Biologically active fibronectin fragments stimulate release of MCP-1 and catabolic cytokines from murine retinal pigment epithelium.

Age-related macular degeneration (AMD) is the most common cause of irreversible visual impairment in patients older than 50.1 As the baby boomer generation ages, the incidence of AMD in the United States is expected to increase to 3 million.2 Despite knowledge about the clinical and pathologic features of AMD, little is known about its underlying cause or causes. Previous attempts to identify an underlying cause for AMD have included genetic studies. More specifically, multiple groups have screened humans with AMD for evidence of restriction fragment length polymorphisms. High-temperature requirement serine protease (HTRA1) polymorphisms have been strongly associated with AMD in diverse human populations.3-30 In human eyes taken from persons with AMD, HTRA1 levels are upregulated in the macular region,6 yet no functional studies have mechanistically linked alterations in HTRA1 expression with ocular changes. Substrates of HTRA1 are extracellular matrix proteins, including fibronectin. Fibronectin is present in Bruch’s membrane, underlying the retinal pigment epithelium (RPE), where it anchors the RPE to the basal lamina through α5β1 integrins.31 Fibronectin is more abundantly expressed by the RPE of donors with AMD than of age-matched controls,32 and fibronectin accumulates in basal linear deposits in the macula of elderly eyes.33 Furthermore, fibronectin is abundant in drusen,34 defined as the abnormal accumulation of extracellular deposits in the basal aspect of the RPE, which is a risk factor for AMD.35,36 We were intrigued by the observation that recombinant HTRA1 can directly digest fibronectin to produce fibronectin fragments (Fnfs)37,38 and reasoned that the Fnfs could be a downstream protagonist of intraocular inflammation and catabolism. Fnfs have been studied extensively in age-related inflammatory diseases outside the eye. It is established that intact fibronectin is capable of binding to many different types of receptors on a diverse range of cells, including fibroblasts, epithelium, endothelium, neural crest, B and T lymphocytes, monocytes, and megakaryocytes. Furthermore, it is known that Fnfs are more potent stimulators of proinflammatory and catabolic cytokines than intact fibronectin. One of the best-studied inflammatory diseases of aging, in which Fnfs play a central role, is arthritis. Two well-studied examples are osteoarthritis and rheumatoid arthritis. Specifically, Fnfs accumulate in synovial fluid in arthritic joints.39-43 The accumulation of Fnfs causes pathology through multiple biochemical mechanisms, including increased release of matrix metalloproteinases44-49 and enhanced accumulation of proinflammatory cytokines, among them IL-6 and TNF-α.33,44,50,51 Given that AMD is also associated with increased levels of matrix metalloproteinases (MMPs) and proinflammatory cytokines, Fnfs may contribute to these changes. Investigators have recently started to consider that drusen accumulation may be secondary to RPE abnormalities involving chronic inflammation.52-57 Therefore, we designed our study to examine the impact of Fnfs on the secretion of proinflammatory and catabolic cytokines from the RPE by testing the hypothesis that exposure of murine RPE cells to high levels of Fnfs stimulates the RPE to release catabolic and proinflammatory cytokines. We show that Fnf treatment of murine RPE cells causes enhanced secretion of IL-6, MCP-1, MMP-3, and MMP-9.