The results from this study provide evidence that excess synthesis of basement membrane components, such as collagen type IV, and the subsequent thickening of the vascular basement membrane may play a far more critical role during the breakdown of blood retinal barrier as seen in diabetic retinopathy than so far suspected. To our knowledge, this is the first study that shows increased protein expression for both LOX and proLOX under HG condition in RRECs and in diabetic rat retinas and that the HG-induced increase in LOX activity could lead to altered cross-linking of collagen fibrils and contribute to excess permeability. Our data also show increased LOX expression in retinas of diabetic rats that supports a previous study reporting increased LOX-dependent cross-linking in skin collagen in diabetes (34
). Although the changes in dermal collagen reported in the earlier study are presumably types I and III, nevertheless, changes in LOX observed in this study suggest that similar biochemical alterations affect different collagen types present in various tissues in diabetes.
A study examining glomerular basement membrane collagen and LOX-mediated cross-links in experimental diabetes reported that the ratio of dihydroxylysinonorleucine to hydroxylysinonorleucine was increased, suggestive of altered cross-linking in diabetes (35
). The development of basement membrane thickening in glomerular and retinal capillaries, a histologic hallmark of diabetic microangiopathy, is associated with increased vascular leakage. In this context it seems quite paradoxical that thickened basement membranes are more permissive to permeance; currently a mechanism for this observation is unknown. Data presented here implicate increased LOX expression and activity as mediating, at least in part, increased permeability through retinal basement membranes. To date, only the expression of LOXL2 seems to be implicated in diabetes and specifically in diabetic nephropathy. Microarray analysis of human biopsy samples from patients with DN suggested that LOXL2 was among the HIF target genes that were found to be upregulated, though the increased expression was confirmed only by real-time PCR analysis (36
). Furthermore LOXL2 has been reported to have amine oxidase activity, but unlike its family members, its enzyme activity was apparently not inhibited by BAPN (30
). There are no reports that lysyl oxidase isoforms LOXL1, LOXL3, and LOXL4 are regulated by HG conditions or diabetes. LOXL1 has been reported to play a role in pseudoexfoliation glaucoma and primary open angle glaucoma. LOXL3 and LOX4 have been implicated in breast cancer invasion and head and neck squamous cell carcinoma (15
). ECM accumulation is now considered to play a more mechanistic role in the development of compromised barrier function. Recent observations point to fibrotic matrices as providing excess ligands for cell surface receptors such as integrins, that in turn modulate cell signaling responses and enhance local synthesis of proteolytic enzymes (13
). Similarly, upregulation of LOX in several invasive cancers has provided additional support for the concept that a fibrotic matrix can be more permeable than a normal matrix (39
). It is possible that elevation of LOX and consequent increased cross-linking may make fibrotic matrices more conducive for permeability of soluble molecules because of strains and resulting gaps between collagen fibrils. Detailed direct ultrastructural analyses of basement membrane integrity after increased LOX-mediated cross-linking may help us understand LOX dependent effects on basement membranes and increased permeability.
Although collagen fibrillar arrangements are stabilized by covalent cross-links, excessive cross-linking could contribute to disorganized assembly of the collagen fibrils. Electron microscopic investigation has revealed fine structural changes in the collagen fibrillar arrangement in diabetes (40
). These differences included increased packing density of collagen fibrils, decreases in fibrillar diameter, and abnormal fibril morphology showing collagen fibrils that appeared twisted, curved, overlapping, and otherwise highly disorganized, suggestive of excess cross-linking that is known to tighten collagen fibrils (40
). Diameter measurements on fibrils obtained during a time course of assembly have demonstrated that a fibril diameter distribution are dependent on late-stage assembly of fibrils (41
) that are in part regulated by LOX activity. Much is still unknown related to the pathways for the secretion and extracellular assembly of collagen molecules into fibrils and the processing enzymes required for converting the insoluble aggregates into mechanically and chemically stable components of the matrices. The principles governing the self-assembly of collagen fibrils are currently not well understood. Further studies are necessary to understand how cells regulate this process, to learn how the deposition of early collagen fibrils is orchestrated in the basement membrane, and to understand the role of other basement membrane components and their interactions in these processes. Thus, the identification of altered activity of a cross-linking enzyme, which is involved in basement membrane organization and ultrastructural assembly of collagen matrices, may provide new mechanistic insights into the relationship between extracellular matrix accumulation and excess vascular permeability in diabetes.
It is of interest that the LOX family of proteins is multifunctional. Although LOX activity, as already noted, is associated with increased metastatic behavior of tumors, the propeptide region of LOX has tumor-suppressor activity (42
). The propeptide region of LOX and LOXL1 are each unique in structure, whereas the pro domains of LOXL2 – LOXL4 contain conserved scavenger receptor cystein-rich domains (SRCR) that in other proteins mediate functional protein interactions (48
). The biologic activities of prodomains of LOX isoforms have not been explored in the context of vascular biology. The present study clearly identifies LOX expression itself, and LOX activity in particular, as being critical for its effects on endothelial barrier function, but additional activities of LOX and LOXL1-LOXL4 that are independent of enzyme activity may also contribute to its biological roles in vivo.
Morphologic abnormalities of retinal capillary basement membrane of diabetic individuals appear to reflect a poorly known process of structural remodeling. These structural abnormalities may be the result of excessive cross-linking represented by the thickened retinal capillary basement membrane, one of the prominent characteristics of diabetic retinopathy (5
). Although upregulation of basement membrane components such as fibronectin, collagen IV, and laminin in diabetes has been established, and recent studies indicate its contributory role to excess retinal vascular permeability (2
), the exact biochemical changes that modify matrix and promote excess permeability are unclear. However, it is clear that increased vascular permeability requires the passage of solutes through two layers inherent in capillaries, the cellular layer and the ECM layer (basement membrane). Although studies have implicated breakdown of tight junctions and increased vacuolar transport to excess permeability (51
), these changes represent abnormalities of the cellular layer. Other mechanisms such as nonenzymatic glycation could also contribute to and potentiate excess permeability (52
). An increase in collagen solubility under elevated glucose concentrations may lead to unbalanced biosynthesis and processing of collagen precursors (54
). This study provides novel information related to the ECM in which biochemical changes of the matrix components may render it more permeable in diabetes.
In this study we observed a modest upregulation of LOX expression by VEGF in retinal endothelial cells. It is, therefore, conceivable that VEGF may partially mediate the regulation of LOX under diabetic conditions, but this requires further study. It is of interest that hypoxia is an upregulator of both VEGF and LOX (55
), and it seems likely that hypoxia-mediated LOX upregulation directly or indirectly increases diabetic microvascular retinal permeability. Increased levels of VEGF and thickening of the vascular basement through upregulation of ECM protein expression in diabetes are among the most distinct characteristics of the disease. Such an increase in ECM expression could lead to retinal vascular basement membrane thickening, and in turn, contribute to increased permeability (49
In summary, our findings indicate that HG-induced increased LOX expression and activity is associated with retinal endothelial cell dysfunction and excess permeability. Successful application of siRNA and BAPN for inhibition of HG-induced upregulation of LOX expression and LOX activity with beneficial outcomes on barrier function opens up the option of reducing LOX overexpression and activity as a potential strategy for preventing increased permeability associated with diabetic retinopathy.