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.

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.

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.

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.

Decorin Concentrations in Aqueous Humor of Patients with Diabetic Retinopathy.

Diabetic retinopathy (DR) is a microvascular complication of diabetes in the retina. Chronic hyperglycemia damages retinal microvasculature embedded into the extracellular matrix (ECM), causing fluid leakage and ischemic retinal neovascularization. Current treatment strategies include intravitreal anti-vascular endothelial growth factor (VEGF) or steroidal injections, laser photocoagulation, or vitrectomy in severe cases. However, treatment may require multiple modalities or repeat treatments due to variable response. Though DR management has achieved great success, improved, long-lasting, and predictable treatments are needed, including new biomarkers and therapeutic approaches. Small-leucine rich proteoglycans, such as decorin, constitute an integral component of retinal endothelial ECM. Therefore, any damage to microvasculature can trigger its antifibrotic and antiangiogenic response against retinal vascular pathologies, including DR. We conducted a cross-sectional study to examine the association between aqueous humor (AH) decorin levels, if any, and severity of DR. A total of 82 subjects (26 control, 56 DR) were recruited. AH was collected and decorin concentrations were measured using an enzyme-linked immunosorbent assay (ELISA). Decorin was significantly increased in the AH of DR subjects compared to controls (p = 0.0034). AH decorin levels were increased in severe DR groups in ETDRS and Gloucestershire classifications. Decorin concentrations also displayed a significant association with visual acuity (LogMAR) measurements. In conclusion, aqueous humor decorin concentrations were found elevated in DR subjects, possibly due to a compensatory response to the retinal microvascular changes during hyperglycemia.

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.

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.

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.

Neutrophils homing into the retina trigger pathology in early age-related macular degeneration.

Age-related macular degeneration (AMD) is an expanding problem as longevity increases worldwide. While inflammation clearly contributes to vision loss in AMD, the mechanism remains controversial. Here we show that neutrophils are important in this inflammatory process. In the retinas of both early AMD patients and in a mouse model with an early AMD-like phenotype, we show neutrophil infiltration. Such infiltration was confirmed experimentally using ribbon-scanning confocal microscopy (RSCM) and IFNλ- activated dye labeled normal neutrophils. With neutrophils lacking lipocalin-2 (LCN-2), infiltration was greatly reduced. Further, increased levels of IFNλ in early AMD trigger neutrophil activation and LCN-2 upregulation. LCN-2 promotes inflammation by modulating integrin ß1 levels to stimulate adhesion and transmigration of activated neutrophils into the retina. We show that in the mouse model, inhibiting AKT2 neutralizes IFNλ inflammatory signals, reduces LCN-2-mediated neutrophil infiltration, and reverses early AMD-like phenotype changes. Thus, AKT2 inhibitors may have therapeutic potential in early, dry AMD.

Correlation between systemic S100A8 and S100A9 levels and severity of diabetic retinopathy in patients with type 2 diabetes mellitus.

AIMS: S100A8 and S100A9 are myeloid-related damage-associated molecular patterns (DAMPs) primarily involved in the modulation of innate immune response to cellular injury. This study evaluated the correlation between circulating concentrations of S100A8 and S100A9 proteins with the severity of diabetic retinopathy (DR) in patients with type 2 diabetes (T2DM). METHODS: T2DM patients with HbA1c levels >7%, fasting blood glucose >126 mg/dl and history of diabetes were included in this study. DR severity was graded based on ETDRS and Gloucestershire classifications. Plasma samples were evaluated for S100A8 and S100A9 levels using ELISA. RESULTS: In this comparative study, DR patients (n = 89) had increased plasma S100A8 and S100A9 proteins compared to age-matched T2DM controls (n = 28), which was directly related to the severity of DR. Female DR subjects had increased S100A8 expression compared to their male counterparts. Substantial retention of S100A8 and S100A9 production was seen in DR patients above 50 years of age. Duration of T2DM was not found to affect protein levels, however T2DM onset at >50 years old significantly increased S100A8 and S100A9 concentrations. CONCLUSIONS: Our findings suggest that systemic circulation levels of S100A8 and S100A9 are correlated with the progression of DR in T2DM patients, indicating their potential role in DR pathogenesis.

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.