunohistochemistry (n = 3). Donors with a history of diabetes, hypertension, carcinoma, and sepsis were not included in the study. The donors had no history of ocular disease. All fine chemicals used were of laboratory grade unless specified.
The eyeball was dissected and iris, ciliary body, (iris and ciliary body were processed separately unlike in activity assay) retina, choroid along with RPE were separated within 2 hours of death. Total RNA was extracted from different ocular tissues by the guanidine isothiocyanate and chloroform method (TRI Reagent; Sigma-Aldrich, St. Louis, MO). All RNA samples were treated with DNase (TURBO; Applied Biosystems/Ambion, Austin, TX) to remove DNA contamination. For all samples, 1 μg of total RNA was used to synthesize first-strand cDNA (SuperScript II reverse transcriptase; Invitrogen, Carlsbad, CA) and random primers.
The present study was prompted by the lack of information on the enzyme LOX in ocular tissues. Collagen, the substrate for LOX is the most abundant connective tissue protein in vertebrates. The metabolism of collagen and its regulation are of vital interest in several clinically important diseases that are characterized by excessive matrix synthesis, degradation, or remodeling. However, there is little knowledge on the levels and distribution of LOX, an enzyme that mediates collagen cross-linking in ocular tissues. Imamura et al.
13 have reported human retina specific amine oxidase in human retina; and, recently, Hewitt et al.
30 have reported expression of LOXL1 mRNA and protein in human donor ocular tissue. In the present study, a detailed distribution profile of LOX activity in ocular tissues from normal donor eyeballs was obtained. This study revealed the presence of LOX in the various ocular tissues in terms of specific activity, localization, and mRNA expression. Vitreous showed the highest specific activity among the ocular tissues studied. This distribution profile of LOX was found to be similar to the collagen content in the ocular tissue reported by Siddiqi et al.
31 The variation in LOX distribution and activity could be reflective of the inherent differences in the collagen structure and composition in these tissues. Therefore, the present study helps in understanding the ocular tissues further with respect to the extent of collagen cross-linking activity. Retina reportedly had the lowest content of soluble and insoluble collagen, which we found to correlate with the lowest LOX expression.
Despite progress in the treatment of PDR and PVR, these vitreoretinal diseases continue to be major causes of visual impairment. Changes in the activity of MMP-2 and -9 have already been documented in both PVR and PDR. Although a low activity of MMP-2 is constitutively present in the vitreous, the MMP-9 activity is reported to be increased in both PDR and PVR.
20 21 22 23 24 The ratio of total MMP-9 to -2 as determined in this study
(Fig. 4)shows that it is similar with that reported by Abu El-Asrar et al.,
23 which was highest in PDR followed by RRD.
Although the depredatory enzyme MMP has been detected in the vitreous, fibrovascular, and neovascular membranes, LOX may also play a crucial role in the ECM changes, this possibility has not been looked into. This study shows that there is a significant decrease in the level of LOX-specific activity that is possibly associated with the ECM remodeling. This is the first report showing a significant decrease in LOX-specific activity in the vitreous of PDR and RRD.
In PDR, immature collagen cross-linking is reported due to the nonenzymatic glycosylation of collagen as sugars can compete for the same lysine and hydroxylysine residues that serve as substrates for LOX, and there could be substrate unavailability.
32 It has been reported that glucose inhibits collagen fibril formation in vitro.
33 The degree of decreased collagen fibril formation has been shown to correlate with the loss of ability of collagen to serve as a substrate for LOX.
33 Despite collagen turnover, the collagen fibrils may not be adequately cross-linked, owing to the decreased LOX activity as observed in the study. However, additional work is needed to determine the relative roles of nonenzymatic glycosylation versus decreased LOX activity, with respect to immature collagen cross-linking in PDR.
Decreased LOX activity was also observed in the vitreous of patients with RRD, compared with control subjects. The decrease in LOX activity in RRD is intriguing, as these patients were nondiabetic. LOX expression has been shown to be regulated by transforming growth factor β,
34 tumor necrosis factor (TNF)-α,
35 platelet-derived growth factor retinoic acid, and fibroblast growth factor.
6 Endothelial dysfunction induced by TNF-α has been shown to be associated with a decrease in LOX expression as studied in human umbilical vein endothelial cells and porcine aortic endothelial cells.
36
Notably Pischon et al.
37 have also found that TNF-α inhibits the expression and activity of LOX in osteoblasts cultures but does not inhibit collagen synthesis, thereby contributing to perturbed collagen cross-linking and accumulation.
37 TNF-α is reported to be increased in the vitreous of PDR and RRD. It is produced locally as an inflammatory response.
38 39 Although PVR and PDR have different causes and clinical characteristics, retinal membranes from both conditions share the features of fibroplasia, excessive matrix protein deposition, and cellular infiltration. The decrease in LOX could be due to growth factors that are common in both PDR and RRD. However, more studies are needed to aid in understanding the regulation of LOX by growth factors in these diseases.
The only other report showing the relevance of LOX in ocular disease comes from a recent study conducted in a LOXL1-knockout mouse model in which there is defective elastin fiber that has been associated with increased susceptibility to laser induced choroidal neovascularization.
40 In the present study, we report that there was decreased LOX activity in the vitreous of eyes with PDR and RRD as part of the altered ECM activity. We hypothesize that the decrease in LOX activity contributes to inadequate or improper collagen cross-linking and increased proteolysis due to elevated levels of MMPs causing a net vitreous degradation leading to liquefaction. For a deeper understanding of the role of LOX, additional structure–function studies have to be performed at the level of collagen in the vitreoretinal diseases such as PDR and RRD.
Supported by the Department of Biotechnology, Government of India Project No. BT/PR4853/BRB/10/358/2004.
Submitted for publication December 4, 2007; revised March 13 and May 4, 2008; accepted September 2, 2008.
Disclosure:
K. Coral, None;
N. Angayarkanni, None;
J. Madhavan, None;
M. Bharathselvi, None;
S. Ramakrishnan, None;
K. Nandi, None;
P. Rishi, None;
N. Kasinathan, None;
S. Krishnakumar, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “
advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Narayanasamy Angayarkanni, Biochemistry Research Department, Sankara Nethralaya, Vision Research Foundation, 18, College Road, Chennai, 600 006;
[email protected].
Table 1. Clinical Details of Patients with PDR
Table 1. Clinical Details of Patients with PDR
Subject | Age (y)/Sex | Stage of the Disease | Duration of Diabetes (y) | Patent Vessels | Vitreous Hemorrhage | Retinal Detachment |
1 | 55/M | Advanced with macular hole | 10 | FVP over retina; active | − | No |
2 | 63/M | Advanced | 30 | FVP over retina; active | + | Combined RD |
3 | 48/M | Advanced with macular hole | 13 | FVP over retina; active | + | No |
4 | 34/M | Active | 10 | No | + | Tractional band |
5 | 67/M | Active | 30 | No | + | No |
6 | 53/M | Active | 24 | FVP over retina; active | + | No |
7 | 64/M | Active | 6 | FVP over retina; active | + | No |
8 | 37/M | Advanced | 7 | FVP present | − | Tractional RD |
9 | 54/M | Inactive | 15 | No | − | No |
10 | 65/F | Active | 10 | FVP over retina; active | + | No |
11 | 65/M | Active | 5 | No | − | Tractional RD |
12 | 48/M | Advanced | 20 | No | + | RD |
13 | 64/M | Advanced | 3 | FVP over retina; active | + | Combined RD |
14 | 52/M | Active | 15 | FVP over disc | − | Tractional RD |
15 | 39/M | Advanced | 6 | FVP over retina; active | + | No |
16 | 68/M | Active | 15 | FVP present; active | + | No |
Table 2. Summary of Patient Profile with RRD
Table 2. Summary of Patient Profile with RRD
Subject | Age (y)/Sex | PVR Grade | Quadrants Involved | Macula Detached | Vitreous Hemorrhage | Complications after Surgery | Resurgery |
1 | 56/M | Not done | 4 | Yes | + | Nil | No |
2 | 38/M | Not done | 4 | Yes | − | Nil | No |
3 | 50/M | Not done | 3 | Yes | − | Preretinal hemorrhage | Had phaco IOL + SOR |
4 | 22/M | Not done | 4 | Yes | − | Nil | No |
5 | 20/M | C | 4 | Yes | − | Nil | No |
6 | 52/M | A | 4 | Yes | − | Nil | No |
7 | 56/F | Not done | 1 | No | − | Nil | No |
8 | 57/M | A | 4 | Yes | − | Nil | No |
9 | 72/M | Not done | 3 | Yes | + | Macular hole | No |
10 | 54/M | Not done | 3 | Yes | − | Redetachment | Yes |
Table 3. Specific Activity of LOX in Vitreous Specimens of PDR and RRD
Table 3. Specific Activity of LOX in Vitreous Specimens of PDR and RRD
Conditions | Protein (mg/mL) | Specific Activity of LOX (μmol/min/mg Protein) | BAPN Inhibited LOX (μmol/min/mg Protein) |
Control (n = 23) | 1.28 ± 0.14 | 0.675 ± 0.173 | 0.336 ± 0.067 |
PDR (n = 16) | 3.30 ± 0.46* | 0.197 ± 0.049, † | 0.114 ± 0.028, † |
| P = 0.002 | P = 0.014 | P = 0.005 |
RRD (n = 10) | 9.10 ± 2.76* | 0.164 ± 0.060* | 0.083 ± 0.030* |
| P = 0.020 | P = 0.010 | P = 0.002 |
The authors acknowledge Venil N. Sumantran for scientific inputs and Ms. Pushparaj Vaijayanthi for technical help.
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