Programmed necrosis, not apoptosis, is a key mediator of cell loss and DAMP-mediated inflammation in dsRNA-induced retinal degeneration.

There is no known treatment for the dry form of an age-related macular degeneration (AMD). Cell death and inflammation are important biological processes thought to have central role in AMD. Here we show that receptor-interacting protein (RIP) kinase mediates necrosis and enhances inflammation in a mouse model of retinal degeneration induced by dsRNA, a component of drusen in AMD. In contrast to photoreceptor-induced apoptosis, subretinal injection of the dsRNA analog poly(I : C) caused necrosis of the retinal pigment epithelium (RPE), as well as macrophage infiltration into the outer retinas. In Rip3(-/-) mice, both necrosis and inflammation were prevented, providing substantial protection against poly(I : C)-induced retinal degeneration. Moreover, after poly(I : C) injection, Rip3(-/-) mice displayed decreased levels of pro-inflammatory cytokines (such as TNF-α and IL-6) in the retina, and attenuated intravitreal release of high-mobility group box-1 (HMGB1), a major damage-associated molecular pattern (DAMP). In vitro, poly(I : C)-induced necrosis were inhibited in Rip3-deficient RPE cells, which in turn suppressed HMGB1 release and dampened TNF-α and IL-6 induction evoked by necrotic supernatants. On the other hand, Rip3 deficiency did not modulate directly TNF-α and IL-6 production after poly(I : C) stimulation in RPE cells or macrophages. Therefore, programmed necrosis is crucial in dsRNA-induced retinal degeneration and may promote inflammation by regulating the release of intracellular DAMPs, suggesting novel therapeutic targets for diseases such as AMD.

Different profiles of Ca2+ responses to endothelin-1 and PDGF in liver myofibroblasts during the process of cell differentiation.

BACKGROUND AND PURPOSE: Hepatic stellate cells play an important role in liver fibrosis but little is known about liver myofibroblasts located around the central vein and in the portal area. In this study, intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured to assess the response to endothelin-1 (ET-1), platelet derived growth factor (PDGF) and ATP in rat liver myofibroblasts. EXPERIMENTAL APPROACH: Rat liver myofibroblasts were compared in 'quiescent' (cultured on Matrigel-coated dishes) and 'activated' (cultured on non-coated plastic dishes) conditions. [Ca(2+)](i) was measured with the fluorescent dye fura-2 and mRNA for ET-1, PDGF and their receptors by RT-PCR. KEY RESULTS: ET-1 increased [Ca(2+)](i) in quiescent cells but not in activated cells, whereas PDGF-BB increased [Ca(2+)](i) in activated cells but not in quiescent cells. However, there was no difference between responses to ATP in quiescent or activated cells. ET-1 (in quiescent cells), PDGF-BB (in activated cells) and ATP (in both cells) all induced transient increases in [Ca(2+)](i) in the absence of extracellular Ca(2+) (with EGTA), indicating the involvement of Ca(2+) release from intracellular Ca(2+) stores. The sustained increase in [Ca(2+)](i) in the presence of external Ca(2+) in activated cells (ATP and PDGF) was significantly reduced by nicardipine, a L-type Ca(2+) channel blocker, but not in quiescent cells (ATP and ET-1). CONCLUSIONS AND IMPLICATIONS: The different pharmacological profiles of [Ca(2+)](i)-response in quiescent and activated myofibroblasts suggest that ET-1 and PDGF contribute differently to myofibroblast activation during the process of liver fibrosis.