Role of lncRNA MALAT1 in UVA-induced corneal endothelial senescence.
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ABSTRACT:
SUBMITTER: Qiao Y
PROVIDER: S-EPMC10363638 | biostudies-literature | 2023 Sep
REPOSITORIES: biostudies-literature
Publications
Role of lncRNA <i>MALAT1</i> in UVA-induced corneal endothelial senescence.
Qiao Yujie Y Zhang Bin B Wang Yani Y Sang Tian T Dou Shengqian S Dong Chunxiao C Wang Qun Q Zhou Qingjun Q
Genes & diseases 20230421 5
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Project description:Long noncoding RNAs (lncRNAs) have important roles in diverse biological processes. Our previous study has revealed that lncRNA-MALAT1 deregulation is implicated in the pathogenesis of diabetes-related microvascular disease, diabetic retinopathy (DR). However, the role of MALAT1 in retinal vasculature remodeling still remains elusive. Here we show that MALAT1 expression is significantly upregulated in the retinas of STZ-induced diabetic rats and db/db mice. MALAT1 knockdown could obviously ameliorate DR in vivo, as shown by pericyte loss, capillary degeneration, microvascular leakage, and retinal inflammation. Moreover, MALAT1 knockdown could regulate retinal endothelial cell proliferation, migration, and tube formation in vitro. The crosstalk between MALAT1 and p38 MAPK signaling pathway is involved in the regulation of endothelial cell function. MALAT1 upregulation represents a critical pathogenic mechanism for diabetes-induced microvascular dysfunction. Inhibition of MALAT1 may serve as a potential target for anti-angiogenic therapy for diabetes-related microvascular complications.
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Project description:Intervertebral degenerative disc disease (IDDD) is a common disease in clinic that causes pain and heavy financial burden on patients with poor prognosis. However, the pathogenesis of IDDD is not clear. Long non-coding RNA (LncRNA) is involved in regulating various body growth and pathological processes by affecting cell proliferation, differentiation, and apoptosis. However, the role of lncRNAs in IDDD is rarely reported. This study aims to investigate the role and mechanism of lncRNA MALAT1 in the development of IDDD. The nucleus pulposus of the intervertebral disc were collected and the primary nucleus pulposus cells were isolated and cultured. The cells were divided into three groups, including IDDD group, empty plasmid group transfected by pcDNA3.1, or MALAT1 group transfected by pcDNA3.1-MALAT1. MALAT1 expression was detected by real-time PCR. Cell proliferation was assessed by MTT assay. Caspase 3 activity was tested by the activity detection kit. IL-1 and IL-6 levels were analyzed by ELISA. The expression of MALAT1 in IDDD nucleus pulposus cells was significantly lower than that in control group (P < 0.05). The expression of MALAT1 was significantly increased after transfection with pcDNA3.1-MALAT1 plasmid in IDDD nucleus pulposus cells, which obviously inhibited cell proliferation, enhanced Caspase 3 activity, and promoted the secretion of IL-1 and IL-6 compared with IDDD group (P < 0.05). MALAT1 level decreased in IDDD nucleus pulposus cells. Upregulation of MALAT1 expression restrained IDDD through suppressing inflammation; inhibiting nucleus pulposus cell apoptosis, and promoting cell proliferation.
| S-EPMC6965762 | biostudies-literature
Project description:Intervertebral degenerative disc disease (IDDD) is a common disease in clinic that causes pain and heavy financial burden on patients with poor prognosis. However, the pathogenesis of IDDD is not clear. Long non-coding RNA (LncRNA) is involved in regulating various body growth and pathological processes by affecting cell proliferation, differentiation, and apoptosis. However, the role of lncRNAs in IDDD is rarely reported. This study aims to investigate the role and mechanism of lncRNA MALAT1 in the development of IDDD. The nucleus pulposus of the intervertebral disc were collected and the primary nucleus pulposus cells were isolated and cultured. The cells were divided into three groups, including IDDD group, empty plasmid group transfected by pcDNA3.1, or MALAT1 group transfected by pcDNA3.1-MALAT1. MALAT1 expression was detected by real-time PCR. Cell proliferation was assessed by MTT assay. Caspase 3 activity was tested by the activity detection kit. IL-1 and IL-6 levels were analyzed by ELISA. The expression of MALAT1 in IDDD nucleus pulposus cells was significantly lower than that in control group (P < 0.05). The expression of MALAT1 was significantly increased after transfection with pcDNA3.1-MALAT1 plasmid in IDDD nucleus pulposus cells, which obviously inhibited cell proliferation, enhanced Caspase 3 activity, and promoted the secretion of IL-1 and IL-6 compared with IDDD group (P < 0.05). MALAT1 level decreased in IDDD nucleus pulposus cells. Upregulation of MALAT1 expression restrained IDDD through suppressing inflammation; inhibiting nucleus pulposus cell apoptosis, and promoting cell proliferation.
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Project description:The human cornea is a tri-laminar structure composed of several cell types with substantial mitotic potential. Age-related changes in the cornea are associated with declining visual acuity and the onset of overt age-related corneal diseases. Corneal transplantation is commonly used to restore vision in patients with damaged or diseased corneas. However, the supply of donor tissue is limited, and thus there is considerable interest in the development of tissue-engineered alternatives. A major obstacle to these approaches is the short replicative lifespan of primary human corneal endothelial cells (HCEC). Accordingly, a comprehensive investigation of the signalling pathways and mechanisms underpinning proliferative lifespan and senescence in HCEC was undertaken. The effects of exogenous human telomerase reverse transcriptase expression, p53 knockdown, disruption of the pRb pathway by over-expression of CDK4 and reduced oxygen concentration on the lifespan of primary HCEC were evaluated. We provide proof-of-principle that forced expression of telomerase, when combined with either p53 knockdown or CDK4 over-expression, is sufficient to produce immortalized HCEC lines. The resultant cell lines express an HCEC-specific transcriptional fingerprint, and retain expression of the corneal endothelial temperature-sensitive potassium channel, suggesting that significant dedifferentiation does not occur as a result of these modes of immortalization. Exploiting these insights into proliferative lifespan barriers in HCEC will underpin the development of novel strategies for cell-based therapies in the human cornea.
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Project description:We have recently found that diabetes-induced premature senescence of retinal endothelial cells is accompanied by NOX2-NADPH oxidase-induced increases in the ureohydrolase enzyme arginase 1 (A1). Here, we used genetic strategies to determine the specific involvement of A1 in diabetes-induced endothelial cell senescence. We used A1 knockout mice and wild type mice that were rendered diabetic with streptozotocin and retinal endothelial cells (ECs) exposed to high glucose or transduced with adenovirus to overexpress A1 for these experiments. ABH [2(S)-Amino-6-boronohexanoic acid] was used to inhibit arginase activity. We used Western blotting, immunolabeling, quantitative PCR, and senescence associated β-galactosidase (SA β-Gal) activity to evaluate senescence. Analyses of retinal tissue extracts from diabetic mice showed significant increases in mRNA expression of the senescence-related proteins p16INK4a, p21, and p53 when compared with non-diabetic mice. SA β-Gal activity and p16INK4a immunoreactivity were also increased in retinal vessels from diabetic mice. A1 gene deletion or pharmacological inhibition protected against the induction of premature senescence. A1 overexpression or high glucose treatment increased SA β-Gal activity in cultured ECs. These results demonstrate that A1 is critically involved in diabetes-induced senescence of retinal ECs. Inhibition of arginase activity may therefore be an effective therapeutic strategy to alleviate diabetic retinopathy by preventing premature senescence.
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Project description:The corneal endothelium is critical for maintaining corneal clarity by mediating hydration through barrier and pump functions. Progressive loss of corneal endothelial cells during aging has been associated with the development of Fuchs endothelial corneal dystrophy (FECD), one of the main causes of cornea-related vision loss. The mechanisms underlying FECD development remain elusive. Single-cell RNA sequencing of isolated healthy human corneas discovered 4 subpopulations of corneal endothelial cells with distinctive signatures. Unsupervised clustering analysis uncovered nuclear enriched abundant transcript 1 (NEAT1), a long non-coding RNA (lncRNA), as the top expressed gene in the C0-endothelial subpopulation, but markedly downregulated in FECD. Consistent with human corneas, a UVA-induced mouse FECD model validated the loss of NEAT1 expression. Loss of NEAT1 function by an in vivo genetic approach reproduced the exacerbated phenotype of FECD by ablating corneal endothelial cells. Conversely, gain of function by a CRISPR-activated adenoviral delivery system protected corneas from UVA-induced FECD. Our findings provide novel mechanistic insights into the development of FECD, and targeting NEAT1 offers an attractive approach for treating FECD.
| S-EPMC8807987 | biostudies-literature
Project description:Senescence is a stress-responsive tumor suppressor mechanism associated with expression of the senescence-associated secretory phenotype (SASP). Through the SASP, senescent cells trigger their own immune-mediated elimination, which if evaded leads to tumorigenesis. Senescent parenchymal cells are separated from circulating immunocytes by the endothelium, which is targeted by microenvironmental signaling. Here we show that SASP induces endothelial cell NF-κB activity and that SASP-induced endothelial expression of the canonical NF-κB component Rela underpins senescence surveillance. Using human liver sinusoidal endothelial cells (LSECs), we show that SASP-induced endothelial NF-κB activity regulates a conserved transcriptional program supporting immunocyte recruitment. Furthermore, oncogenic hepatocyte senescence drives murine LSEC NF-κB activity in vivo. Critically, we show two distinct endothelial pathways in senescence surveillance. First, endothelial-specific loss of Rela prevents development of Stat1-expressing CD4+ T lymphocytes. Second, the SASP up-regulates ICOSLG on LSECs, with the ICOS-ICOSLG axis contributing to senescence cell clearance. Our results show that the endothelium is a nonautonomous SASP target and an organizing center for immune-mediated senescence surveillance.
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Project description:PurposeStress of the endoplasmic reticulum and oxidative stress play critical roles in the pathogenesis of Fuchs Endothelial Corneal Dystrophy (FECD). In the normal aging cornea, cellular stress has been associated with a loss in proliferative capacity (premature senescence) of corneal endothelial cells (CECs). The present study used a transgenic Col8a2(Q455K/Q455K) knock-in mouse model of early-onset FECD to identify the endothelial expression profile of specific cellular stress response-related targets, which may be relevant to late-onset FECD.MethodsThe differential endothelial mRNA levels of cellular stress response-related genes were determined in 12-month-old homozygous Col8a2(Q455K/Q455K) mutant and wild-type mice using customized PCR arrays. Result validation and analysis of additional senescence-related transcripts was performed by real-time PCR. Expression of p53 and p21 was assessed by immunofluorescence. Senescence-associated ?-galactosidase (SA-?-Gal) activity was investigated by histochemical labeling. Human FECD samples and normal controls were examined for p21 expression by immunohistochemistry.ResultsPCR-array analysis showed greater than 2-fold and/or significantly altered endothelial regulation of 19 cellular stress response-related transcripts in Col8a2(Q455K/Q455K) mutant mice; real-time PCR documented statistically significant upregulation of senescence-associated targets Cdkn1a (p21), Serpine1 (PAI-1), Tagln (Sm22), Fn1 and Clu (ApoJ). Immunofluorescence revealed increased expression of nuclear p53 and p21 in mutant animals. SA-?-Gal staining detected increased proportions of senescent CECs in mutant mice. Human FECD endothelium exhibited increased levels of nuclear p21 protein.ConclusionsOur results identify endothelial Cdkn1a (p21) upregulation in a mouse model of early-onset FECD, confirm overexpression of p21 in late-onset human FECD endothelium, and suggest a role for premature senescence in FECD.
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Project description:As a polyphenolic plant flavone, luteolin (Lut) is widely found in many medicinal plants, flowers, and vegetables. Although Lut has been shown to have the effect of preventing and treating skin photoaging, its role in preventing photoaging specifically induced by ultraviolet A (UVA) radiation remains underreported. In vivo, BALB/c mice were used as models for skin photoaging models and treated with Lut. Additionally, NIH-3T3 fibroblasts were utilized in vitro to further investigate whether Lut exerts its anti-photoaging effects by enhancing fibroblast vitality and function. Several biochemical assays (CCK-8, catalase, superoxide dismutase, malondialdehyde, dichloro-dihydro-fluorescein diacetate, quantitative real-time-PCR, gene expression patterns) and histochemical (histological staining, immunofluorescent staining, SA-β-Gal experiments, western blotting analysis) were conducted. The findings demonstrate that the Lut pretreatment could enhance the vitality and function of fibroblasts in both in vitro and in vivo experiments and inhibit UVA-induced collagen degradation, thereby improving the skin's resistance to photoaging. We confirmed that the Lut pretreatment inhibited the expression of UVA-induced senescent factors P21, P16, and pro-inflammatory senescence-associated secretory phenotype (SASP) factors. Additionally, Lut exhibited potent antioxidant effects during UVA exposure. Bioinformatics and network pharmacology analyses revealed that Lut's anti-photoaging effects may be mediated through the regulation of oxidative stress-related pathways and anti-aging genes. Upon utilizing inhibitors and agonists of oxidative stress, we further confirmed that Lut prevents UVA-induced fibroblast senescence by suppressing oxidative stress, and ultimately protects the skin from photoaging damage. These findings indicate that lutein mitigates photoaging caused by UVA-induced fibroblast senescence through the modulation of oxidative stress pathways.
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Project description:Corneal endothelial cells (CECs) do not proliferate or recover after illness or injury, resulting in decreased cell density and loss of pump/barrier function. Considering the shortage of donor cornea, it is vital to establish robust methods to generate CECs from induced pluripotent stem cells (iPSCs). We investigated the efficacy and safety of transplantation of iPSC-derived CECs into a corneal endothelial dysfunction (CED) rabbit model. iPSCs were generated from human fibroblasts. We characterized iPSCs by demonstrating the gene expression of the PSC markers OCT4, SOX2, TRA-1-60, and NANOG, teratoma formation, and differentiation into three germ layers. Differentiation of iPSCs into CECs was induced via neural crest cell (NCC) induction. CEC markers were detected using immunofluorescence and gene expression was analyzed using quantitative real-time PCR (qRT-PCR). After culturing iPSC-derived NCCs, we found the expression of zona occludens-1 (ZO-1) and Na+/K+ ATPase and a hexagonal morphology. ATP1A1, COL8A1, and AQP1 mRNA expression was higher in iPSC-derived CECs than in iPSCs and NCCs. We performed an injection of iPSC-derived CECs into the anterior chamber of a CED rabbit model and found improved levels of corneal transparency. We also found increased numbers of ZO-1- and ATP1A1-positive cells in rabbit corneas in the iPSC-derived CEC transplantation group. Usage of the coating material vitronectin (VTN) and fasudil resulted in good levels of CEC marker expression, demonstrated with Western blotting and immunocytochemistry. Combination of the VTN coating material and fasudil, instead of FNC mixture and Y27632, afforded the best results in terms of CEC differentiation's in vitro and in vivo efficacy. Successful transplantation of CEC-like cells into a CED animal model confirms the therapeutic efficacy of these cells, demonstrated by the restoration of corneal clarity. Our results suggest that iPSC-derived CECs can be a promising cellular resource for the treatment of CED.
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