Current Molecular Medicine - Volume 18, Issue 9, 2018

Purpose: Advanced age is the largest risk factor for age-related macular degeneration (AMD). Sumoylation is a reversible post-translational modification that conjugates small peptide, small ubiquitin-like modifier (SUMO), to a target protein. Dysregulation of sumoylation is recently found to be critically involved in several age-related disorders. However, the effects of sumoylation during retina senescence and aging remains elusive. This study is aimed to investigate the function and regulation of sumoylation pathway in the aging retina and premature senescent retinal pigment epithelial (RPE) cells. Methods: 1.5- and 10-month C57/B6 mice were used for comparative aging study. Both ARPE primary cultures and ARPE-19 cells were used for assay systems. The qRT-PCR was used for analysis of mRNA expression. Western blot and immunofluorescence were used to analyze the protein expression. Cell flow cytometry was used for cell cycle progression analysis. RPE barrier function and senescent-associated β-galactosidase (SA β-gal) activity were analyzed to measure cellular senescence. Results: We show that the expression of SUMO enzymes and global protein sumoylation were downregulated in the aging mouse retina, and in the oxidative stress (OS) -induced premature senescent RPE cells. Dramatical altered distribution of SUMO E1, E2 and E3 enzymes were observed during RPE senescence. Inhibition of sumoylation alleviated OS–induced cell senescence in RPE cells, as indicated by decreased p21 and p53 expression and decreased percentage of cell cycle arrest at G0/G1 phase. Intriguingly, inhibition of SUMO E1 repressed the expression of proinflammatory cytokine and chemokine in the premature senescent RPE cells. However, inhibition of sumoylation did not prevent DNA damage during the OS-induced RPE senescence process. Conclusions: Our data indicate sumoylation critically regulates retina and RPE aging and that targeting sumoylation process may provide potential therapeutic strategy for AMD treatment.

Purpose: Accumulated evidence have well established that protein sumoylation plays multiple roles in various cellular processes. In the vertebrate eye, we and others have demonstrated that sumoylation displays indispensable roles in regulating eye development. Various ocular cell lines including human embryonic cell line (FHL124), the SV40-large T-transformed human lens epithelial cell line (HLE), the SV40-large T-transformed mouse lens epithelial cell line (αTN4-1), the rabbit lens epithelial cell line (N/N1003A) and the human retina pigment epithelial cell line (ARPE-19) have been extensively used for studying various cellular functions and disease processes including sumoylation functions, and mechanisms for cataract and age-related macular degeneration (AMD). However, the sumoylation enzyme systems have not been well established. Methods: FHL124, HLE, αTN4-1, N/N1003A and ARPE-19 were cultured in Dulbecco’s modified eagle medium (DMEM) containing 10% FBS and 1% penicillin & streptomycin. The expression levels of seven SENP mRNAs were analyzed with qRT-PCR, and the expression levels of seven SENP proteins were detected with Western blot analysis. Results: Using both qRT-PCR and Western blot analysis, we have obtained the followings: 1). The 3 human ocular cell lines, FHL124, HLE and ARPE-19 express all types of SENP mRNA and proteins. 2). In mouse lens epithelial cell line αTN4-1, and rabbit lens epithelial cells line N/N1003A, however, only the mRNAs for SENP1, 2, 3, 6 and 7 are expressed. At the protein level, SENP8 was absent in both αTN4-1 and N/N1003A cells; 3). Each cell line has different dominant SENP enzymes. For FHL124, SENP3, 5, 7 and 8 proteins are relatively dominant. SENP3, 5 and 6 are the major de-sumoylation enzymes in HLE cells. Different from human lens epithelial cells, FHL124 and HLE, human retina pigment epithelial cells (ARPE-19) have SENP3, 7, and 8 as the dominant forms of de-sumoylation enzymes. For mouse lens epithelial cells, SENP1, 3 and 7 are the major de-sumoylation enzymes. On the other hand, the rabbit lens epithelial cells have SENP1, 2 and 7 as the major isoforms. Conclusion: Our results for the first time defined the differential expression patterns of the seven types of de-sumoylation enzymes (SENPs) in 5 major ocular cell lines. These results help to establish the basis for the future study of sumoylation functions and the related mechanisms in vertebrate eye.

Background: Dynamic epigenetic alterations accompanying CD4+ T helper cell differentiation have been implicated in multiple autoimmune diseases. The bromodomain and extra-terminal (BET) proteins are epigenetic regulators that recognize and bind to acetylated histones in chromatin and are targets for pharmacological inhibition. In this study we tested a new BET inhibitor under clinical development, OTX015, to interrogate its effects on key CD4+ T cell subsets associated with autoimmunity. Methods: Naïve and memory murine and human CD4+ T cells were isolated and differentiated into populations characterized by the expression of interferon (IFN)-γ and interleukin (IL)-17. Cultured cells were then exposed to varying concentrations of OTX015 in vitro, and its impact on cytokine expression was quantified by flow cytometry. In parallel, the expression of the transcription factors TBX21 and RORC was quantified by PCR. A previously studied BET inhibitor JQ1 was used as a pharmacological control. Results: OTX015 suppressed both murine and human CD4+ T cell proliferation. Its impact on cytokine expression varied in murine and human naïve and memory subsets. OTX015 was similarly effective as JQ1 in the suppression of cytokines and T helper cell proliferation. Higher concentrations of OTX015 also had a greater impact on the viability of murine versus human cells. IL-17 and IFN-γ expression was not altered in murine memory CD4+ T cells, whereas in human memory CD4+ T cells, OTX015 inhibited IL-17, but not IFN-γ. Across all human T cell subsets OTX015 suppressed IL-17 more effectively than IFN-γ. Conclusion: Our studies demonstrate that OTX015 has anti-inflammatory effects by suppressing murine and human CD4+ T cell proliferation and subset-dependent proinflammatory cytokine expression, including the selective suppression of IL-17 in human memory CD4+ T cells.

Objective: Experimental autoimmune uveitis (EAU) represents autoimmune uveitis in humans, among which B10.RIII and C57BL/6 are the frequently used strains in mice, but to date, no study has been reported to compare EAU disease between the two strains. Here we compared the differences in morphology, pathology, visual function of the retinal inflammation and Th1/Th17 immune responses in the EAU models induced by the interphotoreceptor retinoid binding protein (IRBP) between the B10.RIII and C57BL/6 strains, using fundus and histological examinations, optical coherence tomography, electroretinography and immunoassays. Method: EAU induced in B10.RIII mice exhibited a shortterm severe inflammation with massive ocular infiltrates of inflammatory cells and extensive destruction of the retina that culminated in rapid degeneration of the retina and permanent loss of visual function. In contrast, C57BL/6 mice developed chronic inflammation with recurring and persistent retinitis for several months, highlighting moderate scores of disease severity and visual signal in comparison with those in B10.RIII mice. Consistent with the clinical manifestations, increased Th1/Th17 effector responses were detected in the uveitic eyes of B10.RIII strain than those in C57BL/6 strain. These data demonstrate distinguishing features of retinal inflammation and T-cell immune responses involved in IRBP-induced EAU between B10.RIII and C57BL/6 strains. Conclusion: Our findings suggest that the persistent-recurring EAU model induced in C57BL/6 mice may serve as a better tool to represent distinct aspects of human uveitis.

Background: Gamma-ray irradiation could significantly induce widespread apoptosis in corneas and reduced the allogenicity of donor cornea. And the X-rays may have similar biological effects. The feasibility and effects of X-ray-irradiated corneal lamellae have not been assessed yet. Methods: Different doses (10 gray unit (Gy), 20 Gy, 50 Gy, 100 Gy) of X-ray irradiated corneal lamellae were collected from SMILE surgery. These corneal lamellae were assessed by physical characterization, hematoxylin and eosin (H-E) staining, Masson’s staining, TdT-mediated dUTP nick end labeling (TUNEL), cell viability assay and transmission electron microscopy (TEM). We selected the optimum dose (100Gy) to treat the corneal lamellae to be the grafts. The human grafts and fresh allogeneic monkey corneal lamellae were implanted into rhesus monkeys via the small incision femtosecond laser-assisted surgery, respectively. Clinical examinations and the immunostaining were performed after surgery. Results: There were no significant changes in the transparency of the corneal lamellae, but the absorbency of the corneal lamellae was increased. According to the H-E and Masson’s staining results, irradiation had little impact on the corneal collagen. The TUNEL assay and cell viability assay results showed that 100Gy X-ray irradiation resulted in complete apoptosis in the corneal lamellae, which was also confirmed by TEM observations. In the following animal model study, no immune reactions or severe inflammatory responses occurred, and the host corneas maintained transparency for 24 weeks of observation. And the expression of CD4 and CD8 were negative in the all host corneas. Conclusion: X-ray irradiated corneal lamellae could serve as a potential material for xenogeneic inlay, and the small incision femtosecond laser-assisted implantation has the potential to become a new corneal transplantation surgical approach.

Background: Anti-VEGF agent ranibizumab has been extensively used as a standard treatment for wet AMD. We investigated whether traditional Chinese medicine could serve as a complementary therapy for this disease. Methods: 144 patients with neovascular age-related macular degeneration received either intravitreal ranibizumab treatment as needed plus placebo or intravitreal ranibizumab treatment as needed plus an FDA approved traditional Chinese patent medicine named ZQMT. Both groups received treatment for 24 weeks. The primary outcome was the mean change of visual acuity at week 24 as compared to the baseline. Results: We found that intravitreal ranibizumab treatment plus ZQMT was non-inferior to the treatment with intravitreal ranibizumab alone in improving visual acuity scores at week 24 with patients in both groups who gained substantial numbers of letters. In addition, we found that ZQMT treatment resulted in significant improvements in reducing retinal hemorrhage, fluid, and lesion size. Importantly, administration of ZQMT reduced the number of needed ranibizumab injections (P<0.0001, analysis of variance) in wet AMD patients leading to a significant reduction of drug cost. Conclusion: The combinatory therapy of ranibizumab and traditional Chinese patent medicine ZQMT had equivalent effects on visual acuity improvement and safety profiles as the ranibizumab treatment alone. Ranibizumab injections coupled with ZQMT offer therapeutic advantages in terms of reduction of retinal lesions and ease the financial burden of patients undergoing treatment by reducing the frequency of necessary ranibizumab injections.

Background: Overexpression of MDA-9/Syntenin occurs in multiple human cancer cell lines and is associated with higher grade of tumor classification, invasiveness and metastasis. In some cases, its role in cancer biology depends on relationships between MDA-9/Syntenin and NF-ΚB. Objective: This study aims to analyze the presence of a regulation loop like that between MDA-9/Syntenin - NF-ΚB - RKIP in human liver carcinoma. Methods: Transient transfection was performed with siRNA anti-MDA-9/Syntenin. Expression of different factors was evaluated by Real time-PCR and Western blotting, while NF-ΚB activation by TransAM assay. Invasion capacity was analyzed by Matrigel Invasion Assay and the effects of agents on cell viability were examined by MTS assay. Results: We have examined basal expression of MDA-9/Syntenin in three cell lines of human liver carcinoma (HA22T/VGH, Hep3B and HepG2). In all cell lines there was an inverse relationship between MDA-9/Syntenin and RKIP expression levels, and a positive correlation between MDA-9/Syntenin expression and NF-ΚB activation levels. By silencing with a siRNA anti-MDA-9/Syntenin we observed in all cell lines a very strong increase of RKIP at mRNA level. Interestingly, in all cell lines, inhibition of MDA- 9/Syntenin expression induced NF-ΚB downregulation and contemporary a reduction in invasion ability MMP-2 dependent. Finally, we showed a good additive effect of MDA- 9/Syntenin siRNA when associated with Curcumin or Doxorubicin on cell growth inhibition. Conclusion: Our data confirm the key role of MDA-9/Syntenin in HCC biology. The presence of a regulation loop among MDA-9/Syntenin, NF-ΚB and RKIP provide new pharmacological approaches.

Background: Urinary tract infection (UTI) is caused by uropathogenic Escherichia coli (UPEC). The UPEC initiate pathogenesis by expressing type 1 pili, which attach to membrane receptors on the uroepithelial cells. Inhibition of attachment can provide a valuable target for prophylaxis in symptom-free milieu. Methods: The antibacterial efficacy of alcoholic, hydroalcoholic and aqueous extracts of four plants namely Achyranthes aspera, Andrographis paniculata, Artemissia vulgaris and Glycyrrhiza glabra was evaluated against seven isolated bacterial strains and procured E. coli (UTI89/UPEC) strain. Screening of isolated strains was based on morphological characteristics and biofilm forming ability followed by physiological and biochemical analysis. Results: The hydroalcoholic extracts of G. glabra at 50 μg/ml showed an impending antioxidant (DPPH) effect of 95.65% compared to ascorbic acid. The MIC values of all the plant extracts against selected bacterial strains ranged between 125 to 1000 μg/ml. In silico molecular docking performed to make out the antiadhesive role of 115 documented phytochemicals from selected plants identified quercetin-3-glucoside, ethyl caffeate, liquiritoside, liquiritin and isoliquiritigenin as potential phytochemicals. Molecular dynamics simulation performed by PTRAJ module of Amber11 package to monitor the stability of hydrogen bond showed that quercetin-3-glucoside and ethyl caffeate are potential phytochemicals as antiadhesive forming H-bonds with the FimH protein ligand. Conclusions: Aforesaid phytochemicals demonstrate effective antibacterial activity through the anti-adhesion mechanism.