All‐trans retinoic acid upregulates the expression of ciliary neurotrophic factor in retinal pigment epithelial cells

Retinopathy of prematurity, a leading cause of visual impairment in low birth‐weight infants, remains a crucial therapeutic challenge. Ciliary neurotrophic factor (CNTF) is a promyelinating trophic factor that promotes rod and cone photoreceptor survival and cone outer segment regeneration in the degenerating retina. Ciliary neurotrophic factor expression is regulated by many factors such as all‐trans retinoic acid (ATRA). In this study, we found that ATRA increased CNTF expression in mouse retinal pigment epithelial (RPE) cells in a dose‐ and time‐dependent manner, and PKA signaling pathway is necessary for ATRA‐induced CNTF upregulation. Furthermore, we showed that ATRA promoted CNTF expression through CREB binding to its promoter region. In addition, CNTF levels were decreased in serum of retinopathy of prematurity children and in retinal tissue of oxygen‐induced retinopathy mice. In mouse RPE cells cultured with high oxygen, CNTF expression and secretion were decreased, but could be recovered after treatment with ATRA. In conclusion, our data suggest that ATRA administration upregulates CNTF expression in RPE cells.     

Differentiation of human retinal pigment epithelial cells into neuronal phenotype by N-(4-hydroxyphenyl)retinamide

ARPE-19, a human retinal pigment epithelial (RPE) cell line, has been widely used in studies of RPE function as well as gene expression. Here, we report the novel finding that N-(4-hydroxyphenyl)retinamide (fenretinide), a synthetic retinoic acid derivative and a potential chemopreventive agent against cancer, induced the differentiation of ARPE-19 cells into a neuronal phenotype. The treated cells lost their epithelial phenotype and exhibited a typical neuronal shape with long processes (four to five times longer than the cell body). The onset of fenretinide-induced neuronal differentiation was dose and time dependent, started within 1-2 days, and lasted at least 4 weeks. Immunohistochemical studies indicated that the expression of neurofilament proteins (NF160 and NF200), calretinin and neural cell adhesion molecule was increased in these differentiated cells. Western blot analysis indicated that cellular retinaldehyde-binding protein, which is normally expressed in RPE cells, was decreased in treated cells. Protein analysis on a two-dimensional gel followed by matrix-assisted laser desorption ionization-time of flight mass spectrometric analysis demonstrated that heat-shock protein 70 was increased after fenretinide treatment. Thus, fenretinide, a synthetic retinoid, is able to induce neuronal differentiation of human RPE cells in culture.     

Nobiletin protects human retinal pigment epithelial cells from hydrogen peroxide–induced oxidative damage

Nobiletin (3′,4′,5,6,7,8‐hexamethoxyflavone), a dietary polymethoxylated flavonoid found in Citrus fruits, has been reported to have antioxidant effect. However, the effect of nobiletin on human retinal pigment epithelium (RPE) cells induced by hydrogen peroxide (H₂O₂) is still unclear. Therefore, we investigated the protective effect of nobiletin against H₂O₂‐induced cell death in RPE cells. Our results demonstrated that nobiletin significantly increased cell viability from oxidative stress. Nobiletin inhibited H₂O₂‐induced ROS production and caspase‐3/7 activity in ARPE‐19 cells. Furthermore, nobiletin significantly increased Akt phosphorylation in ARPE‐19 cells exposed to H₂O₂. Meanwhile, LY294002, an inhibitor of PI3K/Akt, abolished the protective effect of nobiletin against H₂O₂‐induced decreased cell viability and increased caspase‐3/7 activity in ARPE‐19 cells. In summary, these data show that nobiletin protects RPE cells against oxidative stress through activation of the Akt‐signaling pathway. Thus, nobiletin should be an oxidant that attenuates the development of age‐related macular degeneration.     

Long noncoding RNA BDNF‐AS inversely regulated BDNF and modulated high‐glucose induced apoptosis in human retinal pigment epithelial cells

In this study, we characterized the functional role of long noncoding RNA (lncRNA), brain derived neurotrophic factor anti‐sense (BDNF‐AS) in regulating D‐glucose‐induced (DGI) apoptosis in human retinal pigment epithelial (RPE) cells. Human RPE cell line, ARPE‐19 cells were cultured in vitro and treated with various concentrations of D‐glucose for 24 h. A TUNEL assay was applied with immunohistochemical and quantitative approaches to assess the apoptotic effect of D‐glucose. Under the condition of 50 mM D‐glucose, qPCR was used to assess gene expression of BDNF and BDNF‐AS in ARPE‐19 cells. Using siRNA transfection, BDNF‐AS was endogenously knocked down in ARPE‐19 cells. The effects of BDNF‐AS downregulation on DGI apoptosis and BDNF expression were assessed by TUNEL assay, qPCR, and Western blot, respectively. Furthermore, in BDNF‐AS‐downregulated ARPE‐19 cells, secondary siRNA transfection was conducted to knock down endogenous BDNF expression. Its effect on BDNF‐AS‐associated apoptotic regulation was further evaluated. High concentrations of D‐glucose induced significant apoptosis in ARPE‐19 cells in vitro. With treatment of 50 mM D‐glucose, BDNF was markedly downregulated whereas BDNF‐AS upregulated in ARPE‐19 cells. SiRNA‐mediated BDNF‐AS downregulation ameliorated DGI apoptosis and upregulated BDNF in ARPE‐19 cells. In addition, inhibiting BDNF reversed the protective effect of BDNF‐AS downregulation on DGI apoptosis. Our results suggest that BDNF‐AS, through inverse regulation of BDNF, might play a critical role in the process of DGI apoptosis in diabetic retinopathy.     

A phase I clinical trial of human embryonic stem cell‐derived retinal pigment epithelial cells for early‐stage Stargardt macular degeneration: 5‐years' follow‐up

ObjectivesTo evaluate the long‐term biosafety and efficacy of transplantation of human embryonic stem cells‐derived retinal pigment epithelial (hESC‐RPE) cells in early‐stage of Stargardt macular degeneration (STGD1).Materials and methodsSeven patients participated in this prospective clinical study, where they underwent a single subretinal transplantation of 1 × 10⁵ hESC‐RPE cells in one eye, whereas the fellow eye served as control. These patients were reassessed for a 60‐month follow‐up through systemic and ophthalmic examinations.ResultsNone of the patients experienced adverse reactions systemically or locally, except for two who had transiently high intraocular pressure post‐operation. Functional assessments demonstrated that all of the seven operated eyes had transiently increased or stable visual function 1‐4 months after transplantation. At the last follow‐up visit, two of the seven eyes showed visual function loss than the baseline; however, one of them showed a stable visual acuity when compared with the change of fellow eye. Obvious small high reflective foci in the RPE layer were displayed after the transplantation, and maintained until the last visit. Interestingly, three categories of patients who were classified based on autofluorescence, exhibited distinctive patterns of morphological and functional change.ConclusionsSubretinal transplantation of hESC‐RPE in early‐stage STGD1 is safe and tolerated in the long term. Further investigation is needed for choosing proper subjects according to the multi‐model image and function assessments.     

Trichostatin A,a histone deacetylase inhibitor,suppresses proliferation and epithelial–mesenchymal transition in retinal pigment epithelium cells

The proliferation and epithelial–mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells are the major pathological changes in development of proliferative vitreoretinopathy (PVR), which leads to severe visual impairment. Histone deacetylases (HDACs)‐mediated epigenetic mechanisms play important roles in controlling various physiological and pathological events. However, whether HDACs are involved in the regulation of proliferation and EMT in PRE cells remains unidentified. In this study, we evaluated the expression profile of HDAC family (18 genes) and found that some of class I and class II HDACs were up‐regulated in transforming growth factor‐β2 (TGF‐β2)/TGF‐β1‐stimulated RPE cells. Tricostatin A (TSA), a class I and II HDAC inhibitor, suppressed the proliferation of RPE cells by G1 phase cell cycle arrest through inhibition of cyclin/CDK/p‐Rb and induction of p21 and p27. In the meantime, TSA strongly prevented TGF‐β2–induced morphological changes and the up‐regulation of α‐SMA, collagen type I, collagen type IV, fibronectin, Snail and Slug. We also demonstrated that TSA affected not only the canonical Smad signalling pathway but also the non‐canonical TGF‐β/Akt, MAPK and ERK1/2 pathways. Finally, we found that the underlying mechanism of TSA affects EMT in RPE cells also through down‐regulating the Jagged/Notch signalling pathway. Therefore, this study may provide a new insight into the pathogenesis of PVR, and suggests that epigenetic treatment with HDAC inhibitors may have therapeutic value in the prevention and treatment of PVR.     

Mutual regulation between butyrate and hypoxia‐inducible factor‐1α in epithelial cell promotes expression of tight junction proteins

Inflammatory bowel disease is a kind of multi‐aetiological chronic disease that is driven by multidimensional factors. Hypoxia‐inducible factor‐1α (HIF‐1α) plays an important role in anti‐inflammatory and cellular responses to hypoxia. Previous studies have found that B or T‐cell‐specific HIF‐1α knock out mice exhibit severe colonic inflammation. However, we know very little about other functions of HIF‐1α in intestinal epithelial cells (IECs). In our study, HIF‐1α^ΔIEC mice were used to study the function of HIF‐1α in IECs. HIF‐1α was knocked down in Caco‐2 cells by transfection with a small interfering (si) RNA. Immunohistochemical staining and western blotting were used to detect the expression of zonula occluden‐1 (ZO‐1) and Occludin. The content of colon was harvested for high‐performance liquid chromatography analysis to examine the levels of butyrate in the gut. Our research found that HIF‐1α played a protective role in dextran sulphate sodium‐induced colitis, which was partly due to its regulation of tight junction (TJ) protein expression. Further study revealed that HIF‐1α mediated TJ proteins levels by moderating the content of butyrate. Moreover, we found that butyrate regulated TJ protein expression, which is dependent on HIF‐1α. These results indicated that there is a mutual regulatory mechanism between butyrate and HIF‐1α, which has an important role in the maintenance of barrier function of the gastrointestinal tract.     

Intrinsically altered lung‐resident γδT cells control lung melanoma by producing interleukin‐17A in the elderly

Cancer is an age‐associated disease, potentially related to the altered immune system of elderly individuals. However, cancer has gradually decreased incidence in the eldest globally such as the most common lung cancer, the mechanisms of which remain to be elucidated. In this study, it was found that the number of lung‐resident γδT cells was significantly increased with altered gene expression in aged mice (20–24 months) versus young mice (10–16 weeks). Aged lung Vγ4⁺ and Vγ6⁺ γδT cells predominantly produced interleukin‐17A (IL‐17A), resulting in increased levels in the serum and lungs. Moreover, the aged mice exhibited smaller tumors and reduced numbers of tumor foci in the lungs after challenge with intravenous injection of B16/F10 melanoma cells compared with the young mice. Aged lung Vγ4⁺ and Vγ6⁺ γδT cells were highly cytotoxic to B16/F10 melanoma cells with higher expression levels of CD103. The markedly longer survival of the challenged aged mice was dependent on γδT17 cells, since neutralization of IL‐17A or depletion of indicated γδT cells significantly shortened the survival time. Consistently, supplementation of IL‐17A significantly enhanced the survival time of young mice with lung melanoma. Furthermore, the anti‐tumor activity of aged lung γδT17 cells was not affected by alterations in the load and composition of commensal microbiota, as demonstrated through co‐housing of the aged and young mice. Intrinsically altered lung γδT17 cells underlying age‐dependent changes control lung melanoma, which will help to better understand the lung cancer progression in the elderly and the potential use of γδT17 cells in anti‐tumor immunotherapy.     

Surface expression of CXCR4 in unrestricted somatic stem cells and its regulation by growth factors

Umbilical cord blood‐derived USSCs (unrestricted somatic stem cells) have recently been considered as a potential source for stem cell therapy and transplantation due to their characteristics such as easy accessibility, low immunogenicity, self‐renewing and multilineage differentiation potential. Stem cell homing is a key factor in successful transplantation, which is regulated by CXCR4 in stem cells. In this study, we evaluated the expression of CXCR4 in USSCs different passages. Moreover, the effect of VEGF (vascular endothelial growth factor) and IGF‐1 (insulin‐like growth factor 1) on its expression was assessed. It was shown that the expression of CXCR4 in USSCs decreased with the increase in passage number. It was also revealed that VEGF increased surface expression and mRNA level of CXCR4 in USSCs, while IGF‐1 decreased its expression. When VEGF and IGF‐1 were administered simultaneously, CXCR4 expression was increased, but the expression level was less than VEGF alone. Finally, it was shown that over‐expression of CXCR4 enhanced the migratory capacity of USSCs. The increase of CXCR4 expression, here caused by VEGF in USSCs, can improve the efficacy of stem cell therapy and transplantation after long‐term culture of stem cells before clinical use.     

In vitro enhanced differentiation of neural networks in ES gut-like organ from mouse ES cells by a 5-HT4-receptor activation

Using an embryoid body (EB) culture system, we developed a functional organ-like cluster, a “gut”, from mouse embryonic stem (ES) cells (ES gut). Each ES gut exhibited various types of spontaneous movements. In these spontaneously contracting ES guts, dense distributions of interstitial cells of Cajal (ICC) (c-kit, a transmembrane receptor that has tyrosine kinase activity, positive cells; gut pacemaker cells) and smooth muscle cells were discernibly identified, but enteric neural networks were not identified. In the present study, we succeeded in forming dense enteric neural networks by a 5-HT₄-receptor (SR4) agonist, mosapride citrate (1–10 μM) added only during EB formation. Addition of an SR4-antagonist, GR113808 (10 μM) abolished the SR4-agonist-induced formation of enteric neural networks. The SR4-agonist (1 μM) up-regulated the expression of mRNA of SR4 and the SR4-antagonist abolished this upregulation. 5-HT per se exerted similar effects to those of SR4-agonist, though less potent. These results suggest SR4-agonist differentiated enteric neural networks, mediated via activation of SR4 in the ES gut.