Collagen cross-linking impact on keratoconus extracellular matrix

Abstract

BackgroundKeratoconus (KC) is a common multifactorial ectatic corneal disease with unknown onset. KC most commonly appears in adolescence and affects approximately 1:400 people worldwide. Treatment options, for advanced KC cases, are collagen cross-linking (CXL) and corneal transplants. CXL is a new KC treatment that helps arrest the disease. Unfortunately, only a fraction of KC patients will qualify for CXL treatment. Our goal, in this study, was to begin to understand how CXL affects the corneal microenvironment and pave the way towards a more patient-driven CXL treatment.MethodsPrimary human corneal fibroblasts from healthy and KC donors were plated on transwell polycarbonate membranes and stimulated by a stable vitamin C. At 4 weeks, riboflavin was added followed by UVA irradiation. Transmission Electron Microscopy (TEM) and western blots were used to assess the effect of CXL on the extracellular matrix (ECM) and the resident cells, pre- and post CXL.ResultsData shows CXL improved lamellar organization showing more organized collagen fibrils decorated with proteoglycans (PGs). The distribution of the collagen fibrils and interfibrillar spacing was also visibly improved, post-CXL. Lumican, mimecan, and decorin were the dominant PGs and were significantly upregulated in post-CXL cultures. ECM degradation proteins, matrix metalloproteinases (MMPs), MMP-1, -3, and -9, but not MMP-2, were significantly downregulated post-CXL. TIMP-1 and -2 were not modulated by CXL.ConclusionThe unknown effects of CXL on the human corneal microenvironment have hampered our ability to make CXL available to all KC patients. Our current study provides a deeper understanding on CXL activity, using our unique 3D in vitro model.

Effect of the retention ring-assisted continuous application of riboflavin in pulsed-light accelerated corneal collagen cross-linking on the progression of keratoconus

“…When it is combined with ultraviolet A (UV-A) of 365-nm wavelength, oxygen radicals are produced via photochemical reaction. Oxygen radicals can introduce interfibrillar and intrafibrillar covalent bonds among collagen fibrils [ 2 , 3 ]. Such chemical bonds can increase the corneal rigidity and its resistance to enzymatic degradation of keratoconic cornea [ 4 ], thereby restoring the stability of the cornea and halting the progression of keratoconus.…”

Conjunctival Ultraviolet Autofluorescence as a Measure of Riboflavin and Ultraviolet and Accelerated Cross-Linking Exposure in Keratoconic Patients

“…The dermatological reports reveal that UV-induced changes in the skin tissue structure may be reversible if excessive sun exposure is limited, whereas iterative exposure to UV light promotes accumulative defects [ 22 ]. The observed transient growth of CUVAF area after aCXL surgery can be facilitated by remodeling of collagen and changes in the intracellular content of proteins including cytokines and matrix metalloproteinases [ 21 , 27 ].…”

Mechanisms of Collagen Crosslinking in Diabetes and Keratoconus

“…In terms of downstream effectors modulated by CXL, in vitro studies evaluating the effects of CXL on primary corneal stromal fibroblasts isolated from KC patients have shown that CXL downregulates canonical TGF-β signaling based on lower pSMAD2/3 expression post-CXL in KC [199]. A normalization of lysyl oxidase abundance [199], increased proteoglycan expression (e.g., lumican, mimecan, and decorin) [200], and a reduction in lactate levels in KC-derived corneal fibroblasts following CXL have been reported [200] suggesting that riboflavin/UV-A CXL may modulate ECM deposition and collagen crosslinking at the cellular level in addition to the direct structural modifications to collagen. Given a prominent role for oxidative stress in corneal dystrophies including KC [201], further studies are warranted to distinguish the potent antioxidant properties of riboflavin [202] on the KC phenotype independent of UV-A irradiation.…”