Effects of Lycium barbarum polysaccharide on the photoinduced autophagy of retinal pigment epithelium cells

AIM: To investigate the relationship between autophagy and apoptosis in photoinduced injuries in retinal pigment epithelium (RPE) cells and how Lycium barbarum polysaccharide (LBP) contributes to the increased of RPE cells to photoinduced autophagy. METHODS: In vitro cultures of human RPE strains (ARPE-19) were prepared and randomly divided into the blank control, model, low-dose LBP, middle-dose LBP, high-dose LBP, and 3-methyladenine (3MA) groups. The viability of the RPE cells and apoptosis levels in each group were tested through cell counting kit-8 (CCK8) method with a flow cytometer (Annexin V/PI double staining technique). The expression levels of LC3II, LC3I, and P62 proteins were detected with the immunofluorescence method. The expression levels of beclin1, LC3, P62, PI3K, P-mTOR, mTOR, P-Akt, and Akt proteins were tested through Western blot. RESULTS: LBP considerably strengthens cell viability and inhibits the apoptosis of RPE cells after photoinduction. The PI3K/Akt/mTOR signal pathway is activated because of the upregulation of the phosphorylation levels of Akt and mTOR proteins, and thus autophagy is inhibited. CONCLUSION: LBP can inhibit the excessive autophagy in RPE cells by activating the PI3K/Akt/mTOR signaling pathways and thereby protect RPE cells from photoinduced injuries.

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The cytoskeletal elements of human retinal pigment epithelium: in vitro and in vivo

Journal of Cell Science ◽

10.1242/jcs.91.2.303 ◽

1988 ◽

Vol 91 (2) ◽

pp. 303-312

Author(s):

N.M. McKechnie ◽

M. Boulton ◽

H.L. Robey ◽

F.J. Savage ◽

I. Grierson

Keyword(s):

Retinal Pigment Epithelium ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Cytokeratin 18 ◽

Rpe Cells ◽

Human Retinal Pigment Epithelium ◽

Cytoskeletal Elements

The cytoskeletal elements of normal (in situ) and cultured human retinal pigment epithelium (RPE) were studied by a variety of immunocytochemical techniques. Primary antibodies to vimentin and cytokeratins were used. Positive immunoreactivity for vimentin was obtained with in situ and cultured material. The pattern of reactivity obtained with antisera and monoclonals to cytokeratins was more complex. Cytokeratin immunoreactivity could be demonstrated in situ and in cultured cells. The pattern of cytokeratin expression was similar to that of simple or glandular epithelia. A monoclonal antibody that specifically recognizes cytokeratin 18 identified a population of cultured RPE cells that had particularly well-defined filamentous networks within their cytoplasm. Freshly isolated RPE was cytokeratin 18 negative by immunofluorescence, but upon culture cytokeratin 18 positive cells were identifiable. Cytokeratin 18 positive cells were identified in all RPE cultures (other than early primaries), regardless of passage number, age or sex of the donor. In post-confluent cultures cytokeratin 18 cells were identified growing over cytokeratin 18 negative cells, suggesting an association of cytokeratin 18 immunoreactivity with cell proliferation. Immunofluorescence studies of retinal scar tissue from two individuals revealed the presence of numerous cytokeratin 18 positive cells. These findings indicate that RPE cells can be identified by their cytokeratin immunoreactivity and that the overt expression of cytokeratin 18 may be associated with proliferation of human RPE both in vitro and in vivo.

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JAM-C maintains VEGR2 expression to promote retinal pigment epithelium cell survival under oxidative stress

Thrombosis and Haemostasis ◽

10.1160/th16-11-0885 ◽

2017 ◽

Vol 117 (04) ◽

pp. 750-757

Author(s):

Xin Jia ◽

Chen Zhao ◽

Qishan Chen ◽

Yuxiang Du ◽

Lijuan Huang ◽

...

Keyword(s):

Oxidative Stress ◽

Retinal Pigment Epithelium ◽

Cell Survival ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Retinal Pigment Epithelium Cell ◽

Photoreceptor Cells ◽

Rpe Cells

SummaryJunctional adhesion molecule-C (JAM-C) has been shown to play critical roles during development and in immune responses. However, its role in adult eyes under oxidative stress remains poorly understood. Here, we report that JAM-C is abundantly expressed in adult mouse retinae and choroids in vivo and in cultured retinal pigment epithelium (RPE) and photoreceptor cells in vitro. Importantly, both JAM-C expression and its membrane localisation are downregulated by H2O2-induced oxidative stress. Under H2O2-induced oxidative stress, JAM-C is critically required for the survival of human RPE cells. Indeed, loss of JAM-C by siRNA knockdown decreased RPE cell survival. Mechanistically, we show that JAM-C is required to maintain VEGFR2 expression in RPE cells, and VEGFR2 plays an important role in keeping the RPE cells viable since overexpression of VEGFR2 partially restored impaired RPE survival caused by JAM-C knockdown and increased RPE survival. We further show that JAM-C regulates VEGFR2 expression and, in turn, modulates p38 phosphorylation. Together, our data demonstrate that JAM-C plays an important role in maintaining VEGR2 expression to promote RPE cell survival under oxidative stress. Given the vital importance of RPE in the eye, approaches that can modulate JAM-C expression may have therapeutic values in treating diseases with impaired RPE survival.

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A ROCK Inhibitor Promotes Graft Survival during Transplantation of iPS-Cell-Derived Retinal Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22063237 ◽

2021 ◽

Vol 22 (6) ◽

pp. 3237

Author(s):

Masaaki Ishida ◽

Sunao Sugita ◽

Kenichi Makabe ◽

Shota Fujii ◽

Yoko Futatsugi ◽

...

Keyword(s):

Retinal Pigment Epithelium ◽

Single Cell ◽

Cell Transplantation ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Cell Suspensions ◽

Rpe Cells ◽

Rpe Transplantation

Currently, retinal pigment epithelium (RPE) transplantation includes sheet and single-cell transplantation, the latter of which includes cell death and may be highly immunogenic, and there are some issues to be improved in single-cell transplantation. Y-27632 is an inhibitor of Rho-associated protein kinase (ROCK), the downstream kinase of Rho. We herein investigated the effect of Y-27632 in vitro on retinal pigment epithelium derived from induced pluripotent stem cells (iPS-RPE cells), and also its effects in vivo on the transplantation of iPS-RPE cell suspensions. As a result, the addition of Y-27632 in vitro showed suppression of apoptosis, promotion of cell adhesion, and higher proliferation and pigmentation of iPS-RPE cells. Y-27632 also increased the viability of the transplant without showing obvious retinal toxicity in human iPS-RPE transplantation into monkey subretinal space in vivo. Therefore, it is possible that ROCK inhibitors can improve the engraftment of iPS-RPE cell suspensions after transplantation.

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TRPM7 ameliorates the blue light-induced apoptosis of RPE cells involving PKC/ERK

10.21203/rs.2.24470/v1 ◽

2020 ◽

Author(s):

Luping Hu ◽

Guoxing Xu

Keyword(s):

Blue Light ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Protective Role ◽

Brdu Incorporation ◽

Mrna Levels ◽

Sprague Dawley ◽

Rpe Cells ◽

Expression Levels

Abstract Background Blue light triggers apoptosis of retinal pigment epithelium (RPE) cells and causes retinal damage. The aim of this study was to elucidate the protective role of TRPM7 in photo-damaged RPE cells. Methods RPE cells isolated from Sprague-Dawley (SD) rats were cultured in vitro , and exposed to varying intensities of blue light (500-5000 Lux). Cell proliferation and viability were respectively assessed by BrdU incorporation and MTT assays. Real-time PCR and Western blotting were used to analyze the mRNA and protein expression levels of TRPM7, PKC, ERK and Bax/Bcl-2. The cells were transfected with TRPM7 siRNA to knockdown its mRNA levels, or transduced with TRPM7–overexpressing lentiviruses. Pigment epithelium-derived factor (PEDF) was used in combination to detect the anti apoptosis effect. Results Blue light inhibited the proliferation and viability of RPE cells in an intensity-de pendent manner when compared to non-irradiated controls ( P <0.05). Compared to the control, photo-damaged RPE cells showed decreased levels of TRPM7, PKC, ERK and Bax, increased in Bcl-2 ( P <0.01) . Forced expression of TRPM7 partially ameliorated the reduction of proliferation and viability of RPE cells( P <0.01), alleviated the downregulation of TRPM7, PKC, ERK and Bax expression levels( P <0.01), induced by blue light irradiation, while TRPM7 knockdown had opposite effects( P <0.01). TRPM7 and PEDF synergistically alleviated the damaging effects of blue light. Conclusions The apoptosis of RPE cells induced by blue light was positively correlated with the expression of TRPM7. Forced expression of TRPM7 partially attenuated the deleterious effects of blue-light-demaged RPE cells and showed a synergistic protective effect with PEDF, involving PKC/ERK signaling pathway.

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Overexpression of CERKL Protects Retinal Pigment Epithelium Mitochondria from Oxidative Stress Effects

Antioxidants ◽

10.3390/antiox10122018 ◽

2021 ◽

Vol 10 (12) ◽

pp. 2018

Author(s):

Rocío García-Arroyo ◽

Aleix Gavaldà-Navarro ◽

Francesc Villarroya ◽

Gemma Marfany ◽

Serena Mirra

Keyword(s):

Oxidative Stress ◽

Retinal Pigment Epithelium ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Stress Conditions ◽

Phenotypic Traits ◽

Mitochondrial Network ◽

Causative Gene ◽

Rpe Cells

The precise function of CERKL, a Retinitis Pigmentosa (RP) causative gene, is not yet fully understood. There is evidence that CERKL is involved in the regulation of autophagy, stress granules, and mitochondrial metabolism, and it is considered a gene that is resilient against oxidative stress in the retina. Mutations in most RP genes affect photoreceptors, but retinal pigment epithelium (RPE) cells may be also altered. Here, we aimed to analyze the effect of CERKL overexpression and depletion in vivo and in vitro, focusing on the state of the mitochondrial network under oxidative stress conditions. Our work indicates that the depletion of CERKL increases the vulnerability of RPE mitochondria, which show a shorter size and altered shape, particularly upon sodium arsenite treatment. CERKL-depleted cells have dysfunctional mitochondrial respiration particularly upon oxidative stress conditions. The overexpression of two human CERKL isoforms (558 aa and 419 aa), which display different protein domains, shows that a pool of CERKL localizes at mitochondria in RPE cells and that CERKL protects the mitochondrial network—both in size and shape—against oxidative stress. Our results support CERKL being a resilient gene that regulates the mitochondrial network in RPE as in retinal neurons and suggest that RPE cell alteration contributes to particular phenotypic traits in patients carrying CERKL mutations.

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Grape Seed Proanthocyanindin Extract Moderated Retinal Pigment Epithelium Cellular Senescence Through NAMPT/SIRT1/NLRP3 Pathway

10.21203/rs.3.rs-230519/v1 ◽

2021 ◽

Author(s):

Yan Wu ◽

Sanyou Dai ◽

Yang Long ◽

Hongzhuo Liu ◽

Weiwei Wan ◽

...

Keyword(s):

Retinal Pigment Epithelium ◽

Cellular Senescence ◽

Barrier Function ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Grape Seed ◽

Rpe Cells ◽

Mitochondrial Homeostasis

Abstract Background: Cellular senescence of retinal pigment epithelium (RPE) cell was an important cause of degenerative retinal disorders, however, the potential effects of grape seed proanthocyanindin extract (GSPE) through regulating NAMPT/SIRT1/NLRP3 pathway remained unclear.Methods: The effects of GSPE on the cellular senescence biomarkers as well as NAMPT and NAD+ contents were detected in both in-vivo and in-vitro RPE cell models. The protection of GSPE treatment on the mitochondrial homeostasis and barrier function of RPE cells were detected with mtDNA lesions, JC-1 staining, ZO1 expression, trans-epithelial cell resistance (TEER) as well as senescence-associated secretory phenotype (SASP) expressions. The GSPE treatment with NAMPT inhibitor, Fk866, and SIRT1 inhibitor, EX-527, was used in the potential NAMPT/SIRT1/NLRP3 mechanism detection.Results: GSPE significantly improve the NAMPT and NAD+ content in aging mice and thus alleviated the RPE cellular senescence. In advanced in-vitro studies, GSPE could be an activator of NAMPT and thus relieved H2O2 induced NAD+ depression. In advanced analyses, it was reported that GSPE could alleviate mitochondrial homeostasis, barrier function and SASP of aging RPE cells. Thus, detection the SASP in in-vitro aging model provided us knowledge in the understanding of the anti-aging role of GSPE and following detailed pathological mechanism analyses demonstrated that GSPE demonstrated the protective effects in aging RPE cells through NAMPT/SIRT1/NLRP3 pathway.Conclusions: These findings indicate that GSPE alleviated cellular senescence both in-vivo and in-vitro through NAMPT/SIRT1/NLRP3 pathway. This study highlighted the importance both the potential GSPE in degenerative retinopathy as well as the crosstalk of NAD+ metabolism, SIRT1 function and NLRP3 activation.

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