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Kanae Fukutsu, Kousuke Noda, Miyuki Murata, Shiho Yoshida, Atsuhiro Kanda, Susumu Ishida; Role of acrolein and ROCK1 in retinal glial cells. Invest. Ophthalmol. Vis. Sci. 2020;61(7):311.
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© ARVO (1962-2015); The Authors (2016-present)
Acrolein (ACR) is a highly reactive aldehyde that causes functional dysfunction of proteins. We previously reported that the level of acrolein-conjugated protein was elevated in the vitreous fluid and localized in glial cells of the fibrovascular tissues in patients with proliferative diabetic retinopathy (Murata M et al. Curr Eye Res. 2017). Furthermore, our in vitro assay revealed that ACR increased the migration rate of Müller glial cells; however, its detailed mechanism remains unclear. In the present study, we investigated the roles of ACR and Rho-associated, coiled-coil-containing protein kinase 1 (ROCK1), a widely-known kinase that controls cell migration, in retinal Müller glial cells.
Rat retinal Müller glial cell line, TR-MUL5, was incubated with ACR (5 to 50μM) and the specific ROCK inhibitor ripasudil (0.08 to 10μM) for 6 or 24 hours. The expression and production levels of ROCK1 were measured using real-time PCR, ELISA, and western blotting. Enzymatic activity of ROCK1 was assessed by the phosphorylation level of its downstream molecules, myosin-binding subunit of the myosin light chain phosphatase (MYPT1) and myosin light chain (MLC). Cell migration rate of TR-MUL5 exposed to ACR and ripasudil was measured using Oris cell migration assay.
Expression level of Rock1 mRNA increased 2.3±0.2 fold (25μM) and 7.9±1.0 fold (50μM) by ACR stimulation in TR-MUL5 (n=3, p<0.05). ELISA and western blotting also revealed the increase of ROCK1 protein in TR-MUL5 when stimulated with ACR (n=3, p<0.05). The phosphorylation level of MYPT1 and MLC increased by ACR(25μM) stimulation and ripasudil (0.4 to 2μM) suppressed it. Cell migration rate increased 2.4±0.1 fold in TR-MUL5 by ACR (25μM, n=4, p<0.01), which was abrogated by ripasudil (0.4 to 2.0μM, n=4, p<0.05).
The current study elucidated that ACR enhances the migration of retinal glial cells via induction of ROCK1.
This is a 2020 ARVO Annual Meeting abstract.
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