Profile of Lipoprotein Subclasses in Chinese Primary Open-Angle Glaucoma Patients.

To investigate the plasma lipoprotein subclasses in patients with primary open-angle glaucoma (POAG), a total of 20 Chinese POAG patients on intraocular pressure (IOP)-lowering treatment and 20 age-matched control subjects were recruited. Based on the levels of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C), the study subjects were divided into elevated- and normal-level subgroups. The plasma lipoprotein, lipoprotein subclasses, and oxidized LDL (oxLDL) levels were quantitatively measured. The discrimination potential of the lipoproteins was evaluated using the area under the receiver operating characteristic curve (AUC), and their correlation with clinical parameters was also evaluated. Compared to the control subjects with elevated TC and/or LDL-C levels, the levels of TC, LDL-C, non-high-density lipoprotein cholesterol (non-HDL), LDL subclass LDL3 and small dense LDL (sdLDL), and oxLDL were significantly higher in POAG patients with elevated TC and/or LDL-C levels. No differences in any lipoproteins or the subclasses were found between the POAG patients and control subjects with normal TC and LDL-C levels. Moderate-to-good performance of TC, LDL-C, non-HDL, LDL3, sdLDL, and oxLDL was found in discriminating between the POAG patients and control subjects with elevated TC and/or LDL-C levels (AUC: 0.710-0.950). Significant negative correlations between LDL3 and sdLDL with retinal nerve fiber layer (RNFL) thickness in the superior quadrant and between LDL3 and average RNFL thickness were observed in POAG patients with elevated TC and/or LDL-C levels. This study revealed a significant elevation of plasma lipoproteins, especially the LDL subclasses, in POAG patients with elevated TC and/or LDL-C levels, providing insights on monitoring specific lipoproteins in POAG patients with elevated TC and/or LDL-C.

Oxidized low-density lipoprotein changes the inflammatory status and metabolomics profiles in human and mouse macrophages and microglia.

The conjunctiva of primary open angle glaucoma patients showed high level of oxidized low-density lipoprotein (ox-LDL), which is associated with the inflammatory response. Microglia and macrophages are the immune cells involved in retinal ganglion cell survival regulation; yet, their roles of the ox-LDL-induced inflammation in glaucoma remain elusive. Here we aimed to investigate the lipid uptake, inflammatory cytokine expression, and metabolomics profiles of human and murine-derived microglial and macrophage cell lines treated with ox-LDL. Under the same ox-LDL concentration, macrophages exhibited higher lipid uptake and expression of pro-inflammatory cytokines as compared to microglia. The ox-LDL increased the levels of fatty acid metabolites in macrophages and sphingomyelin metabolites in microglia. In summary, this study revealed the heterogeneity in the inflammatory capacity and metabolic profiles of macrophages and microglia under the stimulation of ox-LDL.

Green tea extract enhances retinal ganglion cell survival and axonal regeneration in rats with optic nerve injury.

Current clinical treatments have not yet effectively cured progressive retinal ganglion cell (RGC) death and axonal degeneration after optic nerve (ON) injury. We previously demonstrated green tea extract (GTE) can reduce RGC death in rats after ischemic injury. Here, we aim to determine the prophylactic and therapeutic effects and mechanisms of GTE on RGC survival and axonal regeneration in rats with ON injury. GTE (275 or 550 mg/kg) was administered intragastrically for 7 d before or 14 d post-ON crush surgery in adult Fischer 344 rats. Rats with pre- or post-operative treatment of 275 mg/kg GTE showed significantly higher numbers of RGCs and regenerated axons post-ON injury with improved pupillary light reflex as compared to saline-treated rats. Akt and Erk p42/44 activation was higher in the retina of rats given 275 mg/kg GTE pre-surgery, whereas Stat3 activation was higher in those with 275 mg/kg GTE post-operation. Less activated microglia were observed in rats with pre-treatment of 275 or 550 mg/kg GTE. RNA sequencing analysis identified the downregulation of inflammation, apoptosis, and microglia activation genes in the retina of rats with pre- or post-treatment with 275 mg/kg GTE as compared to the saline-treated rats. In summary, this study revealed the prophylactic and therapeutic treatment effects of GTE on RGC survival and axonal regeneration in rats with ON injury, indicating a potential alternative treatment for traumatic optic neuropathy.

Reduction of Laser-Induced Choroidal Neovascularization in Mice With Erythropoietin RNA Interference.

Purpose: The purpose of this study was to evaluate the pathological involvement of erythropoietin (EPO) in experimental choroidal neovascularization (CNV) and its association with neovascular age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV) in the Chinese population. Methods: Treatment effect of recombinant EPO protein were assessed by human umbilical vein endothelial cell (HUVEC) proliferation, migration, and tube formation, and ex vivo choroid-sprouting ability. The effect of intravitreal injection of Epo siRNA against neovascularization was evaluated in the laser-induced CNV mouse model. In addition, the association of EPO variants with neovascular AMD and PCV was determined. Results: Exogenous supplementation of EPO significantly enhanced the migration and tube formation of HUVECs and promoted ex vivo choroid sprouting in mouse retinal pigment epithelium (RPE)-choroid-sclera complex culture. In the experimental CNV mouse model, Epo expression was found to be significantly upregulated by 3.5-folds in RPE-choroid-sclera complex at day 10 after laser induction as compared to the baseline. Immunofluorescence analysis showed that Epo was mainly expressed around the vascular endothelial cells in the RPE-choroid-sclera complex. Intravitreal injection of siRNA targeting Epo reduced 40% Epo expression and 40% CNV lesion areas as compared to the scramble control. However, EPO variants were not associated with neovascular AMD nor PCV in the Chinese population. Conclusions: This study revealed the promotion of human endothelial cell tube formation in vitro and choroid sprouting ex vivo by EPO, and the reduction of laser-induced CNV in vivo by Epo RNA interference. Translational Relevance: Targeting EPO could be a potential additional treatment for CNV-related diseases.

Whole exome sequencing analysis identifies novel Stargardt disease-related gene mutations in Chinese Stargardt disease and retinitis pigmentosa patients.

OBJECTIVES: To delineate the disease-causing mutations of the Stargardt disease-related genes in Chinese patients diagnosed with Stargardt disease or retinitis pigmentosa (RP) by whole exome sequencing analysis. METHODS: A total of 123 sporadic RP or Stargardt disease patients and 2 Stargardt disease families were recruited. All sporadic patients and the probands of the families were subjected to whole exome sequencing analysis. The candidate mutations were verified by direct sequencing based on the cosegregation pattern and in 200 control subjects and by the bioinformatics analyses. RESULTS: A total of three reported ABCA4 mutations were identified in the probands of the two Stargardt disease families. The probands and the affected family members with either homozygous or compound heterozygous mutations showed typical Stargardt disease features, which was absent in their unaffected family members. The cosegregation pattern confirmed the mode of recessive inheritance. Moreover, two sporadic Stargardt disease patients were identified to carry two novel ABCA4 and one PROM1 mutations. In addition, 13 novel variants were found in 119 sporadic RP patients in 7 Stargardt disease-related genes, and 8 novel missense variants were conserved across different species and predicted to be damaging to the protein. All 15 novel variants were absent in our 200 control subjects. CONCLUSIONS: This study revealed 22.4% study subjects carrying Stargardt disease-related gene mutations with total 15 novel variants in seven Stargardt disease-related genes, assuring that targeted next-generation sequencing analysis is a high throughput strategy to facilitate the clinical diagnosis from suspicious patients and recommended as a routine examination for inherited retinal dystrophies.

Hydroxychloroquine treatment on SARS-CoV-2 receptor ACE2, TMPRSS2 and NRP1 expression in human primary pterygium and conjunctival cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen for coronavirus disease 2019 (COVID-19) pandemic. Its infection depends on the binding of spike protein to the host cell receptor angiotensin-converting enzyme 2 (ACE2), type II transmembrane serine protease (TMPRSS2) and neuropilin-1 (NRP1). Hydroxychloroquine has been applied as one of the COVID-19 treatment strategies. Here we aimed to evaluate hydroxychloroquine treatment on SARS-CoV-2 receptor expression in human primary pterygium and conjunctival cells and its potential influences. Expression of ACE2, TMPRSS2 and NRP1 proteins were found in the epithelial layer of both primary pterygium and conjunctiva tissues as well as in their isolated fibroblasts. High concentration of hydroxychloroquine treatment significantly reduced the viability of both primary pterygium and conjunctival cells. ACE2 protein expression was significantly decreased in both pterygium and conjunctival cells after hydroxychloroquine treatment. Hydroxychloroquine also reduced NRP1 protein expression in conjunctival cells. In contrast, TMPRSS2 protein expression showed slightly increased in conjunctival cells. Notably, ROS production and SOD2 expression was significantly elevated in both pterygium and conjunctival cells after hydroxychloroquine treatment. In summary, this study revealed the reduction of ACE2 and NRP1 expression by hydroxychloroquine in human primary pterygium and conjunctival fibroblasts; yet with the increase in TMPRSS2 expression and oxidative stress and decrease in cell viability. Implementation of hydroxychloroquine for COVID-19 treatment should be carefully considered with its potential side effects and in combination with TMPRSS2 inhibitor.

Green Tea Catechins Attenuate Human Primary Pterygium Cell Survival and Migration Via Modulation of ERK p42/p44 and p38 Pathways.

Pterygium belongs to an ocular surface disease with triangular-shaped hyperplastic growth, characterized by conjunctivalization, inflammation, and connective tissue remodeling. We previously demonstrated neoplastic-like properties of pterygium cells. Green tea catechin, (-)-epigallocatechin gallate (EGCG), has been shown to possess antitumorigenic properties; herein, we aimed to determine the effects of green tea catechins on human primary pterygium cell survival and migration and compared to that on patients' conjunctival cells. Both human primary pterygium and conjunctival cells expressed EGCG receptor, the 67 kDa laminin receptor. Seven-day treatment of green tea extract (Theaphenon E; 16.25 µg/mL) and EGCG (25 µM) attenuated pterygium cell proliferation by 16.78% (p < 0.001) and 24.09% (p < 0.001) respectively, without significantly influencing conjunctival cells. Moreover, green tea extract (16.25 µg/mL) and EGCG (25 µM) treatments also hindered pterygium cell migration by 35.22% (p < 0.001) and 25.20% (p = 0.019), respectively, but not conjunctival cells. Yet, green tea extract and EGCG treatments did not significantly induce pterygium cell apoptosis. Furthermore, green tea extract and EGCG treatments significantly increased the phosphorylation of p38 protein but reduced the phosphorylation of p42/p44 protein in pterygium cells. In summary, this study revealed that green tea extract and EGCG attenuated human primary pterygium cell survival and migration in vitro without damaging conjunctival cells, suggesting a novel potential therapeutic approach for primary pterygium treatment.

Agonist of growth hormone-releasing hormone enhances retinal ganglion cell protection induced by macrophages after optic nerve injury.

Optic neuropathies are leading causes of irreversible visual impairment and blindness, currently affecting more than 100 million people worldwide. Glaucoma is a group of optic neuropathies attributed to progressive degeneration of retinal ganglion cells (RGCs). We have previously demonstrated an increase in survival of RGCs by the activation of macrophages, whereas the inhibition of macrophages was involved in the alleviation on endotoxin-induced inflammation by antagonist of growth hormone-releasing hormone (GHRH). Herein, we hypothesized that GHRH receptor (GHRH-R) signaling could be involved in the survival of RGCs mediated by inflammation. We found the expression of GHRH-R in RGCs of adult rat retina. After optic nerve crush, subcutaneous application of GHRH agonist MR-409 or antagonist MIA-602 promoted the survival of RGCs. Both the GHRH agonist and antagonist increased the phosphorylation of Akt in the retina, but only agonist MR-409 promoted microglia activation in the retina. The antagonist MIA-602 reduced significantly the expression of inflammation-related genes Il1b, Il6, and Tnf Moreover, agonist MR-409 further enhanced the promotion of RGC survival by lens injury or zymosan-induced macrophage activation, whereas antagonist MIA-602 attenuated the enhancement in RGC survival. Our findings reveal the protective effect of agonistic analogs of GHRH on RGCs in rats after optic nerve injury and its additive effect to macrophage activation, indicating a therapeutic potential of GHRH agonists for the protection of RGCs against optic neuropathies especially in glaucoma.

Corneal curvature-associated MTOR variant differentiates mild myopia from high myopia in Han Chinese population.

BACKGROUND: Myopia is the most prevalent ocular disorder in the world, and corneal parameters have been regarded as key ocular biometric parameters determining the refractive status. Here, we aimed to determine the association of genome-wide association study-identified corneal curvature (CC)-related gene variants with different severity of myopia and ocular biometric parameters in Chinese population. METHODS: Total 2,101 unrelated Han Chinese subjects were recruited, including 1,649 myopia and 452 control subjects. Five previously reported CC-associated gene variants (PDGFRA, MTOR, WNT7B, CMPK1 and RBP3) were genotyped by TaqMan assay, and their association with different myopia severity and ocular biometric parameters were evaluated. RESULTS: Joint additive effect analysis showed that MTOR rs74225573 paired with PDGFRA rs2114039 (P = .009, odds ratio (OR) = 4.91) or CMPK1 rs17103186 (P = .002, OR = 13.03) were significantly associated with higher risk in mild myopia. Critically, mild myopia subjects had significantly higher frequency in MTOR rs74225573 C allele than high myopia subjects (P = .003), especially in male subjects (P = .001, OR = 0.49). High myopia subjects carrying MTOR rs74225573 C allele have significant flatter CC (P = .035) and longer corneal radius (P = .044) than those carrying TT genotype. CONCLUSION: This study revealed that male high myopia subjects are more prone to carry CC-related MTOR rs74225573 T allele, whereas mild myopia subjects are prone to carry the C allele. MTOR rs7422573 variant could be a genetic marker to differentiate mild from high myopia in risk assessment. ABBREVIATIONS: ACD: anterior chamber depth; AL: axial length; AL/CR: axial length/corneal radius ratio; ANOVA: analysis of variance; CC: corneal curvature; CCT: central corneal thickness; C.I.: confidence interval; CMPK1: cytidine/uridine monophosphate kinase 1; CR: corneal radius; D: diopter; GWAS: genome-wide association studies; HWE: Hardy-Weinberg equilibrium; LT: lens thickness; MIPEP: mitochondrial intermediate peptidase; MTOR: mechanistic target of rapamycin kinase; OR: odds ratio; PDGFRA: platelet-derived growth factor receptor-α; RBP3: retinol-binding protein 3; SD: standard deviation; SE: spherical equivalence; SNTB1: syntrophin beta 1; VCD: vitreous chamber depth; VIPR2: vasoactive intestinal peptide receptor 2; WNT7B: wingless/integrated family member 7B.

CXCL5/CXCR2 modulates inflammation-mediated neural repair after optic nerve injury.

BACKGROUND: Previous studies reported that mild inflammation promotes retinal ganglion cell (RGC) survival and axonal regeneration after optic nerve (ON) injury with involvement of infiltrating macrophages and neutrophils. Here we aimed to evaluate the involvement and regulation of the main inflammatory chemokine pathway CXCL5/CXCR2 in the inflammation-mediated RGC survival and axonal regeneration in mice after ON injury. METHODS: The expressions and cellular locations of CXCL5 and CXCR2 were confirmed in mouse retina. Treatment effects of recombinant CXCL5 and CXCR2 antagonist SB225002 were studied in the explant culture and the ON injury model with or without lens injury. The number of RGCs, regenerating axons, and inflammatory cells were determined, and the activation of Akt andSTAT3 signaling pathways were evaluated. RESULTS: Cxcr2 and Cxcl5 expressions were increased after ON and lens injury. Addition of recombinant CXCL5 promoted RGC survival and neurite outgrowth in retinal explant culture with increase in the number of activated microglia, which was inhibited by SB225002 or clodronate liposomes. Recombinant CXCL5 also alleviated RGC death and promoted axonal regeneration in mice after ON injury, and promoted the lens injury-induced RGC protection with increase in the number of activated CD68+ cells. SB225002 inhibited lens injury-induced cell infiltration and activation, and attenuated the promotion effect on RGC survival and axonal regeneration through reduction of lens injury-induced Akt activation. CONCLUSIONS: CXCL5 promotes RGC survival and axonal regeneration after ON injury and further enhances RGC protection induced by lens injury with CD68+ cell activation, which is attenuated by CXCR2 antagonist. CXCL5/CXCR2 could be a potential therapeutic target for RGC survival promotion after ON injury.

Genotype-phenotype correlation and interaction of 4q25, 15q14 and MIPEP variants with myopia in southern Chinese population.

BACKGROUND/AIMS: To determine the association and interaction of genome-wide association study-reported variants for Asian populations with myopia and ocular biometric parameters in southern Chinese population. METHODS: Totally, 1462 unrelated Han Chinese subjects were recruited with complete ophthalmic examinations, including 1196 myopia and 266 control subjects. A total of nine variants were selected for TaqMan genotyping. The genetic association, joint additive effect and genotype-phenotype correlation were investigated. RESULTS: The 4q25 variant rs10034228 (p=0.002, OR=0.56) and MIPEP variant rs9318086 (p=0.004, OR=1.62) were found to be significantly associated with myopia as well as different severity of myopia. Moreover, 15q14 variant rs524952 (p=0.015, OR=1.49) also showed mild association with myopia and high myopia. However, there was no significant association of CTNND2, vasoactive intestinal peptide receptor 2 and syntrophin beta 1 variants with myopia. Joint additive analysis revealed that the subjects carrying 6 risk alleles of the 3 associated variants were 10-fold higher risk predisposed to high myopia. Genotype-phenotype correlation analysis revealed that high myopia subjects carrying 4q25 rs10034228 T allele showed thicker central corneal thickness, whereas high myopia subjects carrying 15q14 rs524952 A allele were associated with longer axial length and larger curvature ratio. CONCLUSION: This study revealed significant association of 4q25, 15q14 and MIPEP variants with myopia and different severity of myopia in southern Chinese population, joint additively enhancing 10-fold of risk predisposing to high myopia. The correlation of these associated variants with axial length and corneal parameters suggests their contribution to the refractive status in high myopia subjects.

Longitudinal evaluation of immediate inflammatory responses after intravitreal AAV2 injection in rats by optical coherence tomography.

Gene therapy has been proposed as a feasible strategy for RGC survival and optic nerve regeneration. Some preclinical and clinical studies revealed intraocular inflammation after intravitreal injection of adeno-associated virus (AAV) by slit-lamp or indirect ophthalmoscope. Here we evaluate the longitudinal profile of immediate inflammatory responses after AAV2 injection in rat retina and vitreous body by optical coherence tomography (OCT). Adult Fischer F344 rats were intravitreally injected once with saline, AAV2 or zymosan. Retinal thickness and cell infiltration were recorded by OCT longitudinally for 2 months and verified by histological analysis. The transduction rate of single intravitreal AAV2 injection was 21.3 ± 4.9% of whole retina, and the transduction efficiency on RGCs was 91.5 ± 2.5% in the transduced area. Significant increase in cell infiltration was observed from Day 1-3 after AAV2 injection, compared to very few infiltrating cells observed in the saline-injected group. The infiltrating cells ceased at Day 5 after intravitreal injection and remained absent at 2 months. The thicknesses of total and inner retina were increased along Day 1-3 after AAV2 injection, but reverted to normal afterwards. The surviving RGCs in the AAV2-injected groups at Day 14 showed no significant difference compared to saline-injected group. In summary, this study revealed the immediate inflammatory responses and retinal edema after intravitreal AAV2 injection in normal rats, without influencing long-term retinal thickness and RGC survival. OCT can be implemented for the time-lapse in vivo evaluation of inflammatory response after AAV-mediated gene therapy through intravitreal injection.

Green Tea Extract Ameliorates Ischemia-Induced Retinal Ganglion Cell Degeneration in Rats.

PURPOSE: Oxidative stress induced by reduced blood circulation is a critical pathological damage to retinal ganglion cells (RGCs) in glaucoma. We previously showed that green tea extract (GTE) and its catechin constituents alleviate sodium iodate-induced retinal degeneration in rats. Here, we investigated the therapeutic effect of GTE on ischemia-induced RGC degeneration in rats. METHODS: RGC degeneration was induced by ischemic reperfusion in adult Fischer F344 rats. Green tea extract (Theaphenon E) was intragastrically administered 4 times within 48 hours after ischemia. RGC survival, pupillary light reflex, expressions of cell apoptosis, oxidative stress, and inflammation-related proteins were studied. RESULTS: Ischemic reperfusion significantly induced apoptotic RGCs, RGC loss, and larger constricted pupil area compared to the untreated normal rats. Expressions of activated caspase-3 and caspase-8, Sod2, and inflammation-related proteins as well as p38 phosphorylation were significantly upregulated in the ischemia-injured rats. Compared to the saline-fed ischemic rats, significantly higher number of surviving RGCs, less apoptotic RGCs, and smaller constricted pupil area were observed in the GTE-fed ischemic rats. GTE also reduced the increased protein expressions caused by ischemic injury but enhanced the Jak phosphorylation in the retina. Notably, green tea extract did not affect the survival of RGCs in the uninjured normal rats. CONCLUSIONS: In summary, GTE offers neuroprotection to RGCs under ischemic challenge, suggesting a potential therapeutic strategy for glaucoma and optic neuropathies.