Long term high glucose exposure induces premature senescence in retinal endothelial cells.
Long term high glucose exposure induces premature senescence in retinal endothelial cells.
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DOI：
10.3389/fphys.2022.929118
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发表时间：
2022
影响因子：
4
通讯作者：
中科院分区：
文献类型：
--
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目的：糖尿病视网膜血管的细胞衰老特征已被描述。本研究旨在探讨高糖微环境对视网膜内皮细胞衰老过程的影响。 研究方法：人视网膜微血管内皮细胞分别在对照和5 mM和25 mM D-葡萄糖的高葡萄糖条件下培养。异构体l-葡萄糖用作渗透控制。使用CASY技术计数细胞，直到它们达到它们的海弗利克极限。衰老相关的β-半乳糖苷酶用于鉴定衰老细胞。通过克隆形成、3D管形成和屏障形成试验评价内皮细胞功能。使用Seahorse Bioanalyzer表征细胞代谢。通过批量RNA测序进行基因表达分析。评价来自db/db和db/+小鼠的视网膜组织是否存在衰老细胞。将来自Akimba和对照小鼠的视网膜的公开可用的scRNA测序数据用于基因集富集分析。 结果如下：与5 mM D-葡萄糖和渗透压对照相比，长期暴露于25 mM D-葡萄糖加速了人视网膜内皮细胞细胞衰老的建立。从4周开始，通过显著较慢的生长、较高的衰老相关β-半乳糖苷酶阳性细胞百分比、细胞大小增加以及较低的pRb和HMGB 2表达来显示这一点。这些衰老特征与克隆形成能力降低、小管形成减少和屏障功能受损有关。长期高糖培养的细胞表现出糖酵解减少，GLUT 1，GLUT 3和PFKFB 3的蛋白表达降低。培养4周后，与对照组相比，用25 mM D-葡萄糖培养的细胞中的转录组学分析鉴定了ALDOC、PFKL和TPI 1的下调。与db/+小鼠相比，db/db小鼠的视网膜显示出与中间和深层视网膜丛中血管密度显著降低相关的无细胞毛细血管显著增加。通过衰老相关β-半乳糖苷酶染色鉴定db/db视网膜血管系统内的衰老内皮细胞。Akimba小鼠视网膜的单细胞转录组学数据的分析突出了与对照小鼠相比时衰老和衰老相关的分泌表型基因特征的富集。 结论：25 mM D-葡萄糖的糖尿病样微环境足以加速人视网膜微血管内皮细胞中细胞衰老的建立。
Purpose: Features of cellular senescence have been described in diabetic retinal vasculature. The aim of this study was to investigate how the high glucose microenvironment impacts on the senescence program of retinal endothelial cells. Methods: Human retinal microvascular endothelial cells were cultured under control and high glucose conditions of 5 mM and 25 mM D-glucose, respectively. Isomeric l-glucose was used as the osmotic control. Cells were counted using CASY technology until they reached their Hayflick limit. Senescence-associated β-Galactosidase was used to identify senescent cells. Endothelial cell functionality was evaluated by the clonogenic, 3D tube formation, and barrier formation assays. Cell metabolism was characterized using the Seahorse Bioanalyzer. Gene expression analysis was performed by bulk RNA sequencing. Retinal tissues from db/db and db/+ mice were evaluated for the presence of senescent cells. Publicly available scRNA-sequencing data for retinas from Akimba and control mice was used for gene set enrichment analysis. Results: Long term exposure to 25 mM D-Glucose accelerated the establishment of cellular senescence in human retinal endothelial cells when compared to 5 mM D-glucose and osmotic controls. This was shown from 4 weeks, by a significant slower growth, higher percentages of cells positive for senescence-associated β-galactosidase, an increase in cell size, and lower expression of pRb and HMGB2. These senescence features were associated with decreased clonogenic capacity, diminished tubulogenicity, and impaired barrier function. Long term high glucose-cultured cells exhibited diminished glycolysis, with lower protein expression of GLUT1, GLUT3, and PFKFB3. Transcriptomic analysis, after 4 weeks of culture, identified downregulation of ALDOC, PFKL, and TPI1, in cells cultured with 25 mM D-glucose when compared to controls. The retina from db/db mice showed a significant increase in acellular capillaries associated with a significant decrease in vascular density in the intermediate and deep retinal plexuses, when compared to db/+ mice. Senescent endothelial cells within the db/db retinal vasculature were identified by senescence-associated β-galactosidase staining. Analysis of single cell transcriptomics data for the Akimba mouse retina highlighted an enrichment of senescence and senescence-associated secretory phenotype gene signatures when compared to control mice. Conclusion: A diabetic-like microenvironment of 25 mM D-glucose was sufficient to accelerate the establishment of cellular senescence in human retinal microvascular endothelial cells.
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影响因子：
64.5
作者：
López-Otín C;Blasco MA;Partridge L;Serrano M;Kroemer G
通讯作者：
Kroemer G
影响因子：
7.7
作者：
Chan M;Yuan H;Soifer I;Maile TM;Wang RY;Ireland A;O'Brien JJ;Goudeau J;Chan LJG;Vijay T;Freund A;Kenyon C;Bennett BD;McAllister FE;Kelley DR;Roy M;Cohen RL;Levinson AD;Botstein D;Hendrickson DG
通讯作者：
Hendrickson DG
影响因子：
3.9
作者：
Fang J;Chang X
通讯作者：
Chang X
影响因子：
29
作者：
Crespo-Garcia, Sergio;Tsuruda, Pamela R.;Sapieha, Przemyslaw
通讯作者：
Sapieha, Przemyslaw
DOI：
10.1152/ajpendo.00323.2005
发表时间：
2006-06-01
影响因子：
5.1
作者：
Ola, M. Shamsul;Berkich, Deborah A.;LaNoue, Kathryn F.
通讯作者：
LaNoue, Kathryn F.