Simon Trusen , Julia Sarah Alexandra Zimmermann , Fabian Norbert Fries , Zhen Li , Ning Chai , Berthold Seitz , Shweta Suiwal , Maryam Amini , Nóra Szentmáry , Tanja Stachon
{"title":"人类虹膜睫状体成纤维细胞更易受到氧化应激的影响。","authors":"Simon Trusen , Julia Sarah Alexandra Zimmermann , Fabian Norbert Fries , Zhen Li , Ning Chai , Berthold Seitz , Shweta Suiwal , Maryam Amini , Nóra Szentmáry , Tanja Stachon","doi":"10.1016/j.exer.2024.110105","DOIUrl":null,"url":null,"abstract":"<div><div>Aniridia-associated keratopathy originates from a haploinsufficiency of the transcription factor PAX6 (PAX6<sup>+/−</sup>). In the corneal epithelium of PAX6<sup>+/−</sup> mice, a significant increase in oxidized proteins was observed, accompanied by impaired compensation for elevated oxidative stress (OS). The extent to which limbal fibroblast cells (LFCs) are affected by an increased susceptibility to OS in cases of congenital aniridia (AN) has not been determined, yet. Our aim was to examine the impact of OS on antioxidant enzyme expression in normal and AN-LFCs. Following isolation and culture of primary LFCs (n = 8) and AN-LFCs (n = 8), cells were treated with cobalt chloride for 48 h to chemically induce hypoxic conditions and OS. Subsequently, HIF-1α/-2α, PHD1/2, Nrf2, CAT, SOD1, PRDX6, and GPX1 gene expression was examined by qPCR. SOD1, PRDX6, and GPX1 protein levels were assessed from the cell lysate by Western blot. The induction of hypoxia led to reduced HIF-1α gene expression in both fibroblast groups (<em>p</em><em>≤</em> <em>0.</em>008), while the decrease in PHD1 was limited to AN-LFCs (<em>p</em> = 0.0007). On the other hand, under hypoxic conditions, PHD2 showed higher mRNA expression in AN-LFCs compared to normal LFCs (<em>p</em> = 0.013). As a result of OS, the mRNA levels of Nrf2 (<em>p</em><em><</em>0.0001) and the antioxidant enzymes CAT (<em>p</em> = 0.005), SOD1 (<em>p</em> = 0.005), GPX1 (<em>p</em> = 0.002) decreased in AN-LFCs. This was accompanied by an increased protein expression of SOD1 (<em>p</em> = 0.019) and PRDX6 (<em>p</em><em>=</em>0.0009). In the normal LFC group, the induced extent of OS had no impact on the gene (<em>p</em><em>≥</em>0.151) and protein expression (<em>p</em> ≥ 0.629) of antioxidant enzymes, except for the GPX1 mRNA level (<em>p</em> = 0.027). AN-LFCs exhibit higher susceptibility to OS than normal LFCs. Therefore, in AN-LFCs, there are sustained alterations in gene and protein expression of antioxidative enzymes even after 48 h of CoCl<sub>2</sub> treatment.</div></div>","PeriodicalId":12177,"journal":{"name":"Experimental eye research","volume":"248 ","pages":"Article 110105"},"PeriodicalIF":3.0000,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Increased susceptibility of human limbal aniridia fibroblasts to oxidative stress\",\"authors\":\"Simon Trusen , Julia Sarah Alexandra Zimmermann , Fabian Norbert Fries , Zhen Li , Ning Chai , Berthold Seitz , Shweta Suiwal , Maryam Amini , Nóra Szentmáry , Tanja Stachon\",\"doi\":\"10.1016/j.exer.2024.110105\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><div>Aniridia-associated keratopathy originates from a haploinsufficiency of the transcription factor PAX6 (PAX6<sup>+/−</sup>). In the corneal epithelium of PAX6<sup>+/−</sup> mice, a significant increase in oxidized proteins was observed, accompanied by impaired compensation for elevated oxidative stress (OS). The extent to which limbal fibroblast cells (LFCs) are affected by an increased susceptibility to OS in cases of congenital aniridia (AN) has not been determined, yet. Our aim was to examine the impact of OS on antioxidant enzyme expression in normal and AN-LFCs. Following isolation and culture of primary LFCs (n = 8) and AN-LFCs (n = 8), cells were treated with cobalt chloride for 48 h to chemically induce hypoxic conditions and OS. Subsequently, HIF-1α/-2α, PHD1/2, Nrf2, CAT, SOD1, PRDX6, and GPX1 gene expression was examined by qPCR. SOD1, PRDX6, and GPX1 protein levels were assessed from the cell lysate by Western blot. The induction of hypoxia led to reduced HIF-1α gene expression in both fibroblast groups (<em>p</em><em>≤</em> <em>0.</em>008), while the decrease in PHD1 was limited to AN-LFCs (<em>p</em> = 0.0007). On the other hand, under hypoxic conditions, PHD2 showed higher mRNA expression in AN-LFCs compared to normal LFCs (<em>p</em> = 0.013). As a result of OS, the mRNA levels of Nrf2 (<em>p</em><em><</em>0.0001) and the antioxidant enzymes CAT (<em>p</em> = 0.005), SOD1 (<em>p</em> = 0.005), GPX1 (<em>p</em> = 0.002) decreased in AN-LFCs. This was accompanied by an increased protein expression of SOD1 (<em>p</em> = 0.019) and PRDX6 (<em>p</em><em>=</em>0.0009). In the normal LFC group, the induced extent of OS had no impact on the gene (<em>p</em><em>≥</em>0.151) and protein expression (<em>p</em> ≥ 0.629) of antioxidant enzymes, except for the GPX1 mRNA level (<em>p</em> = 0.027). AN-LFCs exhibit higher susceptibility to OS than normal LFCs. Therefore, in AN-LFCs, there are sustained alterations in gene and protein expression of antioxidative enzymes even after 48 h of CoCl<sub>2</sub> treatment.</div></div>\",\"PeriodicalId\":12177,\"journal\":{\"name\":\"Experimental eye research\",\"volume\":\"248 \",\"pages\":\"Article 110105\"},\"PeriodicalIF\":3.0000,\"publicationDate\":\"2024-09-19\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Experimental eye research\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0014483524003270\",\"RegionNum\":2,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"OPHTHALMOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Experimental eye research","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0014483524003270","RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"OPHTHALMOLOGY","Score":null,"Total":0}
Increased susceptibility of human limbal aniridia fibroblasts to oxidative stress
Aniridia-associated keratopathy originates from a haploinsufficiency of the transcription factor PAX6 (PAX6+/−). In the corneal epithelium of PAX6+/− mice, a significant increase in oxidized proteins was observed, accompanied by impaired compensation for elevated oxidative stress (OS). The extent to which limbal fibroblast cells (LFCs) are affected by an increased susceptibility to OS in cases of congenital aniridia (AN) has not been determined, yet. Our aim was to examine the impact of OS on antioxidant enzyme expression in normal and AN-LFCs. Following isolation and culture of primary LFCs (n = 8) and AN-LFCs (n = 8), cells were treated with cobalt chloride for 48 h to chemically induce hypoxic conditions and OS. Subsequently, HIF-1α/-2α, PHD1/2, Nrf2, CAT, SOD1, PRDX6, and GPX1 gene expression was examined by qPCR. SOD1, PRDX6, and GPX1 protein levels were assessed from the cell lysate by Western blot. The induction of hypoxia led to reduced HIF-1α gene expression in both fibroblast groups (p≤0.008), while the decrease in PHD1 was limited to AN-LFCs (p = 0.0007). On the other hand, under hypoxic conditions, PHD2 showed higher mRNA expression in AN-LFCs compared to normal LFCs (p = 0.013). As a result of OS, the mRNA levels of Nrf2 (p<0.0001) and the antioxidant enzymes CAT (p = 0.005), SOD1 (p = 0.005), GPX1 (p = 0.002) decreased in AN-LFCs. This was accompanied by an increased protein expression of SOD1 (p = 0.019) and PRDX6 (p=0.0009). In the normal LFC group, the induced extent of OS had no impact on the gene (p≥0.151) and protein expression (p ≥ 0.629) of antioxidant enzymes, except for the GPX1 mRNA level (p = 0.027). AN-LFCs exhibit higher susceptibility to OS than normal LFCs. Therefore, in AN-LFCs, there are sustained alterations in gene and protein expression of antioxidative enzymes even after 48 h of CoCl2 treatment.
期刊介绍:
The primary goal of Experimental Eye Research is to publish original research papers on all aspects of experimental biology of the eye and ocular tissues that seek to define the mechanisms of normal function and/or disease. Studies of ocular tissues that encompass the disciplines of cell biology, developmental biology, genetics, molecular biology, physiology, biochemistry, biophysics, immunology or microbiology are most welcomed. Manuscripts that are purely clinical or in a surgical area of ophthalmology are not appropriate for submission to Experimental Eye Research and if received will be returned without review.