Biologically active fibronectin fragments stimulate release of MCP-1 and catabolic cytokines from murine retinal pigment epithelium.
Biologically active fibronectin fragments stimulate release of MCP-1 and catabolic cytokines from murine retinal pigment epithelium.
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DOI：
10.1167/iovs.08-2495
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发表时间：
2009-06
影响因子：
4.4
通讯作者：
Nussenblatt RB
中科院分区：
文献类型：
--
作者：
Austin BA;Liu B;Li Z;Nussenblatt RB
在多项人类遗传学研究中，高温必需丝氨酸蛋白酶（HTRA1）被确定为年龄相关性黄斑变性的候选基因。迄今为止，尚无功能研究表明 HTRA1 引发眼组织异常的机制。在本研究中，作者重点关注 HTRA1 的底物纤连蛋白，因为纤连蛋白片段 (Fnfs) 会刺激其他与年龄相关的退行性疾病中的生化事件，这些事件类似于与年龄相关的黄斑变性 (AMD) 相关的变化。该研究的目的是确定 Fnfs 是否刺激小鼠视网膜色素上皮 (RPE) 释放促炎和分解代谢细胞因子。通过明胶交联琼脂糖色谱法从鼠血清中纯化纤连蛋白，随后用 α-胰凝乳蛋白酶进行酶消化。通过测量暴露于 Fnf 的小鼠脾细胞中 IL-6 和 TNF-α 的水平来验证 Fnfs 的生物活性。为了分析Fnfs对RPE的影响，通过ELISA测定RPE培养物上清液中的细胞因子和趋化因子水平。 IL-6 和 TNF-α 促炎细胞因子从原代小鼠脾细胞中释放，与 Fnf 治疗的剂量和时间成比例，表明纤连蛋白的 α-胰凝乳蛋白酶消化物具有生物活性。 Fnf 处理小鼠 RPE 细胞刺激释放微克和纳克水平的 IL-6、MMP-3、MMP-9 和 MCP-1，而在未处理的细胞中仅检测到皮克水平。 Fnfs 刺激鼠 RPE 细胞释放促炎细胞因子、基质金属蛋白酶和单核细胞趋化蛋白。这一观察结果表明，Fnfs 可能通过促进炎症、分解代谢和单核细胞化学吸引而导致眼部异常。
High-temperature requirement serine protease (HTRA1) was identified as a candidate age-related macular degeneration gene in multiple genetic studies in humans. To date, no functional studies have shown a mechanism for HTRA1 to instigate ocular tissue abnormalities. In the present study, the authors focused on a substrate of HTRA1, fibronectin, because fibronectin fragments (Fnfs) stimulate biochemical events in other age-related degenerative diseases that are analogous to changes associated with age-related macular degeneration (AMD). The purpose of the study was to determine whether Fnfs stimulate the release of proinflammatory and catabolic cytokines from murine retinal pigment epithelium (RPE). Fibronectin was purified from murine serum by gelatin cross-linked agarose chromatography and subsequently was enzymatically digested with α-chymotrypsin. The bioactivity of Fnfs was verified by measuring levels of IL-6 and TNF-α in Fnf-exposed murine splenocytes. To analyze the effect of Fnfs on RPE, cytokine and chemokine levels in RPE culture supernatants were assayed by ELISA. IL-6 and TNF-α proinflammatory cytokines were released from primary murine splenocytes in proportion to the dose and length of Fnf treatment, indicating that α-chymotryptic digests of fibronectin are biologically active. Fnf treatment of murine RPE cells stimulated the release of microgram and nanogram levels of IL-6, MMP-3, MMP-9, and MCP-1, whereas only picogram levels were detected in untreated cells. Fnfs stimulate the release of proinflammatory cytokines, matrix metalloproteinases, and monocyte chemoattractant protein from murine RPE cells. This observation indicated that Fnfs could contribute to ocular abnormalities by promoting inflammation, catabolism, and monocyte chemoattraction.
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