Early detection and correlation of tear fluid inflammatory factors that influence angiogenesis in premature infants with and without retinopathy of prematurity.

Purpose: To measure the levels of inflammatory factors in tear fluid of pre-term infants with and without retinopathy of prematurity (ROP). Methods: The cross-sectional pilot study included 29 pre-term infants undergoing routine ROP screening. Pre-term infants were grouped as those without ROP (no ROP; n = 14) and with ROP (ROP; n = 15). Sterile Schirmer's strips were used to collect the tear fluid from pre-term infants. Inflammatory factors such as interleukin (IL)-6, IL-8, MCP1 (Monocyte Chemoattractant Protein 1; CCL2), RANTES (Regulated on Activation, Normal T Cell Expressed and Secreted; CCL5), and soluble L-selectin (sL-selectin) were measured by cytometric bead array using a flow cytometer. Results: Birth weight (BW) and gestation age (GA) were significantly (P < 0.05) lower in pre-term infants with ROP compared with those without ROP. Higher levels of RANTES (P < 0.05) and IL-8 (P = 0.09) were observed in the tear fluid of pre-term infants with ROP compared with those without ROP. Lower levels of tear fluid IL-6 (P = 0.14) and sL-selectin (P = 0.18) were measured in pre-term infants with ROP compared with those without ROP. IL-8 and RANTES were significantly (P < 0.05) higher in the tear fluid of pre-term infants with stage 3 ROP compared with those without ROP. Tear fluid RANTES level was observed to be inversely associated with GA and BW of pre-term infants with ROP and not in those without ROP. Furthermore, the area under the curve and odds ratio analysis demonstrated the relevance of RANTES/BW (AUC = 0.798; OR-7.2) and RANTES/MCP1 (AUC = 0.824; OR-6.8) ratios in ROP. Conclusions: Distinct changes were observed in the levels of tear inflammatory factors in ROP infants. The status of RANTES in ROP suggests its possible role in pathobiology and warrants further mechanistic studies to harness it in ROP screening and management.

Correlation between tear levels of vascular endothelial growth factor and vitamin D at retinopathy of prematurity stages in preterm infants.

Deregulation of vascular endothelial growth factor (VEGF) levels leads to retinopathy of prematurity (ROP). Vitamin D (VIT-D) is known to regulate VEGF in an oxygen dependent manner. The purpose of this study was to correlate tear levels of VEGF and VIT-D with different ROP stages in preterm infants. In this prospective cross-sectional study, we enrolled 104 pre-term infants. They were grouped into: Group-1 (Classical ROP) and Group-2 (Aggressive ROP), which were further subdivided into Group-1A (progressing), Group-1B (regressing), Group-2A (pre-treatment), and Group-2B (post-treatment). Tear VEGF and VIT-D levels and their association with different ROP stages were assessed. Stage 1 and stage 2 had higher whereas stage 3 had lower VEGF levels in Group-1B compared to Group-1A. Stage 1 and stage 3 showed higher levels of VIT-D with no difference in stage 2 in Group-1B compared to Group-1A., Group-2B showed higher VEGF and lower VIT-D levels compared to Group-2A. Presence of a positive correlation at an early stage (stage 1) of ROP and a negative correlation at a more advanced stage (stage 3) of ROP with VIT-D and VEGF implies stage-specific distinct signaling crosstalk. These findings suggest that VIT-D supplementation may have the potential to modify the course and outcome of ROP.

Depleted hexokinase1 and lack of AMPKα activation favor OXPHOS-dependent energetics in retinoblastoma tumors.

Lack of retinoblastoma (Rb) protein causes aggressive intraocular retinal tumors in children. Recently, Rb tumors have been shown to have a distinctly altered metabolic phenotype, such as reduced expression of glycolytic pathway proteins alongside altered pyruvate and fatty acid levels. In this study, we demonstrate that loss of hexokinase 1(HK1) in tumor cells rewires their metabolism allowing enhanced oxidative phosphorylation-dependent energy production. We show that rescuing HK1 or retinoblastoma protein 1 (RB1) in these Rb cells reduced cancer hallmarks such as proliferation, invasion, and spheroid formation and increased their sensitivity to chemotherapy drugs. Induction of HK1 was accompanied by a metabolic shift of the cells to glycolysis and a reduction in mitochondrial mass. Cytoplasmic HK1 bound Liver Kinase B1 and phosphorylated AMP-activated kinase-α (AMPKα Thr172), thereby reducing mitochondria-dependent energy production. We validated these findings in tumor samples from Rb patients compared to age-matched healthy retinae. HK1 or RB1 expression in Rb-/- cells led to a reduction in their respiratory capacity and glycolytic proton flux. HK1 overexpression reduced tumor burden in an intraocular tumor xenograft model. AMPKα activation by AICAR also enhanced the tumoricidal effects of the chemotherapeutic drug topotecan in vivo. Therefore, enhancing HK1 or AMPKα activity can reprogram cancer metabolism and sensitize Rb tumors to lower doses of existing treatments, a potential therapeutic modality for Rb.

Activated cGAS/STING signaling elicits endothelial cell senescence in early diabetic retinopathy.

Diabetic retinopathy (DR) is a leading cause of blindness in working-age adults and remains an important public health issue worldwide. Here we demonstrate that the expression of stimulator of interferon genes (STING) is increased in patients with DR and animal models of diabetic eye disease. STING has been previously shown to regulate cell senescence and inflammation, key contributors to the development and progression of DR. To investigate the mechanism whereby STING contributes to the pathogenesis of DR, diabetes was induced in STING-KO mice and STINGGT (loss-of-function mutation) mice, and molecular alterations and pathological changes in the retina were characterized. We report that retinal endothelial cell senescence, inflammation, and capillary degeneration were all inhibited in STING-KO diabetic mice; these observations were independently corroborated in STINGGT mice. These protective effects resulted from the reduction in TBK1, IRF3, and NF-κB phosphorylation in the absence of STING. Collectively, our results suggest that targeting STING may be an effective therapy for the early prevention and treatment of DR.

Ocular surface immune cell diversity in dry eye disease.

Dry eye disease (DED) is a multifactorial chronic ocular surface inflammatory condition. Disease severity has been directly related to the immuno-inflammatory status of the ocular surface. Any perturbation in the orchestrated functional harmony between the ocular surface structural cells and immune cells, both resident and trafficking ones, can adversely affect ocular surface health. The diversity and contribution of ocular surface immune cells in DED have been of interest for over a couple of decades. As is true with any mucosal tissue, the ocular surface harbors a variety of immune cells of the innate-adaptive continuum and some of which are altered in DED. The current review curates and organizes the knowledge related to the ocular surface immune cell diversity in DED. Ten different major immune cell types and 21 immune cell subsets have been studied in the context of DED in human subjects and in animal models. The most pertinent observations are increased ocular surface proportions of neutrophils, dendritic cells, macrophages, and T cell subsets (CD4+; CD8+; Th17) along with a decrease in T regulatory cells. Some of these cells have demonstrated disease-causal association with ocular surface health parameters such as OSDI score, Schirmer's test-1, tear break-up time, and corneal staining. The review also summarizes various interventional strategies studied to modulate specific immune cell subsets and reduce DED severity. Further advancements would enable the use of ocular surface immune cell diversity, in patient stratification, i.e. DED-immunotypes, disease monitoring, and selective targeting to resolve the morbidity related to DED.

Assessing clinical and molecular outcomes of prophylactic thermal pulsation therapy on ocular surface health following refractive surgery.

Purpose: To study ocular surface signs, symptoms, and tear film composition following prophylactic thermal pulsation therapy (TPT) prior to refractive surgery, and to compare these outcomes with those who underwent TPT after refractive surgery. Methods: Patients with mild-to-moderate evaporative dry eye disease (DED) and/or meibomian gland dysfunction (MGD) undergoing refractive surgery were included. Group 1 patients received TPT (LipiFlow) prior to laser-assisted in situ keratomileusis (LASIK; n = 32, 64 eyes), and Group 2 patients received TPT three months after LASIK (n = 27, 52 eyes). Ocular Surface Disease Index (OSDI) score, Schirmer's test (ST1, ST2), Tear Breakup Time (TBUT), meibography, and tear fluid were obtained preoperatively and at three months postoperatively in Groups 1 and 2. Additional postoperative evaluation was performed three months after TPT in Group 2. Tear soluble factor profile was measured by multiplex enzyme-linked immunosorbent assay (ELISA) using flow cytometry. Results: Postoperative OSDI score was significantly lower and TBUT was significantly higher when compared with matched preoperative values of Group 1 participants. On the other hand, the postoperative OSDI score was significantly higher and TBUT significantly lower when compared with matched preoperative values of Group 2 participants. TPT significantly reduced the postoperative elevation in OSDI and significantly reduced the postoperative reduction in TBUT in Group 2 participants. Tear Matrix metalloproteinase-9/ Tissue inhibitor matrix metalloproteinase 1 (MMP-9/TIMP1) ratio was significantly higher, postoperatively, when compared with matched preoperative levels in Group 2. However, MMP9/TIMP1 ratio remained unaltered in Group 1 participants. Conclusion: TPT prior to refractive surgery improved postsurgical ocular surface signs and symptoms and reduced tear inflammatory factors, thereby suggesting the plausibility of reduced post-refractive surgery DED in patients.

Increased LCN2 (lipocalin 2) in the RPE decreases autophagy and activates inflammasome-ferroptosis processes in a mouse model of dry AMD.

In dry age-related macular degeneration (AMD), LCN2 (lipocalin 2) is upregulated. Whereas LCN2 has been implicated in AMD pathogenesis, the mechanism remains unknown. Here, we report that in retinal pigmented epithelial (RPE) cells, LCN2 regulates macroautophagy/autophagy, in addition to maintaining iron homeostasis. LCN2 binds to ATG4B to form an LCN2-ATG4B-LC3-II complex, thereby regulating ATG4B activity and LC3-II lipidation. Thus, increased LCN2 reduced autophagy flux. Moreover, RPE cells from cryba1 KO, as well as sting1 KO and Sting1Gt mutant mice (models with abnormal iron chelation), showed decreased autophagy flux and increased LCN2, indicative of CGAS- and STING1-mediated inflammasome activation. Live cell imaging of RPE cells with elevated LCN2 also showed a correlation between inflammasome activation and increased fluorescence intensity of the Liperfluo dye, indicative of oxidative stress-induced ferroptosis. Interestingly, both in human AMD patients and in mouse models with a dry AMD-like phenotype (cryba1 cKO and KO), the LCN2 homodimer variant is increased significantly compared to the monomer. Sub-retinal injection of the LCN2 homodimer secreted by RPE cells into NOD-SCID mice leads to retinal degeneration. In addition, we generated an LCN2 monoclonal antibody that neutralizes both the monomer and homodimer variants and rescued autophagy and ferroptosis activities in cryba1 cKO mice. Furthermore, the antibody rescued retinal function in cryba1 cKO mice as assessed by electroretinography. Here, we identify a molecular pathway whereby increased LCN2 elicits pathophysiology in the RPE, cells known to drive dry AMD pathology, thus providing a possible therapeutic strategy for a disease with no current treatment options.Abbreviations: ACTB: actin, beta; Ad-GFP: adenovirus-green fluorescent protein; Ad-LCN2: adenovirus-lipocalin 2; Ad-LCN2-GFP: adenovirus-LCN2-green fluorescent protein; LCN2AKT2: AKT serine/threonine kinase 2; AMBRA1: autophagy and beclin 1 regulator 1; AMD: age-related macular degeneration; ARPE19: adult retinal pigment epithelial cell line-19; Asp278: aspartate 278; ATG4B: autophagy related 4B cysteine peptidase; ATG4C: autophagy related 4C cysteine peptidase; ATG7: autophagy related 7; ATG9B: autophagy related 9B; BLOC-1: biogenesis of lysosomal organelles complex 1; BLOC1S1: biogenesis of lysosomal organelles complex 1 subunit 1; C57BL/6J: C57 black 6J; CGAS: cyclic GMP-AMP synthase; ChQ: chloroquine; cKO: conditional knockout; Cys74: cysteine 74; Dab2: DAB adaptor protein 2; Def: deferoxamine; DHE: dihydroethidium; DMSO: dimethyl sulfoxide; ERG: electroretinography; FAC: ferric ammonium citrate; Fe2+: ferrous; FTH1: ferritin heavy chain 1; GPX: glutathione peroxidase; GST: glutathione S-transferase; H2O2: hydrogen peroxide; His280: histidine 280; IFNL/IFNλ: interferon lambda; IL1B/IL-1ß: interleukin 1 beta; IS: Inner segment; ITGB1/integrin ß1: integrin subunit beta 1; KO: knockout; LC3-GST: microtubule associated protein 1 light chain 3-GST; C-terminal fusion; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LCN2: lipocalin 2; mAb: monoclonal antibody; MDA: malondialdehyde; MMP9: matrix metallopeptidase 9; NLRP3: NLR family pyrin domain containing 3; NOD-SCID: nonobese diabetic-severe combined immunodeficiency; OS: outer segment; PBS: phosphate-buffered saline; PMEL/PMEL17: premelanosome protein; RFP: red fluorescent protein; rLCN2: recombinant LCN2; ROS: reactive oxygen species; RPE SM: retinal pigmented epithelium spent medium; RPE: retinal pigment epithelium; RSL3: RAS-selective lethal; scRNAseq: single-cell ribonucleic acid sequencing; SD-OCT: spectral domain optical coherence tomography; shRNA: small hairpin ribonucleic acid; SM: spent medium; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; STAT1: signal transducer and activator of transcription 1; STING1: stimulator of interferon response cGAMP interactor 1; TYR: tyrosinase; VCL: vinculin; WT: wild type.

Ocular Surface Immune Cell Profiles in Contact Lens-Induced Discomfort.

Purpose: Contact lens-induced discomfort (CLD) remains a primary factor in discontinuation or prevention of contact lens wear. Thus, we investigated the role of ocular surface immune cells in subjects with CLD. Methods: Habitual contact lens (CL) wearers with CLD (n = 19; 38 eyes) and without CLD (n = 21; 42 eyes) as determined by the Contact Lens Dry Eye Questionnaire-8 was included in a trial. Enrolled subjects used either of the two types of CL (designated as CL-A or CL-D). Ocular surface cells from the bulbar conjunctiva were obtained by impression cytology. The collected cells were phenotyped using fluorochrome-conjugated antibodies specific for leukocytes (CD45+), neutrophils (CD66b+,High,Low), macrophages (CD163+), T cells (CD3+CD4+, CD3+CD8+), natural killer (NK) cells (CD56+, High, Low), natural killer T (NKT) cells (CD3+CD56+), and gamma delta T (γδT) cells (CD3+γδTCR+) by flow cytometry. Further, corneal dendritic cell density (cDCD) was also determined using in vivo confocal microscopy. Results: Significantly higher proportions of CD45+ cells were observed in subjects with CLD compared to those without CLD. The percentages of CD66bTotal,Low, CD163+, pan T cells, CD4+T cells, CD8+T cells, CD56Total,High,Low (NK) cells, and NKT cells, as well as the CD4/CD8 ratio, were significantly higher in CLD subjects. The proportion of T cells (CD4, CD8, CD4/CD8 ratio, NKT cells) and macrophages exhibited a direct association with discomfort score. The percentages of CD45+, CD66bTotal,Low, CD163+, CD3+, CD56Total,High,Low, and NKT cells and cDCD were significantly higher in CLD subjects wearing CL-D. The percentages of CD66bHigh, CD4+T cells, CD8+T cells, NKT cells, and CD4/CD8 ratio were significantly higher in CLD subjects wearing CL-A. Conclusions: Increased proportions of ocular surface immune cells are observed in CLD, and the lens type could impact the immune cells associated with CLD. Translational Relevance: The association between the proportion of altered ocular surface immune cell subsets and contact lens-induced discomfort underpins the importance of considering immune-related aspects during contact lens development and in the clinical management of ocular surface pain.

Integrated Analysis of Cancer Tissue and Vitreous Humor from Retinoblastoma Eyes Reveals Unique Tumor-Specific Metabolic and Cellular Pathways in Advanced and Non-Advanced Tumors.

Retinoblastoma (Rb) is a pediatric intraocular malignancy that is proposed to originate from maturing cone cell precursors in the developing retina. The molecular mechanisms underlying the biological and clinical behaviors are important to understand in order to improve the management of advanced-stage tumors. While the genetic causes of Rb are known, an integrated understanding of the gene expression and metabolic processes in tumors of human eyes is deficient. By integrating transcriptomic profiling from tumor tissues and metabolomics from tumorous eye vitreous humor samples (with healthy, age-matched pediatric retinae and vitreous samples as controls), we uncover unique functional associations between genes and metabolites. We found distinct gene expression patterns between clinically advanced and non-advanced Rb. Global metabolomic analysis of the vitreous humor of the same Rb eyes revealed distinctly altered metabolites, indicating how tumor metabolism has diverged from healthy pediatric retina. Several key enzymes that are related to cellular energy production, such as hexokinase 1, were found to be reduced in a manner corresponding to altered metabolites; notably, a reduction in pyruvate levels. Similarly, E2F2 was the most significantly elevated E2F family member in our cohort that is part of the cell cycle regulatory circuit. Ectopic expression of the wild-type RB1 gene in the Rb-null Y79 and WERI-Rb1 cells rescued hexokinase 1 expression, while E2F2 levels were repressed. In an additional set of Rb tumor samples and pediatric healthy controls, we further validated differences in the expression of HK1 and E2F2. Through an integrated omics analysis of the transcriptomics and metabolomics of Rb, we uncovered a significantly altered tumor-specific metabolic circuit that reduces its dependence on glycolytic pathways and is governed by Rb1 and HK1.

Corneal Confocal Microscopy Features and Tear Molecular Profile in Study Participants with Discordance between Ocular Surface Disease Clinical Signs and Discomfort.

Various ocular surface conditions such as dry eye disease can present with severe discomfort and pain. However, it is clinically challenging to establish etiology and prescribe correct treatment in patients who have a lot of discordance between symptoms and signs. To understand the basis of such discordance, we stratified subjects with ocular surface pain based on concordance between the severity of signs and symptoms and evaluated corneal structural features and tear molecular factors. All subjects underwent slit lamp examination, dry eye evaluation, and ocular surface disease index (OSDI) scoring. Subjects were stratified into group 1­without symptoms or clinical signs; group 2­without symptoms but with signs; group 3­with similar severity of symptoms and signs; and group 4­with symptom severity greater than that of the signs. Laser scanning in vivo confocal microscopy (IVCM) and tear fluid analysis for soluble factors by multiplex ELISA was performed for all subjects. Patients with a higher grade of symptoms and signs showed increased corneal dendritic cell (cDC) density (p < 0.05) which was more pronounced in subjects with discordance between the symptoms and signs (group 4). A significantly higher proportion of microneuroma-like structures and cDC were observed in group 4. IL-17A levels were significantly elevated in the tears of subjects with more discomfort. Our results demonstrate that corneal IVCM and the measurement of tear film factors can help clinicians improve diagnosis and treatment choice. Stratifying patients with ocular surface discomfort on the basis of discordance between symptoms and clinical signs may help identify patients who need additional adjunctive targeted therapy to resolve their condition.

Altered Ocular Surface Health Status and Tear Film Immune Profile Due to Prolonged Daily Mask Wear in Health Care Workers.

Prolonged daily face mask wearing over several months might affect health of the ocular surface and is reported to be associated with complaints of discomfort and dry-eye-like symptoms. We studied the ocular surface clinical parameters, tear soluble factors and immune cell proportions in ophthalmologists practicing within similar environmental conditions (n = 17) at two time points: pre-face-mask period (Pre-FM; end of 2019) and post-face-mask-wearing period (Post-FM; during 2020 COVID-19 pandemic), with continuous (~8 h/day) mask wear. A significant increase in ocular surface disease index (OSDI) scores without changes in tear breakup time (TBUT), Schirmer's test 1 (ST1) and objective scatter index (OSI) was observed Post-FM. Tear soluble factors (increased-IL-1ß, IL-33, IFNß, NGF, BDNF, LIF and TSLP; decreased-IL-12, IL-13, HGF and VEGF-A) and mucins (MUC5AC) were significantly altered Post-FM. Ex vivo, human donor and corneoscleral explant cultures under elevated CO2 stress revealed that the molecular profile, particularly mucin expression, was similar to the Post-FM tear molecular profile, suggesting hypercapnia is a potential contributor to ocular surface discomfort. Among the immune cell subsets determined from ocular surface wash samples, significantly higher proportions of leukocytes and natural killer T cells were observed in Post-FM compared to Pre-FM. Therefore, it is important to note that the clinical parameters, tear film quality, tear molecular factors and immune cells profile observed in prolonged mask-wear-associated ocular surface discomfort were distinct from dry eye disease or other common ocular surface conditions. These observations are important for differential diagnosis as well as selection of appropriate ocular surface treatment in such subjects.

Keratoconus patients exhibit a distinct ocular surface immune cell and inflammatory profile.

Inflammatory factors have been considered to contribute to keratoconus (KC) pathogenesis. This study aims to determine the immune cells subsets and soluble inflammatory factor profile on the ocular surface of KC patients. 32 KC subjects (51 eyes) across different grades of severity and 15 healthy controls (23 eyes) were included in the study. Keratometry and pachymetry measurements were recorded. Ocular surface immune cells (collected by ocular surface wash) immunophenotyped using flow cytometry include leukocytes, neutrophils, macrophages, natural killer (NK) cells, pan-T cells, gamma delta T (γδT) cells and NKT cells. Tear fluid collected using Schirmer's strip was used to measure 50 soluble factors by multiplex ELISA. Proportions of activated neutrophils, NK cells and γδT cells were significantly increased in KC patients. Significantly higher levels of tear fluid IL-1ß, IL-6, LIF, IL-17A, TNFα, IFNα/ß/γ, EPO, TGFß1, PDGF-BB, sVCAM, sL-selectin, granzyme-B, perforin, MMP2, sFasL and IgE, along with significantly lower levels of IL-1α and IL-9 were observed in KC patients. Alterations observed in few of the immuno-inflammatory parameters correlated with grades of disease, allergy, eye rubbing and keratometry or pachymetry measurements. The observation implies a distinct immuno-inflammatory component in KC pathogenesis and its potential as an additional therapeutic target in KC management.

Altered ocular surface immune cell profile in patients with dry eye disease.

PURPOSE: Aberrant inflammation and immune dysregulation are known pathogenic contributors in dry eye disease (DED). Aim of the study was to determine the proportions of immune cell subsets on the ocular surface (OS) of DED patients. METHODS: 15 healthy controls (22 eyes) and 48 DED subjects (36 eyes with evaporative DED - EDED; 60 eyes with aqueous deficient DED - ADED) were included in the study. Tear break up time (TBUT), Schirmer's test 1 (ST1), corneal staining (CS) and ocular surface disease index (OSDI) scoring were recorded. OS wash was used to collect immune cells on the OS of study subjects. The cells immunophenotyped using flow cytometry include leukocytes, neutrophils, macrophages, natural killer-NK cells and T cell subsets (CD4; CD8; double positive-DP; gamma delta-γδ and NK T cells). RESULTS: Significantly higher proportions of leukocytes, neutrophils, CD4 T cells, CD8 T cells, DP T cells and CD4/CD8 T cells ratio were observed in EDED and/or ADED patients. Significantly higher proportions of neutrophils and lower proportions of NK cells were observed in ADED subjects with corneal staining compared to those without and controls. Neutrophils/NK cells ratio was significantly higher in EDED and ADED subjects compared to controls. Correlation analysis revealed pathological relationships between proportions of leukocytes, neutrophils, CD4 T cells and Neutrophil/NK cells ratio with DED clinical parameters. CONCLUSION: OS immune cell subset proportion changes in DED patients were associated with DED types and severity. The data suggests the potential for a new generation of therapies targeting immune cells on the ocular surface.

Tear Fluid Angiogenic Factors: Potential Noninvasive Biomarkers for Retinopathy of Prematurity Screening in Preterm Infants.

Purpose: To determine the status of proangiogenic factors in the tear fluid of preterm infants with and without retinopathy of prematurity (ROP). Methods: Preterm infants (n = 36) undergoing routine ROP screening included in the prospective study were categorized as No-ROP (n = 13, no ROP at any visits), ROP (if ROP was present at first visit; n = 18), or No-ROP to ROP (no disease at first visit, but developed ROP subsequently; n = 5). Infants with ROP were also grouped as progressing (n = 7) and regressing (n = 16) based on ROP evolution between the first and subsequent visits. Schirmer's strips were used to collect tear fluid and proangiogenic factors (VEGF, angiogenin, soluble vascular cell adhesion molecule, and fractalkine) levels (in picograms per milliliter) in tear fluid were measured by multiplex ELISA. Results: Lower levels of VEGF (135 ± 69; mean ± standard deviation) and higher levels of angiogenin (6568 ± 4975) were observed in infants with ROP compared with infants without ROP (172.5 ± 54.0; 4139 ± 3909) at the first visit. Significantly lower levels of VEGF were observed in the No-ROP to ROP group compared with the No-ROP and ROP groups. The VEGF and angiogenin levels at the first visit were significantly lower in infants with ROP with progressing disease. Angiogenin levels negatively correlated with birth weight and gestational age in ROP. The area under the curve (AUC) and odds ratio (OR) analysis demonstrated that angiogenin/birth weight (AUC = 0.776; OR, 8.6); angiogenin/gestational age (AUC = 0.706; OR, 7.3) and Angiogenin/VEGF (AUC = 0.806; OR, 14.3) ratios were able to differentiated preterm infants with and without ROP. Conclusions: The association between angiogenin and ROP suggests its possible role in ROP. The ratio of angiogenin level with birth weight, gestational age, and/or VEGF could serve as a potential noninvasive screening biomarker for ROP.

ßA1-crystallin regulates glucose metabolism and mitochondrial function in mouse retinal astrocytes by modulating PTP1B activity.

ßA3/A1-crystallin, a lens protein that is also expressed in astrocytes, is produced as ßA3 and ßA1-crystallin isoforms by leaky ribosomal scanning. In a previous human proteome high-throughput array, we found that ßA3/A1-crystallin interacts with protein tyrosine phosphatase 1B (PTP1B), a key regulator of glucose metabolism. This prompted us to explore possible roles of ßA3/A1-crystallin in metabolism of retinal astrocytes. We found that ßA1-crystallin acts as an uncompetitive inhibitor of PTP1B, but ßA3-crystallin does not. Loss of ßA1-crystallin in astrocytes triggers metabolic abnormalities and inflammation. In CRISPR/cas9 gene-edited ßA1-knockdown (KD) mice, but not in ßA3-knockout (KO) mice, the streptozotocin (STZ)-induced diabetic retinopathy (DR)-like phenotype is exacerbated. Here, we have identified ßA1-crystallin as a regulator of PTP1B; loss of this regulation may be a new mechanism by which astrocytes contribute to DR. Interestingly, proliferative diabetic retinopathy (PDR) patients showed reduced ßA1-crystallin and higher levels of PTP1B in the vitreous humor.

Treatment of glaucoma by prostaglandin agonists and beta-blockers in combination directly reduces pro-fibrotic gene expression in trabecular meshwork.

Prostaglandin analogues (PG), beta-blockers (BB) or their combination (PG+BB) are used primarily to reduce the intraocular pressure (IOP) pathologically associated with glaucoma. Since, fibrosis of the trabecular meshwork (TM) is a major aetiological factor in glaucoma, we studied the effect of these drugs on fibrosis-associated gene expression in TM of primary glaucoma patients. In the present study, TM and iris of primary open-angle (n = 32) and angle-closure (n = 37) glaucoma patients were obtained surgically during trabeculectomy and categorized based on the type of IOP-lowering medications use as PG, BB or PG+BB. mRNA expression of pro-fibrotic and anti-fibrotic genes was quantified using qPCR in these tissues. The gene expression levels of pro-fibrotic genes were significantly lower in PG+BB as compared to other groups. These observations and underlying signalling validated in vitro in human TM cells also showed reduced fibrotic gene and protein expression levels following PG+BB treatment. In conclusion, it is observed that PG+BB combination rather than their lone use renders a reduced fibrotic status in TM. This further suggests that IOP-lowering medications, in combination, would also modulate fibrosis-associated molecular changes in the TM, which may be beneficial for maintaining aqueous out-flow mechanisms over the clinical treatment duration.

Distinct ocular surface soluble factor profile in human corneal dystrophies.

PURPOSE: Corneal dystrophies (CD) are classified as rare eye diseases that results in visual impairment and requires corneal transplant in advanced stages. Ocular surface inflammatory status in different types of CD remains underexplored. Hence, we studied the levels of tear soluble factors in the tears of patients with various types of corneal dystrophies. METHODS: 17 healthy subjects and 30 CD subjects (including epithelial, stromal and endothelial CD) were included in the study. Schirmer's strips were used to collect the tear fluid in all subjects. 27 soluble factors including cytokines, chemokines, soluble cell adhesion molecules and growth factors were measured in the eluted tears by multiplex ELISA or single analyte sandwich ELISA. RESULTS: Percentages of subjects with detectable levels of tear soluble factors were significantly higher in CD compared to controls. Significant higher level of IL-2 was observed in both epithelial and stromal CD. IL-4, TGFß1 and IgE were significantly higher in stromal CD. VCAM, IL-13 and Fractalkine were significantly elevated in epithelial and macular CD. IL-1α, IL-8, IL-12, ANG, Eotaxin, MCP1, RANTES, ICAM1, L-selectin and P-selectin were significantly higher in epithelial CD. TGFBIp was significantly elevated in lattice CD and endothelial CD. CONCLUSION: Distinct set of the tear soluble factors were dysregulated in various types of CD. Increase in tear inflammatory factors was observed in majority of the CD subjects depending on their sub-types. This suggests a plausible role of aberrant inflammation in CD pathobiology. Hence, modulating inflammation could be a potential strategy in improving the prognosis of CD.

Dysregulated Tear Fluid Nociception-Associated Factors, Corneal Dendritic Cell Density, and Vitamin D Levels in Evaporative Dry Eye.

Purpose: The purpose of this study was to study the status and association among tear-soluble factors, corneal dendritic cell density, vitamin D, and signs and symptoms in dry eye disease (DED). Methods: A total of 33 control subjects and 47 evaporative dry eye patients were included in the study. DED diagnosis and classification was based on the 2017 Report of the Tear Film & Ocular Surface Society International Dry Eye Workshop (TFOS DEWS II). DED workup, including tear film break-up time (TBUT), Schirmer's test I (STI), corneal and conjunctival staining, ocular surface disease index (OSDI) scoring, and in vivo confocal microscopy (to assess corneal dendritic cell density [cDCD] and subbasal nerve plexus [SBNP] features) was performed in the study subjects. Tear fluid using Schirmer's strip and serum were collected from the subjects. Multiplex ELISA or single analyte ELISA was performed to measure 34 tear-soluble factors levels including vitamin D. Results: Significantly higher OSDI discomfort score, lower TBUT, and lower STI were observed in DED patients. cDCD was significantly higher in DED patients. No significant difference was observed in SBNP features. Tear fluid IL-1ß, IL-17A, MMP9, MMP10, MMP9/TIMP ratio, and VEGF-B were significantly higher in DED patients. Significantly lower tear fluid IL-2, IP-10, NPY, VEGF-A, and vitamin D was observed in DED patients. These dysregulated tear factors showed significant associations with DED signs and symptoms. Conclusions: Altered tear fluid soluble factors with potential to modulate nociception exhibited a distinct association with ocular surface discomfort status, TBUT, STI, and cDCD. This implies a functional relationship between the various tear-soluble factors and dry eye pathogenesis, indicating new molecular targets for designing targeted therapies.

Pain management after photorefractive keratectomy.

PURPOSE: To evaluate the effect of a bandage contact lens soaked in ketorolac ophthalmic 0.45% solution (Acuvail) on pain modulation in patients having transepithelial photorefractive keratectomy (PRK). DESIGN: Prospective case series. SETTING: Narayana Nethralaya Superspeciality Eye Hospital, Bangalore, India. METHODS: Eyes of patients were divided into 2 groups. After transepithelial PRK, a regular soft bandage contact lens was placed in Group 1 and a ketorolac-soaked bandage contact lens was placed in Group 2. The patients were matched for age and sex in the 2 groups. Postoperative pain was compared using the validated Wong-Baker pain scale. Before placement of a ketorolac-soaked bandage contact lens in patients' eyes, the safety of the procedure was checked using cultures from the bandage contact lens and measuring the quantity of the drug adsorbed and the elution profile over time with ultra-high-performance liquid chromatography (UHPLC). RESULTS: Each group comprised 35 eyes of 35 patients. The mean pain score was 7.95 ± 2.12 (SD) in Group 1, which was significantly higher than in Group 2 (2.76 ± 0.85), which received the ketorolac-soaked bandage contact lens. The UHPLC results showed that the soaked bandage contact lens acted as a depot for ketorolac, which was released onto the ocular surface over time, providing postoperative pain relief without causing adverse events. CONCLUSION: A bandage contact lens soaked in ketorolac 0.45% solution can act as a potential drug depot that can reduce pain after transepithelial PRK.

Dopamine levels in human tear fluid.

PURPOSE: To determine the levels of dopamine in tear fluid and demonstrate the use of tear fluid as a non-invasive source for dopamine measurements in humans. METHODS: The study cohort included 30 clinically healthy individuals without any pre-existing ocular or systemic conditions. Matched tear fluid (using Schirmer's strips and capillary tubes) and plasma were collected from the subjects. Dopamine levels were evaluated using direct competitive chemiluminescent enzyme-linked immunosorbent assay (ELISA), dopamine kit (Cloud Clone Corp, TX, USA). RESULTS: Significantly higher dopamine levels were found in the tear fluid compared to plasma in the study subjects. The level of dopamine was 97.2 ± 11.80 pg/ml (mean ± SEM), 279 ± 14.8 pg/ml (mean ± SEM), and 470.4 ± 37.64 pg/ml (mean ± SEM) in the plasma and in the tears collected using Schirmer's strips and capillary tubes, respectively. CONCLUSION: Dopamine was detectable in all the tear fluid samples tested and was also found to be at a higher concentration than in plasma samples. Tear fluid can be used as a non-invasive sample source to monitor dopamine levels.