Current Molecular Medicine - Volume 17, Issue 4, 2017

Background: The sampling of tear fluids is essential for the reproducibility of tear assays. But, an operable, efficient and stable method for tear collection has not been widely established. This study evaluated the utility of polyethersulfone membranes (PESms) for tear collection and compared it with two frequently employed approaches: the use of Schirmer test paper (STP) and capillary tube (CaT). Methods: STP and PESms (0.45 and 0.65 μm) were examined using scanning electron microscopy(SEM), applied to soak up water, bovine albumin and cellular lysates, and employed to collect tear fluids from rabbits. The proteins in the cellular lysates and tear fluid were characterized through band profiling of SDS-polyacrylamide electrophoresis (PAGE) gels. Western blot analyses with antibodies against vinculin and lysozyme C were used to compare rabbit tear fluids sampling with CaT, STP and PESm. Rabbit ocular surfaces and eye blinking were evaluated under different conditions. Results: The SEM examination showed that PESms exhibited much smoother surfaces and smaller pores than STP. PESm65 exhibited the highest water absorption and water recovery in vitro. Although SDS-PAGE revealed no obvious differences in the cellular or tear protein band patterns, the highest Dice's similarity coefficient and lowest variance in the differences in peak heights(i.e. band intensities) were observed in the PESm65 samples. The Western blot showed that the band intensities of vinculin protein in tear fluids obtained using CaT, STP and PESm65 are in order of CaTPESm65>STP. PESm65 exhibited less blinks and conjunctival injuries than STP during sampling-rabbit tears. Conclusion: Tear collection using PESm has less variance in protein components and is easily operable than traditional STP. PESm possesses the potential for clinical utilization in diagnostic tear assays.

Background: Glutathione is a small antioxidant peptide in cells and it plays an important role in maintaining a reducing intracellular environment. Glutathione is also involved in the dynamic regulation of specific protein functions by reversible glutathiolation of certain proteins in response to oxidative stress. Objective: The purpose of this work is to mechanistically investigate the effects of glutathiolation on the susceptibility of proteins to degradation by the ubiquitinproteasome pathway (UPP). Methods and Results: The data show that γC-crystallin and carbonic anhydrase III were barely degraded by the UPP without modifications, but both were rapidly degraded by the UPP after glutathiolation. Modifications of sulfhydryls by other thiol-modification reagents, such as iodoacetamide, also increased the degradation of γC-crystallin, but not as effectively as glutathiolation. Biophysical analysis showed that glutathiolation caused reversible conformational changes of these proteins, including a significant increase in protein surface hydrophobicity and a decrease in thermal stability. The modified protein regained its native conformation and its resistance to degradation upon removal of the glutathione moiety. A cataract-causing T5P mutant γC-crystallin shares many biophysical characteristics as glutathiolated γC-crystallin, including increased surface hydrophobicity and decreased thermal stability. T5P mutant γC-crystallin was also rapidly degraded. Comparison of the conformational changes and the susceptibility to degradation of glutathiolated γC-crystallin with other forms of modified γC-crystallin suggests that the glutathiolation-induced exposure of hydrophobic patches, rather than the modification per se, serves as the signal for degradation by the UPP. Consistent with this hypothesis, masking the surface hydrophobicity of glutathiolated and T5P mutant γC-crystallins significantly reduced their susceptibility to degradation by the UPP. Conclusion: This work demonstrates that glutathiolation is a novel mechanism for the UPP to recognize substrates in response to oxidative stress.

Background and Objective: Lens regeneration is an optimal strategy for cataract patients to regain visual acuity with accommodation. We recently designed a novel, minimally invasive capsulorhexis surgical method for cataract removal that achieved functional lens regeneration in human infants. However, small anterior capsulorhexis requires advanced surgical expertise. To examine whether the quality of the regenerated lens can be maintained with enlarged anterior capsulorhexis, we investigated the shape and transparency of the regenerated lenses with different anterior capsulorhexis diameters (ACDs). Methods: Thirty-six 4-week-old New Zealand albino rabbits were randomly divided into three groups which underwent lens extraction with different ACDs (Group A: 2.0±0.5 mm, Group B: 4.0±0.5 mm, Group C: 6.0±0.5 mm). The anterior capsule opening area (ACOA) was quantified, and the morphology, weight, and histological characteristics of the regenerated lenses were examined. Results: Lens regeneration was observed in all three groups. In Group A, the regenerated lenses were relatively complete and transparent. In Groups B and C, the regenerated lenses were doughnut-shaped and opaque. The speed of lens regeneration in Group A was significantly faster than that in Groups B and C. The ACOA in Group A healed quickly and completely approximately 2 weeks after surgery. However, in Groups B and C, ACOA did not heal completely until 12 weeks after surgery. Histological examination showed that in Group A, most of the lens epithelial cells differentiated into well-organized lens fibers. However, in Groups B and C, the regenerated lens fibers were disorganized. Conclusion: Capsulorhexis size is a critical determinant of integrity and transparency in lens regeneration.

Objective: To explore the expression profile of angiogenic factors associated with proliferative diabetic retinopathy (PDR). Methods: Undiluted vitreous humor samples were obtained from 10 diabetic patients with PDR (10 eyes) and 9 non-diabtic patients (9 eyes). The concentrations of 60 angiogenic factors in the vitreous humor samples were measured by RayBio Angiogenic Cytokine Antibody Array. Some differentially expressed factors were further confirmed in vitreous humor by enzyme-linked immunosorbent assay (ELISA). Results: Compared with the non-diabetic controls, 20 differentially expressed factors with more than 1.50 fold changes were detected in patients with PDR. The median concentration of vascular endothelial growth factor (VEGF), interleukin 6 (IL-6), angiopoietin (ANG)-1, ANG-2, urokinase-type plasminogen activator receptor (uPAR), Follistatin and matrix metalloproteinases 9 (MMP-9) was significantly increased in vitreous samples from PDR compared to controls (P < 0.05). However, (MCP)-1, Angiogenin and Leptin was significantly lower in PDR eyes compared to controls (P < 0.05). In the verification assay using ELISA, ANG-1, ANG-2, IL-6, VEGF, MMP-9, hepatocyte growth factor (HGF) and placenta growth factor (PIGF) concentration were increased in patients with PDR compared to controls (all P-values < 0.05). Conclusion: This is the first report of a comprehensive multiplex analysis to identify angiogenic factors associated with PDR. These angiogenic factors may contribute to the pathogenesis of PDR and may be targets for therapeutic strategies of PDR.

Objective: Diabetes mellitus (DM) and diabetic retinopathy (DR) are associated with oxidative stress and carotenoids have antioxidant properties. This study aimed to test the relationship between serum carotenoid concentrations and the risk for DM and DR. Methods: This is a cross-sectional study of the Chinese urban population. A total of 747 subjects, consisting of 272 DR patients, 190 diabetic patients without retinopathy, and 285 non-diabetes mellitus healthy controls, were recruited to this study. Demographic and lifestyle characteristics were ascertained by questionnaire. General physical and ophthalmic examinations were completed for all participants. Serum carotenoids were measured by high-performance liquid chromatography (HPLC). The associations of serum carotenoids with DM and DR were assessed by logistic regression with adjustment of known risk factors. The correlation analyses of serum carotenoids with the candidate influence factors were assessed using the single variable linear regression. Results: Both pro-vitamin A (PVA) carotenoids and non-PVA carotenoids in the serum were measured and compared between different groups. Levels of α-carotene were significantly lower in DR patients and β-carotene were significantly lower in DM patients as compared to non DM healthy control group. In contrast, levels of β-cryptoxanthin, lycopene, lutein and zeaxanthin were comparable among different groups. After adjusting for confounding factors, β-carotene concentration was associated with reduced risk for DM (OR (95%CI): 0.56 (0.34, 0.91), P=0.02) and α-carotene was associated with reduced risk for DR in non-smokers (OR (95%CI): 0.41 (0.17, 0.99), P=0.048). No significant association was found between hemoglobin A1c and any carotenoids (P>0.05). Significantly associations with serum carotenoids were found in age, sex, BMI, smoking, and exercise (P<0.05). Conclusion: Serum β-carotene may have a protective effect on DM and α-carotene may be a protective factor for DR in non-smokers.

Background: The compromise of blood brain barrier (BBB) integrity is often associated with human hemorrhage stroke and neurodegeneration diseases, including retina diseases, such as age-related macular degeneration and diabetic retinopathy. Brain pericytes play pivotal roles in regulation and maintenance of BBB integrity. However, the mechanisms underlying brain pericyte development to establish BBB integrity remain unclear. Methods: Zebrafish transgenic lines Tg(flk1:GFP; gata1:dsRed), Tg(flk1:GFP), Tg(fli1:GFP) and Tg(BRE:GFP) were used in this work. The functional studies of bmp3 were performed by mopholino oligonucleotide (MO) injection, dye-based permeability assay, RT-PCR, in vivo imaging, immunofluorescence staining and statics analysis. Results: Here we report that bmp3 regulates BBB integrity in zebrafish brain by promoting pericyte development. Knockdown of bmp3 with injection of bmp3-MO causes intracerebral hemorrhage in zebrafish embryos. Meanwhile, disruption of bmp3 function by bmp3-MO injection impairs cerebral pericyte coverage in zebrafish embryos. Mechanistically, knockdown of bmp3 disrupts the pattern and activities of BMP signaling in zebrafish brain, thus probably disrupting the balance of TGFβ/BMP signaling in zebrafish embryos. Conclusion: In summary, our data shows that bmp3 regulates BBB integrity potentially by promoting pericyte development.

Background: Macrophages undergo polarization or activation in response to environmental stimuli, an essential process for proper immune response. Meanwhile, excessive activation of macrophages causes autoimmune diseases. It is therefore crucial to prevent over-activation of macrophage in order to maintain the proper immune response. Arginase 1 (Arg-1) plays a critical role in coordinating the immune response by regulating availability of arginine. Objective: To understand the mechanism of Arg-1 regulation. Methods: Real-time PCR and Western Blot analysis were utilized to examine the Arg-1 levels expressed from the VEGFR1-deleted and VEGFR1-TK-deficient bone marrowderived macrophages (BMDMs). Results: The VEGFR1-mediated signaling suppressed IL-4-induced Arg-1 expression. Deletion of VEGFR1 resulted in elevated Arg-1 expression and the tyrosine kinase domain of VEGFR1 was required for the suppression. Each of three ligands of VEGFR1, VEGF-A, VEGF-B and PIGF, mediated the inhibition to the similar degree. Conclusion: Our findings identified a novel function of the VEGFR1 signaling in avoiding over-expression of Arginase 1 potentially to maintain the proper innate immune response.

Background: Proliferative vitreoretinopathy (PVR) is a disease caused by dedifferentiation, translocation and proliferation of several types of local cells. These cells form fibrocellular membranes resulting in detachment of retinal and vision loss. PVR occurs in 8%-10% of patients undergoing primary retinal detachment (RD) surgery and becomes a major obstacle for successful RD repair. Retinal pigment epithelial (RPE) cells are among the major cells which consist of fibrocellular membranes. Reproliferation and Epithelial-mesenchymal transition (EMT) are the primary pathological alteration of RPE cells in PVR. Methods: RPE cells were treated with curcumin at different concentrations for 24, 48 and 72 hours. The viable cells were detected by MTT assay. The apoptosis of RPE was stained by Multicaspase/7-AAD and detected using flow cytometry. Cell cycle analysis was quantified by PI staining. The mRNA levels were detected by real-time PCR. The protein levels were detected by western blot. Results: We found a compound curcumin significantly inhibited proliferation and EMT of RPE cells in vitro. Further study showed curcumin induced cell cycle arrest by activating G2 checkpoint through p53 pathway. Meanwhile, we found that curcumin suppressed the AKT, MAPK and TGF-β pathways in RPE cells which may also affect proliferation and EMT. Our research identified curcumin a potential novel agent for the PVR prevention and treatment. Curcumin induces cell cycle arrest by activating G2 checkpoint. Conclusion: Our results in this study also provide the insights to broaden the application of curcumin in research and probably clinics.