Molecular Vision最新文献

Purpose: To investigate the therapeutic effects of eye drops, namely, timolol maleate, a β-adrenergic receptor antagonist, and latanoprost, a prostaglandin F2α analog, on retinal edema in a murine retinal vein occlusion (RVO) model.

Methods: An RVO model was established using laser-induced RVO in mice, which were administered timolol maleate and latanoprost eye drops several times after venous occlusion. Subsequently, the thickness of the inner nuclear layer (INL) and the expression levels of such genes as Vegf and Atf4, which are stress markers of the endoplasmic reticulum, were examined. Primary human cultured retinal microvascular endothelial cells (HRMECs) were treated with timolol under hypoxic conditions, after which the gene expression pattern was investigated. Importantly, an integrated stress response inhibitor (ISRIB) was used in the RVO model, he known ISRIB, which suppresses the expression of ATF4 in retinal edema.

Results: Increased INL thickness was suppressed by timolol eye drops, as were the expressions of Vegf and Atf4, in the RVO model. However, latanoprost eye drops did not induce any change in INL thickness. In HRMECs, hypoxic stress and serum deprivation increased the Vegf and Atf4 expressions; in response, treatment with timolol suppressed the Vegf expression. Furthermore, the ISRIB decreased the Vegf expression pattern and edema formation, which are associated with RVO.

Conclusions: These results indicate that timolol eye drops may be a potential option for RVO treatment.

Purpose: To evaluate the frequency of LTBP2 mutations and to elaborate on LTBP2-related clinical phenotypes in a Chinese congenital ectopia lentis (CEL) cohort.

Methods: In total, 145 Chinese probands with CEL were recruited for this study and underwent ocular and systemic examinations. Whole-exome sequencing was used to identify mutations, and Sanger sequencing and bioinformatics analysis were further performed to verify pathogenic mutations.

Results: Overall, biallelic mutations in LTBP2 involving eight novel mutations (c.4370-7_4370-9delTCT, c.4370-5C>G, c.3452G>A, c.2253delG, c.4114T>C, c.1251G>A, c.4760G>A, and c.620G>A) were identified in four CEL probands (4/145, 2.76%). Patients with LTBP2 mutations were characterized by a megalocornea, spherophakia, high myopia, and glaucoma instead of a flat cornea, high corneal astigmatism, cardiovascular and skeletal abnormalities that were reported in other gene mutations. A novel homozygous frameshift mutation was detected, and this type of mutation was found to cause more complicated ocular symptoms than others, ranging from the anterior segment to the fundus.

Conclusion: This study reported the mutation frequency of the LTBP2 gene in a Chinese CEL cohort and provided novel insight into LTBP2-related genotype-phenotype associations in CEL.

Purpose: To determine the expression levels of SIRT6 and NMNAT2 in diabetic retinopathy (DR).

Methods: We obtained peripheral blood mononuclear cells (PBMCs) and vitreous samples from 77 patients with type 2 diabetes mellitus: 52 with DR and 25 without DR, and 27 healthy control subjects. Western blot analysis and qRT-PCR were performed to evaluate the expression of SIRT6 and NMNAT2 in their PBMCs. The levels of IL-1β, IL-6, and TNF-α in the vitreous fluid were determined by ELISA. Immunohistochemistry was performed to detect the expression of SIRT6 and NMNAT2 in proliferative DR (PDR) and the control subjects.

Results: The expression of SIRT6 and NMNAT2 was markedly downregulated in DR patients, which was negatively correlated with the increased expression of IL-1β, IL-6 and TNF-α. Additionally, we observed decreased expression of SIRT6 and NMNAT2 in the fibrovascular membranes of PDR patients.

Conclusions: The downregulated expression of SIRT6 and NMNAT2 in PDR patients reveals a potential pathogenic association; more extended studies could verify them as potential therapeutic targets.

Purpose: To evaluate the serum lipid levels, including total cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) of patients with normal tension glaucoma (NTG) and to investigate the relationship between serum HDL levels and the severity of NTG.

Methods: In this cross-sectional, case-control study, 282 NTG subjects and 202 control subjects were enrolled from the outpatient clinic of the Department of Ophthalmology at Taichung Veterans General Hospital in central Taiwan from 2015 to 2021. Fasting cholesterol, HDL, and LDL levels were evaluated using a biochemical analyzer (ARCHITECT c16000). Glaucoma severity was classified by visual field test as mild (mean deviation [MD] ≥ -6.0dB), moderate (-12dB ≤ MD < -6 dB), and severe (MD < -12dB), based on the mean deviation.

Results: HDL levels were significantly lower in the NTG group compared with the control subjects (47 ± 18mg/dl versus 53 ± 18mg/dl; p = 0.03). There were no statistically significant differences in total cholesterol or LDL levels between the NTG and control subjects (total cholesterol levels: 194 ± 39mg/dl versus 190 ± 32mg/dl; p > 0.05; LDL levels: 113 ± 30mg/dl versus 110 ± 29mg/dl; p > 0.05). The mean serum HDL levels were lowest in the severe group (41 ± 11mg/dl) followed by the moderate (45 ± 16mg/dl) and mild (50 ± 15mg/dl) groups, with significant differences among the three groups (p = 0.02). The multivariate regression analysis revealed a statistically significant negative correlation between HDL and vertical cup-to-disc ratio (VCDR; B =-0.16, p = 0.03) among all NTG patients and a positive correlation between HDL and retinal nerve fiber layer (RNFL; r = 0.34, p = 0.03) among all NTG patients.

Conclusions: A significantly lower serum HDL concentration was found in the NTG patients, which was negatively associated with disease severity. The findings warrant further study to elucidate the role of these phenomena in the pathogenesis of glaucoma.

Purpose: Retinoblastoma (RB) caused by the mutation of the RB1 gene is one of the most common ocular malignancies in children The propeptide region of lysyl oxidase (LOX), the enzyme involved in the cross-linking of collagen and elastin, has been identified to be anti-tumorigenic in various cancers. However, this role of lysyl oxidase propeptide (LOX-PP) in RB is still elusive. This study aims to identify the anti-tumorigenic effect of LOX-PP in human Y79 RB cells.

Methods: LOX-PP was overexpressed in Y79 RB cells, and differential gene expression was assessed by microarray followed by pathway analysis using transcriptome analysis console (TAC) software. Additionally, cell proliferation was studied by PrestoBlue assay, and DNA content was evaluated by cell cycle and apoptosis assays. The pro-apoptotic and anti-proliferative mechanisms induced by the overexpression of/exogenously added LOX-PP was evaluated by western blotting and real-time PCR.

Results: The expression of the LOX-PP transcript was significantly decreased in Y79 RB cells compared to human retinal endothelial cells. Gene expression analysis in LOX-PP overexpressed Y79 RB cells showed deregulation of pathways involved in apoptosis, cell cycle, focal adhesion-PI3K-AKT signaling, and DNA repair mechanisms. Interestingly, LOX-PP overexpressed Y79 RB cells showed significantly increased apoptosis, decreased proliferation, and cell cycle arrest at S-phase with a concordant reduction of proliferative cell nuclear antigen and Cyclin D1 protein expressions. Moreover, pAKT (S473) was significantly downregulated in Y79 RB cells, which decreased NFκB leading to significantly reduced BCL2 expression.

Conclusions: Our results demonstrate the anti-tumorigenic effect of LOX-PP in Y79 RB cells by inducing apoptosis and decreasing proliferation. This effect was mediated by the downregulation of AKT signaling. These results suggest that LOX-PP can be explored as a therapeutic molecule in RB.

Purpose: The purpose of this study was to evaluate the elastic modulus, keratocyte-fibroblast-myocyte transformation, and haze formation of the corneal stroma following combined phototherapeutic keratectomy (PTK) and epithelium-off UV-A/riboflavin corneal collagen crosslinking (CXL) using an in vivo rabbit model.

Methods: Rabbits underwent PTK and CXL, PTK only, or CXL 35 days before PTK. Rebound tonometry, Fourier-domain optical coherence tomography, and ultrasound pachymetry were performed on days 7, 14, 21, 42, 70, and 90 post-operatively. Atomic force microscopy, histologic inflammation, and immunohistochemistry for α-smooth muscle actin (α-SMA) were assessed post-mortem.

Results: Stromal haze formation following simultaneous PTK and CXL was significantly greater than in corneas that received PTK only and persisted for more than 90 days. No significant difference in stromal haze was noted between groups receiving simultaneous CXL and PTK and those receiving CXL before PTK. Stromal inflammation did not differ between groups at any time point, although the intensity of α-SMA over the number of nuclei was significantly greater at day 21 between groups receiving simultaneous CXL and PTK and those receiving CXL before PTK. The elastic modulus was significantly greater in corneas receiving simultaneous CXL and PTK compared with those receiving PTK alone.

Conclusions: We showed that stromal haze formation and stromal stiffness is significantly increased following CXL, regardless of whether it is performed at or before the time of PTK. Further knowledge of the biophysical cues involved in determining corneal wound healing duration and outcomes will be important for understanding scarring following CXL and for the development of improved therapeutic options.

Purpose: Positron emission tomography (PET) is widely used in high-precision imaging, which may provide a simple and noninvasive method for the detection of pathology and therapeutic effects. [¹⁸F]-DPA-714 is a second-generation translocator protein (TSPO) positron emission tomography radiotracer that shows great promise in a model of neuroinflammation. In this study, [¹⁸F]-DPA-714 micro-PET imaging was used to evaluate retinal inflammation in mice exposed to blue light, a well-established model of age-related macular degeneration (AMD) for molecular mechanism research and drug screening.

Methods: C57BL/6J melanized mice were subjected to 10,000, 15,000, and 20,000 lux blue light for 5 days (8 h/day) to develop the retinal injury model, and the structure and function of the retina were assessed using hematoxylin-eosin (HE) staining, electroretinography (ERG), and terminal-deoxynucleotidyl transferase (TdT)-mediated nick-end labeling (TUNEL) immunostaining. Then, [¹⁸F]-DPA-714 was injected approximately 100 μCi through each tail vein, and static imaging was performed 1 h after injection. Finally, the mice eyeballs were collected for biodistribution and immune analysis.

Results: The blue light exposure significantly destroyed the structure and function of the retina, and the uptake of [¹⁸F]-DPA-714 in the retinas of the mice exposed to blue light were the most significantly upregulated, which was consistent with the biodistribution data. In addition, the immunohistochemical, western blot, and immunofluorescence data showed an increase in microglial TSPO expression.

Conclusions: [¹⁸F]-DPA-714 micro-PET imaging might be a good method for evaluating early inflammatory status during retinal pathology.

Purpose: The retinal pigment epithelium (RPE) is an important tissue for maintaining a healthy retina. Retinal pigment epithelial cells help regulate nutrient transport to photoreceptors and are heavily pigmented to prevent light scattering. These cells also have junction proteins to form monolayers. Monolayers are key players in pathologies such as age-related macular degeneration (AMD), a leading cause of vision loss in older adults. During AMD, RPE cell detachment can occur, resulting in a loss of junctions. Losing junctions can increase the expression of pro-angiogenic vascular endothelial growth factor (VEGF). This overexpression can cause abnormal blood vessel growth or angiogenesis in the retina. Age-related macular degeneration treatments target VEGF to slow angiogenesis progression. However, other proteins, such as angiopoietin-2 (Ang-2) and the tissue inhibitor of metalloproteinase-1 (TIMP-1), may also play important roles, making them potential targets for treatment. Controlling RPE junction formation will help elucidate the relationship between RPE cell detachment and additional angiogenic factor secretion, lead to more therapeutics, and increase the efficacy of current treatments.

Methods: Micropatterning was used to control the spatial arrangement of primary porcine RPE cells using polydimethylsiloxane (PDMS) stencils. Patterns were formed into PDMS stencils to mimic 10%, 25%, and 50% overall detachment of the RPE monolayer. Zonula-occludens-1 (ZO-1), Ang-2, and VEGF were visualized using immunocytochemical (ICC) staining. An enzyme-linked immunosorbent assay (ELISA) was used to quantify extracellular Ang-2, VEGF, TIMP-1, and TIMP-2 levels. A rod outer segment (OS) phagocytosis assay was performed to determine how RPE junction loss directly affects photoreceptor support.

Results: The growth of primary porcine RPE cells was successfully controlled using stencils. Morphological changes and a decrease in pigmentation were observed, showing a decline in barrier and light absorption functions as degeneration increased. One day after stencil removal, junction proteins were delocalized, and angiogenic factor secretions were correlated with increased levels of detachment. Secretion levels of Ang-2 and TIMP-1 were significantly increased, whereas VEGF and TIMP-2 concentrations were not as affected by varying levels of detachment. OS phagocytosis appeared lower in RPE cells when ZO-1 was affected.

Conclusions: These results suggest a correlation between loss of junctions, abnormal angiogenic protein secretion, and reduced OS phagocytosis. Furthermore, Ang-2 and TIMP-1 proteins might be beneficial targets for AMD treatments, and their roles in retinal diseases deserve further investigation.

Purpose: To understand which cell types, either alone or in combination, contribute to the assembly of the epithelial basement membrane (BM) during corneal wound healing.

Methods: A 3D corneal organotypic model and an in situ rabbit photorefractive keratectomy (PRK) model were used in this study. The 3D corneal organotypic model was established by culturing the rabbit corneal epithelial cells with either corneal fibroblasts or myofibroblasts embedded in collagen type I for 18 days. Corneal fibroblasts were isolated from fresh rabbit corneas, and the myofibroblasts were derived either directly from bone marrow or differentiated from corneal fibroblasts. Immunocytochemistry for alpha-smooth muscle actin (SMA), vimentin, desmin, and vinculin markers confirmed well-differentiated myofibroblasts. Immunohistochemistry was performed in cryofixed sections for BM markers, including laminin alpha-5, laminin beta-3, perlecan, nidogen-1, and collagen type IV. Specimens were also examined with transmission electron microscopy (TEM). Corneas were collected from rabbits after -3 diopter (D) PRK at different time points after surgery, with four corneas at each time point in each group. Cryofixed corneal sections were stained for vimentin, alpha-SMA, and nidogen-1.

Results: The formation of an epithelial BM with expression of laminin alpha-5, laminin beta-3, perlecan, nidogen-1, and collagen IV was observed at the interface between the corneal epithelial cells and corneal fibroblasts. TEM images further confirmed the presence of epithelial BM in organotypic cultures of epithelial cells and corneal fibroblasts. No epithelial BM was observed in cultures of corneal epithelial cells and myofibroblasts (cornea or bone marrow derived), corneal epithelial cells alone, or corneal fibroblasts alone. In rabbit corneas after -3D PRK, a strong association was observed between the regenerating epithelial BM and the presence of corneal fibroblasts at the site of epithelial BM generation.

Conclusions: The corneal epithelial BM assembly is mediated by epithelial cells in coordination with corneal fibroblasts during wound healing.

Purpose: Myopia, or nearsightedness, is the most common form of refractive error and is increasing in prevalence. While significant efforts have been made to identify genetic variants that predispose individuals to myopia, these variants are believed to account for only a small portion of the myopia prevalence, leading to a feedback theory of emmetropization, which depends on the active perception of environmental visual cues. Consequently, there has been renewed interest in studying myopia in the context of light perception, beginning with the opsin family of G-protein coupled receptors (GPCRs). Refractive phenotypes have been characterized in every opsin signaling pathway studied, leaving only Opsin 3 (OPN3), the most widely expressed and blue-light sensing noncanonical opsin, to be investigated for function in the eye and refraction.

Methods: Opn3 expression was assessed in various ocular tissues using an Opn3eGFP reporter. Weekly refractive development in Opn3 retinal and germline mutants from 3 to 9 weeks of age was measured using an infrared photorefractor and spectral domain optical coherence tomography (SD-OCT). Susceptibility to lens-induced myopia was then assessed using skull-mounted goggles with a -30 diopter experimental and a 0 diopter control lens. Mouse eye biometry was similarly tracked from 3 to 6 weeks. A myopia gene expression signature was assessed 24 h after lens induction for germline mutants to further assess myopia-induced changes.

Results: Opn3 was found to be expressed in a subset of retinal ganglion cells and a limited number of choroidal cells. Based on an assessment of Opn3 mutants, the OPN3 germline, but not retina conditional Opn3 knockout, exhibits a refractive myopia phenotype, which manifests in decreased lens thickness, shallower aqueous compartment depth, and shorter axial length, atypical of traditional axial myopias. Despite the short axial length, Opn3 null eyes demonstrate normal axial elongation in response to myopia induction and mild changes in choroidal thinning and myopic shift, suggesting that susceptibility to lens-induced myopia is largely unchanged. Additionally, the Opn3 null retinal gene expression signature in response to induced myopia after 24 h is distinct, with opposing Ctgf, Cx43, and Egr1 polarity compared to controls.

Conclusions: The data suggest that an OPN3 expression domain outside the retina can control lens shape and thus the refractive performance of the eye. Prior to this study, the role of Opn3 in the eye had not been investigated. This work adds OPN3 to the list of opsin family GPCRs that are implicated in emmetropization and myopia. Further, the work to exclude retinal OPN3 as the contributing domain in this refractive phenotype is unique and suggests a distinct mechanism when compared to other opsins.