Determination of the Trans-Lamina Cribrosa Pressure Difference in a Community-Based Population and its Association with Open-Angle Glaucoma.

PURPOSE: To determine the trans-lamina cribrosa pressure difference (TLCPD) in a cohort of normal community-based patients and the relationship to primary open-angle glaucoma (POAG) and normal-tension glaucoma (NTG). DESIGN: Retrospective cohort study of the Mayo Clinic Study of Aging. PARTICIPANTS: The Mayo Clinic Study of Aging is a prospective study evaluating the normal aging population. METHODS: Mayo Clinic Study of Aging patients who underwent routine lumbar puncture (LP) studies with eye examinations were reviewed. The trans-lamina cribrosa pressure difference was calculated in 2 contexts of intraocular pressure (IOP): (1) maximum IOP at eye visit closest in time to the LP (closest-in-time TLCPD); and (2) IOP before IOP-lowering treatment (pretreatment IOP and pretreatment TLCPD) in POAG and NTG patients. Glaucoma patients without POAG or NTG were excluded. Regression analyses were performed to determine the relationship with glaucoma. MAIN OUTCOME MEASURES: IOP, intracranial pressure, TLCPD, POAG, normal-tension glaucoma (NTG) diagnosis, glaucoma parameters. RESULTS: Five hundred forty-eight patients were analyzed. Of these, there were 38 treated glaucoma patients (14 POAG and 24 NTG) and 510 nonglaucomatous patients. Cerebral spinal fluid (CSF) opening pressure was 155.0 ± 42.2 mmH2O in nonglaucomatous patients, 144.0 ± 34.0 mmH2O in POAG (P = 0.15 vs. nonglaucomatous patients), and 136.6 ± 29.3 mmH2O in NTG (P = 0.017 vs. nonglaucomatous patients). Intraocular pressure was 15.47 ± 2.9 mmHg in nonglaucomatous patients, 26.6 ± 3.7 mmHg in POAG, and 17.4 ± 3.4 mmHg in NTG. The closest-in-time TLCPD in the nonglaucomatous cohort was 4.07 ± 4.22 mmHg, which was lower than both the POAG cohort (7.19 ± 3.6 mmHg) and the NTG cohort (5.79 ± 4.5 mmHg, P = 0.04). Pretreatment TLCPD for the overall glaucoma cohort was 10.57 ± 6.1 mmHg. The POAG cohort had a higher pretreatment TLCPD (16.05 ± 5.2 mmHg) than the NTG cohort (7.37 ± 4.1 mmHg; P < 0.0001). Closest-in-time TLCPD for the nonglaucoma cohort (4.07± 4.2 mmHg) was significantly lower than pretreatment TLCPDs for both POAG (16.05 ± 5.2 mmHg; P < 0.0001) and NTG (7.37 ± 4.1 mmHg; P < 0.0001) cohorts. CONCLUSIONS: This study establishes the baseline TLCPD in a large cohort of normal, community-based patients. The differences in regression analysis between TLCPD and IOP suggests NTG pathophysiology is partially driven by TLCPD, but is also likely multifactorial. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Direct early growth response-1 knockdown decreases melanoma viability independent of mitogen-activated extracellular signal-related kinase inhibition.

To investigate downstream molecular changes caused by mitogen-activated protein kinase (MEK) inhibitor treatment and further explore the impact of direct knockdown of early growth response-1 ( EGR1 ) in melanoma cell culture. RNA-sequencing (RNA-Seq) was performed to determine gene expression changes with MEK inhibitor treatment. Treatment with MEK inhibitor (trametinib) was then assessed in two cutaneous (MEL888, MEL624) and one conjunctival (YUARGE 13-3064) melanoma cell line. Direct knockdown of EGR1 was accomplished using lentiviral vectors containing shRNA. Cell viability was measured using PrestoBlueHS Cell Viability Reagent. Total RNA and protein were assessed by qPCR and SimpleWestern. RNA-Seq demonstrated a profound reduction in EGR1 with MEK inhibitor treatment, prompting further study of melanoma cell lines. Following trametinib treatment of melanoma cells, viability was reduced in both cutaneous (MEL888 26%, P  < 0.01; MEL624 27%, P  < 0.001) and conjunctival (YUARGE 13-3064 33%, P  < 0.01) melanoma compared with DMSO control, with confirmed EGR1 knockdown to 0.04-, 0.01-, and 0.16-fold DMSO-treated levels (all P  < 0.05) in MEL888, MEL624, and YUARGE 13-3064, respectively. Targeted EGR1 knockdown using shRNA reduced viability in both cutaneous (MEL624 78%, P  = 0.05) and conjunctival melanoma (YUARGE-13-3064 67%, P  = 0.02). RNA-Sequencing in MEK inhibitor-treated cells identified EGR1 as a candidate effector molecule of interest. In a malignant melanoma cell population, MEK inhibition reduced viability in both cutaneous and conjunctival melanoma with a profound downstream reduction in EGR1 expression. Targeted knockdown of EGR1 reduced both cutaneous and conjunctival melanoma cell viability independent of MEK inhibition, suggesting a key role for EGR1 in melanoma pathobiology.

Preclinical Pharmacokinetic Profile of Topical Ophthalmic and Intravenous Delivery of QLS-101, a Novel ATP-Sensitive Potassium Channel Opening Ocular Hypotensive Agent.

Purpose: To evaluate the pharmacokinetic profiles of the ocular hypotensive agent QLS-101, a novel ATP-sensitive potassium channel opening prodrug, and its active moiety levcromakalim, following topical ophthalmic and intravenous dosing of normotensive rabbits and dogs. Methods: Dutch belted rabbits (n = 85) and beagle dogs (n = 32) were dosed with QLS-101 (0.16-3.2 mg/eye/dose) or formulation buffer for 28 days. Pharmacokinetic profiles of QLS-101 and levcromakalim were evaluated in ocular tissues and blood by LC-MS/MS. Tolerability was assessed by clinical and ophthalmic examinations. Maximum systemic tolerated dose was evaluated in beagle dogs (n = 2) following intravenous bolus administrations of QLS-101 (0.05 to 5 mg/kg). Results: Plasma analysis following topical dosing of QLS-101 (0.8-3.2 mg/eye/dose) for 28 days indicated an elimination half-life (T1/2) of 5.50-8.82 h and a corresponding time (Tmax) range of 2-12 h in rabbits, and a T1/2 of 3.32-6.18 h with a Tmax range of 1-2 h in dogs. Maximum tissue concentration (Cmax) values ranged from 54.8-540 (day 1) to 50.5-777 ng/mL (day 28) in rabbits, and 36.5-166 (day 1) to 47.0-147 ng/mL (day 28) in dogs. Levcromakalim plasma T1/2 and Tmax were similar to QLS-101, while Cmax was consistently lower. Topical ophthalmic delivery of QLS-101 was well tolerated in both species, with sporadic mild ocular hyperemia noted in the group treated with the highest concentration (3.2 mg/eye/dose). Following topical ophthalmic dosing, QLS-101 and levcromakalim were found primarily in the cornea, sclera, and conjunctiva. Maximum tolerated dose was determined to be 3 mg/kg. Conclusions: QLS-101 was converted to its active moiety levcromakalim and showed characteristic absorption, distribution, and safety profiles of a well-tolerated prodrug.

Isolation and Culture of Vascular Distal Outflow Pathway (VDOP) Cells From Human Donor Eyes.

Glaucoma, a progressive neurodegenerative ocular disease, is the leading cause of irreversible blindness worldwide. Elevated intraocular pressure (IOP) is the most common-and the only treatable-risk factor for glaucoma. IOP is generated by the balance between production and removal of aqueous humor in the anterior part of the eye, and the latter happens either through the uveoscleral or the conventional pathway. Although both pathways are important for aqueous humor removal, dysfunction within the conventional pathway is more commonly associated with increased resistance leading to elevated IOP and glaucoma. The conventional pathway can be separated into proximal (trabecular meshwork and inner wall of Schlemm's canal) and distal (outer wall of Schlemm's canal, collector channels, and episcleral vasculature) regions. Both regions contribute resistance to aqueous humor removal, but the proximal region has been studied more extensively due to the availability of model systems. In contrast, little is known about the role of the distal region in outflow resistance, largely due to the lack of suitable in vitro models. To address this, we have developed a novel method of isolating and culturing vascular distal outflow pathway (VDOP) cells from the distal outflow region of human eyes. VDOP cells can be used to study the physiological and molecular functions of cells in the distal outflow region and can help in the development of ocular hypotensive drugs that specifically target this area. We also provide a protocol describing immunohistochemical methods to validate the molecular profile of these cells, utilizing cell surface markers that distinguish them from adjacent cells. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Isolation and culture of VDOP cells Basic Protocol 2: Analysis of cell surface markers.

Evaluation of neural innervation in the human conventional outflow pathway distal to Schlemm's canal.

The distal outflow pathway of the human eye consists of the outer wall of Schlemm's canal, collector channels, and the deep-scleral, mid-scleral and episcleral vessels. It is the last region of transit for aqueous humor before returning to the venous system. While the trabecular meshwork, scleral spur, and inner wall of Schlemm's canal have been extensively analyzed to define their contributions to aqueous outflow, the role of the distal outflow pathway is not completely understood. Collector channels, emanating from Schlemm's canal were previously thought to be passive conduits for aqueous humor. However, recent studies have shown many collector channels contain flap-like appendages which move with changes in pressure. These findings, along with studies demonstrating innervation of episcleral vessels, have led to questions regarding whether other structures in the distal outflow pathway are under neural regulation and how this may influence aqueous humor outflow. This study evaluates the innervation of the outer wall of Schlemm's canal and collector channels, along with the deep-scleral, mid-scleral and episcleral vasculature with microcomputed tomography and 3-dimensional reconstruction, correlative light microscopy, immunohistochemistry, and transmission electron microscopy. Peripheral, autonomic, and sensory nerve fibers were found to be present adjacent to Schlemm's canal outer wall endothelium, collector channel endothelium, and in the different regions of the distal outflow vasculature. Nerves were more commonly identified in regions that contained collector channels when compared to regions without collector channels. These findings regarding the neural anatomy suggest an active neural regulation of aqueous humor outflow throughout the proximal and distal regions of the conventional outflow pathway.

Transgene expression of Stanniocalcin-1 provides sustained intraocular pressure reduction by increasing outflow facility.

Glaucoma is the leading cause of irreversible blindness worldwide. Therapies for glaucoma are directed toward reducing intraocular pressure (IOP), the leading risk factor and only reliable therapeutic target via topical medications or with procedural intervention including laser or surgery. Though topical therapeutics are typically first line, less than 50% of patients take drops as prescribed. Sustained release technologies that decrease IOP for extended periods of time are being examined for clinical use. We recently identified Stanniocalcin-1, a naturally occurring hormone, as an IOP-lowering agent. Here, we show that a single injection into the anterior chamber of mice with an adeno-associated viral vector containing the transgene of stanniocalcin-1 results in diffuse and sustained expression of the protein and produces IOP reduction for up to 6 months. As the treatment effect begins to wane, IOP-lowering can be rescued with a repeat injection. Aqueous humor dynamic studies revealed an increase in outflow facility as the mechanism of action. This first-in-class therapeutic approach has the potential to improve care and reduce the rates of vision loss in the 80 million people worldwide currently affected by glaucoma.

Cancer Risk in Patients With Fuchs Endothelial Corneal Dystrophy.

PURPOSE: The purpose of this study is to quantify cancer risk in patients with Fuchs endothelial corneal dystrophy (FECD). METHODS: Using the 2014 to 2016 Medicare Limited 5% Data Sets-Carrier Line File, US Medicare fee-for-service beneficiaries (aged 65 years or older) with FECD and cancer were identified through International Classification of Diseases , ninth and 10th Revision diagnostic codes from January 1, 2014, to December 31, 2016. The main outcome measures were odds ratios (ORs) of cancer at various anatomic locations in patients with versus without FECD. RESULTS: Of the 1,462,740 Medicare beneficiaries, 15,534 patients (1.1%) had an International Classification of Disease code for FECD. Compared with US Medicare beneficiaries without FECD, patients with FECD were at increased risk for the following malignancies: breast [OR: 1.32; 95% confidence interval (CI): 1.22-1.43; P < 0.001], cutaneous basal cell (OR: 1.42; 95% CI: 1.35-1.49; P < 0.001), cutaneous melanoma (OR: 1.20; 95% CI: 1.03-1.40; P = 0.02), cutaneous squamous cell (OR: 1.45; 95% CI: 1.38-1.53; P < 0.001), ovarian (OR: 1.84; 95% CI: 1.48-2.30; P < 0.001), and thyroid (OR: 1.32; 95% CI: 1.04-1.68; P = 0.02). By contrast, FECD cases were at lower odds of having lung (OR: 0.81; 95% CI: 0.71-0.93; P = 0.003) and prostate cancer diagnoses (OR: 0.88; 95% CI: 0.81-0.96; P = 0.002). CONCLUSIONS: Patients with FECD aged 65 years or older may be at increased risk for cancer at several anatomic locations. Follow-up studies are needed to further explore the association of FECD and malignancy, elucidate potential disease mechanisms, and identify genetic and/or environmental risk factors.

Ocular Hypotensive Properties and Biochemical Profile of QLS-101, a Novel ATP-Sensitive Potassium (KATP) Channel Opening Prodrug.

Purpose: To characterize the ocular hypotensive and pharmacological properties of QLS-101, a novel ATP-sensitive potassium (KATP) channel opening prodrug. Methods: Ocular hypotensive properties of QLS-101 were evaluated by measuring IOP with a handheld rebound tonometer after daily topical ocular instillation of 0.2% (n = 5) or 0.4% QLS-101 (n = 10) in C57BL/6J mice. KATP channel specificity was characterized in HEK-293 cells stably expressing human Kir6.2/SUR2B subunits and assessed for off-target interactions using a receptor binding screen. Conversion of QLS-101 prodrug to its active moiety, levcromakalim, was evaluated in vitro using human ocular tissues and plasma samples and after incubation with human phosphatase enzymes (2.0 nM-1.0 µM). Results: C57BL/6J mice treated once daily with 0.2% QLS-101 exhibited significant (P < 0.01) IOP reductions of 2.1 ± 0.4 mmHg after five days; however, a daily attenuation of the effect was noted by 23h post-dose. By comparison, treatment with 0.4% QLS-101 lowered IOP by 4.8 ± 0.7 mm Hg (P < 0.0001) which was sustained for 24 hours. Unlike levcromakalim, QLS-101 failed to induce KATP channel activity in HEK-Kir6.2/SUR2B cells consistent with its development as a prodrug. No off-target receptor effects were detected with either compound. In vitro ocular tissue conversion of QLS-101 prodrug was identified in human iris, ciliary body, trabecular meshwork, and sclera. Alkaline phosphatase was found to convert QLS-101 (mean Km = 630 µM, kcat = 15 min-1) to levcromakalim. Conclusions: QLS-101 is a novel KATP channel opening prodrug that when converted to levcromakalim shows 24-hour IOP lowering after once-daily topical ocular administration.

Effect of ATP-sensitive Potassium Channel Openers on Intraocular Pressure in Ocular Hypertensive Animal Models.

Purpose: To evaluate the effect of ATP-sensitive potassium channel openers cromakalim prodrug 1 (CKLP1) and diazoxide on IOP in three independent mouse models of ocular hypertension. Methods: Baseline IOP was measured in TGFß2 overexpression, steroid-induced, and iris dispersion (DBA/2J) ocular hypertension mouse models, followed by once daily eyedrop administration with CKLP1 (5 mM) or diazoxide (5 mM). The IOP was measured in conscious animals with a handheld rebound tonometer. Aqueous humor dynamics were assessed by a constant perfusion method. Effect of treatment on ocular tissues was evaluated by transmission electron microscopy. Results: CKLP1 decreased the IOP by 20% in TGFß2 overexpressing mice (n = 6; P < 0.0001), 24% in steroid-induced ocular hypertensive mice (n = 8; P < 0.0001), and 43% in DBA/2J mice (n = 15; P < 0.0001). Diazoxide decreased the IOP by 32% in mice with steroid-induced ocular hypertension (n = 13; P < 0.0001) and by 41% in DBA/2J mice (n = 4; P = 0.005). An analysis of the aqueous humor dynamics revealed that CKLP1 decreased the episcleral venous pressure by 29% in TGFß2 overexpressing mice (n = 13; P < 0.0001) and by 72% in DBA/2J mice (n = 4 control, 3 treated; P = 0.0002). Diazoxide lowered episcleral venous pressure by 35% in steroid-induced ocular hypertensive mice (n = 3; P = 0.03). Tissue histology and cell morphology appeared normal when compared with controls. Accumulation of extracellular matrix was reduced in CKLP1- and diazoxide-treated eyes in the steroid-induced ocular hypertension model. Conclusions: ATP-sensitive potassium channel openers CKLP1 and diazoxide effectively decreased the IOP in ocular hypertensive animal models by decreasing the episcleral venous pressure, supporting a potential therapeutic application of these agents in ocular hypertension and glaucoma.

Comparison of TCF4 repeat expansion length in corneal endothelium and leukocytes of patients with Fuchs endothelial corneal dystrophy.

Expansion of CTG trinucleotide repeats (TNR) in the transcription factor 4 (TCF4) gene is highly associated with Fuchs Endothelial Corneal Dystrophy (FECD). Due to limitations in the availability of DNA from diseased corneal endothelium, sizing of CTG repeats in FECD patients has typically been determined using DNA samples isolated from peripheral blood leukocytes. However, it is non-feasible to extract enough DNA from surgically isolated FECD corneal endothelial tissue to determine repeat length based on current technology. To circumvent this issue, total RNA was isolated from FECD corneal endothelium and sequenced using long-read sequencing. Southern blotting of DNA samples isolated from primary cultures of corneal endothelium from these same affected individuals was also assessed. Both long read sequencing and Southern blot analysis showed significantly longer CTG TNR expansion (>1000 repeats) in the corneal endothelium from FECD patients than those characterized in leukocytes from the same individuals (<90 repeats). Our findings suggest that the TCF4 CTG repeat expansions in the FECD corneal endothelium are much longer than those found in leukocytes.

Characterization of a dual media system for culturing primary normal and Fuchs endothelial corneal dystrophy (FECD) endothelial cells.

Primary cultures of human corneal endothelial cells (HCECs) are an important model system for studying the pathophysiology of corneal endothelium. The purpose of this study was to identify and validate an optimal primary culture model of normal and Fuchs endothelial corneal dystrophy (FECD) endothelial cells by comparing cell morphology and marker expression under different media conditions to in vivo donor tissues. Primary and immortalized HCECs, isolated from normal and FECD donors, were cultured in proliferation media (Joyce, M4, Bartakova) alone or sequentially with maturation media (F99, Stabilization 1, M5). CD56, CD73 and CD166 expressions were quantified in confluent and matured cell lines by flow cytometry. HCECs that were allowed to proliferate in Joyce's medium followed by maturation in low-mitogen containing media yielded cells with similar morphology to corneal endothelial tissues. Elevated expression of CD56 and CD166 and low expression of CD73 correlated with regular, hexagonal-like HCEC morphology. CD56:CD73 > 2.5 was most consistent with normal HCEC morphology and mimicked corneal endothelial tissue. Immortalization of normal HCECs by hTERT transduction showed morphology and CD56:CD73 ratios similar to parental cell lines. HCECs established from FECD donors showed reduced CD56:CD73 ratios compared to normal HCECs which coincided with reduced uniformity and regularity of cell monolayers. Overall, a dual media system with Joyce's medium for proliferation and a low-mitogen media for maturation, provided normal cultures with regular, hexagonal-like cell morphologies consistent with corneal endothelial cells in vivo. CD56:CD73 expression ratio >2.5 was predictive of in vivo-like cellular morphology.

Pharmacological Profile and Ocular Hypotensive Effects of Cromakalim Prodrug 1, a Novel ATP-Sensitive Potassium Channel Opener, in Normotensive Dogs and Nonhuman Primates.

Purpose: To evaluate pharmacokinetic parameters and ocular hypotensive effects of cromakalim prodrug 1 (CKLP1) in normotensive large animal models. Methods: Optimal CKLP1 concentration was determined by dose response and utilized in short- (5-8 days) and long-term (60 days) evaluation in hound dogs (n = 5) and African Green Monkeys (n = 5). Blood pressure was recorded 3-5 times per week with a tail cuff. Concentrations of CKLP1 and the parent compound levcromakalim were assessed in hound dog plasma and select tissues by LC-MS/MS after bilateral ocular treatment with CKLP1 for 8 days. Pharmacokinetic parameters were calculated from days 1, 4, and 8 data. After necropsy, histology was assessed in 43 tissue samples from each animal. Results: In hound dogs and African Green monkeys, 10 mM CKLP1 (optimal concentration) significantly lowered intraocular pressure (IOP) by 18.9% ± 1.1% and 16.7% ± 6.7%, respectively, compared with control eyes (P < 0.05). During treatment, no significant change in systolic or diastolic blood pressure was observed in either species (P > 0.1). Average values for half-life of CKLP1 was 295.3 ± 140.4 min, Cmax, 10.5 ± 1.6 ng/mL, and area under the concentration vs. time curve (AUClast) 5261.4 ± 918.9 ng·min/mL. For levcromakalim, average values of half-life were 96.2 ± 27 min, Cmax 1.2 ± 0.2 ng/mL, and AUClast 281.2 ± 110.8 ng·min/mL. No significant pathology was identified. Conclusions: CKLP1 lowered IOP in hound dogs and African green monkeys with no effect on systemic blood pressure. Ocular topical treatment of CKLP1 showed excellent tolerability even after extended treatment periods.

Stanniocalcin-1 Reduced Intraocular Pressure in Two Models of Ocular Hypertension.

Purpose/Aim: Glaucomatous optic neuropathy (GON) remains the world's leading cause of irreversible blindness. Treatments including topical medications are directed at reducing intraocular pressure (IOP), the most significant risk factor for GON. Current medications, while generally effective, are limited by insufficient response and side-effects in some patients. In search of a more targeted therapy that acts downstream of existing medications that has a potential for a lower side effect profile, our laboratory has identified Stanniocalcin-1 (STC-1), a multifunctional hormone, as an effector molecule in latanoprost-mediated IOP reduction with similar IOP-lowering efficacy as latanoprost in normotensive mice.Materials and methods: To investigate whether STC-1 can also reduce IOP in ocular hypertensive mice, we used a steroid-induced ocular hypertensive mouse model characterized by trabecular meshwork dysfunction as well as the DBA/2J mouse as an inherited model of pigment dispersion and secondary angle closure. Steroid-induced ocular hypertension was induced by weekly injections of dexamethasone into the conjunctival fornix of wild-type C57BL/6J mice (6-8 months old). After confirmation of the steroid response, mice were administered STC-1 or phosphate buffered saline (PBS) topically once daily for six weeks. For DBA/2J mice (14 months old), after baseline IOP measurements, mice were treated topically once daily with STC-1 or PBS for 5 days and IOP was assessed twice daily.Results: In steroid-induced ocular hypertensive mice, STC-1 lowered IOP by 26% (P < .001, week three) and maintained this level of IOP reduction throughout the remainder of the treatment period (P < .001, week six). In DBA/2J mice, STC-1 lowered IOP by 37% (P < .001).Conclusions: Together, these data show that STC-1 reduced IOP in two models of ocular hypertension with different mechanisms of outflow obstruction.

Relationship of Body Mass Index With Fuchs Endothelial Corneal Dystrophy Severity and TCF4 CTG18.1 Trinucleotide Repeat Expansion.

PURPOSE: To investigate the association of body mass index (BMI) with Fuchs endothelial corneal dystrophy (FECD) severity and TCF4 CTG18.1 expansion. METHODS: A total of 343 patients with FECD were enrolled from the Mayo Clinic. FECD severity was graded by slit-lamp biomicroscopy. BMI values were obtained from the electronic medical records. DNA extracted from leukocytes was analyzed for CTG18.1 expansion length, with ≥40 repeats considered expanded. Wilcoxon signed-rank tests were used to compare FECD grade and CTG18.1 expansion length in patients by BMI (<25, ≥25 to <30, and ≥30 kg/m2). FECD grade was regressed on age, sex, BMI, and CTG18.1 expansion and, separately, BMI on CTG18.1 expansion. Models were investigated for effect modification by age and sex with an interaction term of P < 0.05 considered statistically significant. RESULTS: When examining the association between BMI and FECD, there was a significant interaction between BMI and sex (P for interaction = 0.004). When controlling for age and CTG18.1 expansion, a positive association was observed between BMI and FECD grade in women, but not in men. In addition, BMI was not associated with CTG18.1 expansion when controlling for age and sex. CONCLUSIONS: BMI was positively associated with FECD severity among women but not men. There was no significant association between BMI and CTG18.1 expansion. These findings suggest that increased BMI is potentially a modifiable risk factor for FECD disease progression among women.

Isolation and characterization of novel primary cells from the human distal outflow pathway.

Ocular hypertension occurs due to increased resistance to aqueous humor removal through the conventional outflow pathway. Unlike the proximal region of the conventional outflow pathway, the distal region has not been well studied, mostly due to lack of model systems. Here we describe isolation and characterization of human primary vascular distal outflow pathway (VDOP) cells from the distal region of the conventional outflow pathway. Tissue from the distal region was isolated from human corneo-scleral rims, digested with collagenase type I (100 U/ml) and placed on gelatin coated plates to allow cellular growth in Dulbecco's Modified Eagle's Medium (low glucose) containing fetal bovine serum and antibiotic/antimycotic. VDOP cells showed consistent proliferation for up to 7 passages, retained endothelial-like nature of the parent tissues and showed a unique marker phenotype of Lectin+VEGFR2-CD34-NG2- that was distinct from neighboring trabecular meshwork (Lectin+VEGFR2-CD34-NG2+) and Schlemm's canal (Lectin+VEGFR2+CD34+NG2+) cells. Dexamethasone treated VDOP cells did not express myocilin and did not form cross-linked actin networks, in contrast to trabecular meshwork cells. These data show that VDOP cells are unique to the distal outflow region and can be used as a viable in vitro model system to understand the biology of the distal outflow pathway and intraocular pressure regulation.

Effects of Intense Pulsed Light on Tear Film TGF-ß and Microbiome in Ocular Rosacea with Dry Eye.

PURPOSE: To assess tear film transforming growth factor-beta (TGF-ß) and ocular microbiome changes after intense pulsed light with meibomian gland expression (IPL-MGX) vs only MGX in treating ocular rosacea with dry eye symptoms. METHODS: Twenty patients were randomly assigned to IPL-MGX or MGX. Patients were examined, treated, and administered the ocular surface disease index (OSDI) survey every 4-6 weeks for four total treatments. Tear film and conjunctival samples were collected at first and last visits, and analyzed for TGF-ß concentration and 16s rRNA amplicon sequencing of ocular microbiome. Wilcoxon Rank Sum and Sign-Rank were used to examine changes from baseline. RESULTS: OSDI revealed significantly greater improvement in symptoms after IPL-MGX (p=0.030) compared to MGX. There was no significant difference in mean TGF-ß1, 2, or 3 concentration after IPL-MGX (p=0.385, 0.709, 0.948, respectively). Quantities of Clostridium, Klebsiella, Brevibacterium, Lactobacillus, Neisseria, Streptococcus, Corynebacterium, Butyricicoccus, and Actinomyces were significantly reduced from baseline in both groups but without a significant difference between the two treatment groups. CONCLUSION: IPL-MGX improved dry eye symptoms more than MGX alone. IPL treatment offered no additional benefit to MGX in decreasing virulent bacteria present on the ocular surface and did not influence TGF-ß levels in tears. Prospective studies on IPL-MGX with larger sample sizes are needed to further investigate cytokines and IPL in patients suffering from ocular rosacea with dry eye symptoms. CLINICALTRIALSGOV IDENTIFIER: NCT03194698.

Disease Expression and Familial Transmission of Fuchs Endothelial Corneal Dystrophy With and Without CTG18.1 Expansion.

Purpose: To characterize inheritance, penetrance, and trinucleotide repeat expansion stability in Fuchs endothelial corneal dystrophy (FECD). Methods: One thousand unrelated and related subjects with and without FECD were prospectively recruited. CTG18.1 repeat length (CTG18.1L) was determined via short tandem repeat assay and Southern blotting of leukocyte DNA. Multivariable logistic regression and generalized estimating equation models were employed. Results: There were 546 unrelated FECD cases (67.6% female; 70 ± 10 years) and 235 controls (63.8% female; 73 ± 8 years; all ≥ 50 years). CTG18.1 expansion (CTG18.1exp+) was observed in 424 (77.7%) cases and 18 (7.7%) controls (P = 2.48 × 10-44). CTG18.1 expansion was associated with FECD severity (P = 5.62 × 10-7). The family arm of the study included 331 members from 112 FECD-affected families; 87 families were CTG18.1exp+. Autosomal dominant inheritance with variable expression of FECD was observed, regardless of expansion status. FECD penetrance of CTG18.1 expansion increased with age, ranging from 44.4% in the youngest (19-46 years) to 86.2% in the oldest (64-91 years) age quartiles. Among 62 parent-offspring transmissions of CTG18.1exp+, 48 (77.4%) had a change in CTG18.1L ≤ 10 repeats, and eight (12.9%) were ≥50 repeats, including five large expansions (∼1000-2000 repeats) that contracted. Among 44 offspring who did not inherit the CTG18.1exp+ allele, eight (18.2%) exhibited FECD. Conclusions: CTG18.1 expansion was highly associated with FECD but demonstrated incomplete penetrance. CTG18.1L instability occurred in a minority of parent-offspring transmissions, with large expansions exhibiting contraction. The observation of FECD without CTG18.1 expansion among family members in CTG18.1exp+ families highlights the complexity of the relationship between the FECD phenotype and CTG18.1 expansion.

TCF4-mediated Fuchs endothelial corneal dystrophy: Insights into a common trinucleotide repeat-associated disease.

Fuchs endothelial corneal dystrophy (FECD) is a common cause for heritable visual loss in the elderly. Since the first description of an association between FECD and common polymorphisms situated within the transcription factor 4 (TCF4) gene, genetic and molecular studies have implicated an intronic CTG trinucleotide repeat (CTG18.1) expansion as a causal variant in the majority of FECD patients. To date, several non-mutually exclusive mechanisms have been proposed that drive and/or exacerbate the onset of disease. These mechanisms include (i) TCF4 dysregulation; (ii) toxic gain-of-function from TCF4 repeat-containing RNA; (iii) toxic gain-of-function from repeat-associated non-AUG dependent (RAN) translation; and (iv) somatic instability of CTG18.1. However, the relative contribution of these proposed mechanisms in disease pathogenesis is currently unknown. In this review, we summarise research implicating the repeat expansion in disease pathogenesis, define the phenotype-genotype correlations between FECD and CTG18.1 expansion, and provide an update on research tools that are available to study FECD as a trinucleotide repeat expansion disease. Furthermore, ongoing international research efforts to develop novel CTG18.1 expansion-mediated FECD therapeutics are highlighted and we provide a forward-thinking perspective on key unanswered questions that remain in the field.

Stanniocalcin-1 (STC-1), a downstream effector molecule in latanoprost signaling, acts independent of the FP receptor for intraocular pressure reduction.

Prostaglandin F2 alpha (PGF2α) analogues such as latanoprost are common first-line intraocular pressure (IOP) lowering medications. However, their clinical use is limited in some patient populations due to minimal or no IOP lowering response or side effects. In searching for a more targeted approach for IOP reduction, our lab recently identified Stanniocalcin-1 (STC-1) as a molecule that was required for latanoprost-mediated IOP reduction and also acted as a stand-alone IOP lowering agent. In order to determine whether latanoprost and STC-1 were equivalent and/or additive for IOP reduction, we treated C57BL/6J mice with one or a combination of these agents and measured IOP. Importance of the FP receptor for latanoprost- and STC-1-mediated IOP reduction was examined in C57BL/6J mice utilizing the pharmacologic FP receptor inhibitor AL-8810 as well as FP receptor knockout mice generated in our laboratory. Latanoprost-free acid (LFA) and STC-1 reduced IOP to a similar degree and were non-additive in C57BL/6J mice. As expected, the IOP lowering effects of LFA were abrogated by pharmacologic inhibition of the FP receptor with AL-8810 and in FP receptor knockout mice. In contrast, STC-1 maintained IOP-lowering effects in the presence of AL-8810 and also in FP receptor knockout mice. These results suggest that LFA and STC-1 show equivalent and non-additive IOP reduction in C57BL/6J mice and that unlike LFA, STC-1-mediated IOP reduction occurs independent of the FP receptor.

Pharmacological and pharmacokinetic profile of the novel ocular hypotensive prodrug CKLP1 in Dutch-belted pigmented rabbits.

Elevated intraocular pressure is the only treatable risk factor for glaucoma, an eye disease that is the leading cause of irreversible blindness worldwide. We have identified cromakalim prodrug 1 (CKLP1), a novel water-soluble ATP-sensitive potassium channel opener, as a new ocular hypotensive agent. To evaluate the pharmacokinetic and safety profile of CKLP1 and its parent compound levcromakalim, Dutch-belted pigmented rabbits were treated intravenously (0.25 mg/kg) or topically (10 mM; 4.1 mg/ml) with CKLP1. Body fluids (blood, aqueous and vitreous humor) were collected at multiple time points and evaluated for the presence of CKLP1 and levcromakalim using a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) based assay. Histology of tissues isolated from Dutch-belted pigmented rabbits treated once daily for 90 days was evaluated in a masked manner by a certified veterinary pathologist. The estimated plasma parameters following intravenous administration of 0.25 mg/kg of CKLP1 showed CKLP1 had a terminal half-life of 61.8 ± 55.2 min, Tmax of 19.8 ± 23.0 min and Cmax of 1968.5 ± 831.0 ng/ml. Levcromakalim had a plasma terminal half-life of 85.0 ± 37.0 min, Tmax of 61.0 ± 32.0 min and Cmax of 10.6 ± 1.2 ng/ml. Topical CKLP1 treatment in the eye showed low levels (<0.3 ng/mL) of levcromakalim in aqueous and vitreous humor, and trace amounts of CKLP1 and levcromakalim in the plasma. No observable histological changes were noted in selected tissues that were examined following topical application of CKLP1 for 90 consecutive days. These results suggest that CKPL1 is converted to levcromakalim in the eye and likely to some extent in the systemic circulation.