Stop codon variant in EFEMP1 is associated with primary open-angle glaucoma due to impaired regulation of aqueous humor outflow.

It is known that the actin cytoskeleton and its associated cellular interactions in the trabecular meshwork (TM) and juxtacanalicular tissues mainly contribute to the formation of resistance to aqueous outflow of the eye. Fibulin-3, encoded by EFEMP1 gene, has a role in extracellular matrix (ECM) modulation, and interacts with enzymatic ECM regulators, but the effects of fibulin-3 on TM cells has not been explored. Here, we report a stop codon variant (c.T1480C, p.X494Q) of EFEMP1 that co-segregates with primary open angle glaucoma (POAG) in a Chinese pedigree. In the human TM cells, overexpression of wild-type fibulin-3 reduced intracellular actin stress fibers formation and the extracellular fibronectin levels by inhibiting Rho/ROCK signaling. TGFß1 up-regulated fibulin-3 protein levels in human TM cells by activating Rho/ROCK signaling. In rat eyes, overexpression of wild-type fibulin-3 decreased the intraocular pressure and the fibronectin expression of TM, however, overexpression of mutant fibulin-3 (c.T1480C, p.X494Q) showed opposite effects in cells and rat eyes. Taken together, the EFEMP1 variant may impair the regulatory capacity of fibulin-3 which has a role for modulating the cell contractile activity and ECM synthesis in TM cells, and in turn may maintain normal resistance of aqueous humor outflow. This study contributes to the understanding of the important role of fibulin-3 in TM pathophysiology and provides a new possible POAG therapeutic approach.

Evaluation of trabecular meshwork-specific promoters in vitro and in vivo using scAAV2 vectors expressing C3 transferase.

AIM: To evaluate the potential of two trabecular meshwork (TM)-specific promoters, Chitinase 3-like 1 (Ch3L1) and matrix gla protein (MGP), for improving specificity and safety in glaucoma gene therapy based on self-complementary AAV2 (scAAV2) vector technologies. METHODS: An scAAV2 vector with C3 transferase (C3) as the reporter gene (scAAV2-C3) was selected. The scAAV2-C3 vectors were driven by Ch3L1 (scAAV2-Ch3L1-C3), MGP (scAAV2-MGP-C3), enhanced MGP (scAAV2-eMGP-C3) and cytomegalovirus (scAAV2-CMV-C3), respectively. The cultured primary human TM cells were treated with each vector at different multiplicities of infections. Changes in cell morphology were observed by phase contrast microscopy. Actin stress fibers and Rho GTPases/Rho-associated protein kinase pathway-related molecules were assessed by immunofluorescence staining, real-time quantitative polymerase chain reaction and Western blot. Each vector was injected intracamerally into the one eye of each rat at low and high doses respectively. In vivo green fluorescence was visualized by a Micron III Retinal Imaging Microscope. Intraocular pressure (IOP) was monitored using a rebound tonometer. Ocular responses were evaluated by slit-lamp microscopy. Ocular histopathology analysis was examined by hematoxylin and eosin staining. RESULTS: In TM cell culture studies, the vector-mediated C3 expression induced morphologic changes, disruption of actin cytoskeleton and reduction of fibronectin expression in TM cells by inhibiting the Rho GTPases/Rho-associated protein kinase signaling pathway. At the same dose, these changes were significant in TM cells treated with scAAV2-CMV-C3 or scAAV2-Ch3L1-C3, but not in cells treated with scAAV2-eMGP-C3 or scAAV2-MGP-C3. At low-injected dose, the IOP was significantly decreased in the scAAV2-Ch3L1-C3-injected eyes but not in scAAV2-MGP-C3-injected and scAAV2-eMGP-C3-injected eyes. At high-injected dose, significant IOP reduction was observed in the scAAV2-eMGP-C3-injected eyes but not in scAAV2-MGP-C3-injected eyes. Similar to scAAV2-CMV-C3, scAAV2-Ch3L1-C3 vector showed efficient transduction both in the TM and corneal endothelium. In anterior segment tissues of scAAV2-eMGP-C3-injected eyes, no obvious morphological changes were found except for the TM. Inflammation was absent. CONCLUSION: In scAAV2-transduced TM cells, the promoter-driven efficiency of Ch3L1 is close to that of cytomegalovirus, but obviously higher than that of MGP. In the anterior chamber of rat eye, the transgene expression pattern of scAAV2 vector is presumably affected by MGP promoter, but not by Ch3L1 promoter. These findings would provide a useful reference for improvement of specificity and safety in glaucoma gene therapy using scAAV2 vector.

Compound Heterozygous Variants of the CPAMD8 Gene Co-Segregating in Two Chinese Pedigrees With Pigment Dispersion Syndrome/Pigmentary Glaucoma.

The molecular mechanisms underlying the pathogenesis of pigment dispersion syndrome and pigmentary glaucoma remain unclear. In pedigree-based studies, familial aggregation and recurrences in relatives suggest a strong genetic basis for pigmentary glaucoma. In this study, we aimed to identify the genetic background of two Chinese pedigrees with pigmentary glaucoma. All members of these two pedigrees who enrolled in the study underwent a comprehensive ophthalmologic examination, and genomic DNA was extracted from peripheral venous blood samples. Whole-exome sequencing and candidate gene verifications were performed to identify the disease-causing variants; in addition, screening of the CPAMD8 gene was performed on 38 patients of sporadic pigmentary glaucoma. Changes in the structure and function of abnormal proteins caused by gene variants were analyzed with a bioinformatics assessment. Pigmentary glaucoma was identified in a total of five patients from the two pedigrees, as were compound heterozygous variants of the CPAMD8 gene. No signs of pigmentary glaucoma were found in carriers of monoallelic CPAMD8 variant/variants. All four variants were inherited in an autosomal recessive mode. In addition to the 38 patients of sporadic pigmentary glaucoma, 13 variants of the CPAMD8 gene were identified in 11 patients. This study reported a possible association between CPAMD8 variants and pigment dispersion syndrome/pigmentary glaucoma.

Forkhead Domain Inhibitor-6 Suppresses Corneal Neovascularization and Subsequent Fibrosis After Alkali Burn in Rats.

Purpose: The purpose of this study was to investigate the effects of Forkhead Domain Inhibitor-6 (FDI-6) on regulating inflammatory corneal angiogenesis and subsequent fibrosis induced by alkali burn. Methods: A corneal alkali burn model was established in Sprague Dawley rats using NaOH and the rat eyes were topically treated with FDI-6 (40 µM) or a control vehicle four times daily for 7 days. Corneal neovascularization, inflammation and epithelial defects were observed on days 1, 4, and 7 under a slit lamp microscope after corneal alkali burn. Analysis of angiogenesis-, inflammation-, and fibrosis-related indicators was conducted on day 7. Murine macrophages (RAW264.7 cells) and mouse retinal microvascular endothelial cells (MRMECs) were used to examine the effects of FDI-6 on inflammatory angiogenesis in vitro. Results: Topical delivery of FDI-6 significantly attenuated alkali burn-induced corneal inflammation, neovascularization, and fibrosis. FDI-6 suppressed the expression of angiogenic factors (vascular epidermal growth factor, CD31, matrix metalloproteinase-9, and endothelial NO synthase), fibrotic factors (α-smooth muscle actin and fibronectin), and pro-inflammatory factor interleukin-6 in alkali-injured corneas. FDI-6 downregulated the expression of monocyte chemotactic protein-1, pro-inflammatory cytokines (interleukin-1ß and tumor necrosis factor-alpha), nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3, and vascular endothelial growth factor in RAW264.7 cells and inhibited the proliferation, migration, and tube formation of MRMECs in vitro. Conclusions: FDI-6 can attenuate corneal neovascularization, inflammation, and fibrosis in alkali-injured corneas.

A missense mutation in Pitx2 leads to early-onset glaucoma via NRF2-YAP1 axis.

Glaucoma is a leading cause of blindness, affecting 70 million people worldwide. Owing to the similarity in anatomy and physiology between human and mouse eyes and the ability to genetically manipulate mice, mouse models are an invaluable resource for studying mechanisms underlying disease phenotypes and for developing therapeutic strategies. Here, we report the discovery of a new mouse model of early-onset glaucoma that bears a transversion substitution c. G344T, which results in a missense mutation, p. R115L in PITX2. The mutation causes an elevation in intraocular pressure (IOP) and progressive death of retinal ganglion cells (RGC). These ocular phenotypes recapitulate features of pathologies observed in human glaucoma. Increased oxidative stress was evident in the inner retina. We demonstrate that the mutant PITX2 protein was not capable of binding to Nuclear factor-like 2 (NRF2), which regulates Pitx2 expression and nuclear localization, and to YAP1, which is necessary for co-initiation of transcription of downstream targets. PITX2-mediated transcription of several antioxidant genes were also impaired. Treatment with N-Acetyl-L-cysteine exerted a profound neuroprotective effect on glaucoma-associated neuropathies, presumably through inhibition of oxidative stress. Our study demonstrates that a disruption of PITX2 leads to glaucoma optic pathogenesis and provides a novel early-onset glaucoma model that will enable elucidation of mechanisms underlying the disease as well as to serve as a resource to test new therapeutic strategies.

Forkhead domain inhibitory-6 attenuates subconjunctival fibrosis in rabbit model with trabeculectomy.

Antiproliferative therapies are crucially important for improving the success rate of the glaucoma filtration surgeries. In this study, we investigated the potential efficacy of Forkhead Domain Inhibitory-6 (FDI-6) in inhibiting post-trabeculectomy subconjunctival fibrosis. In vitro, the effect of FDI-6 (10 µM) on fibrotic response and its underlying mechanism were investigated in rabbit tenon's fibroblasts (RTFs) treated with or without transforming growth factor-ß1 (TGF-ß1, 20 ng/mL). In vivo, FDI-6 (40 µM) was injected subconjunctivally to a rabbit trabeculectomy model. Intraocular pressure (IOP) changes were monitored within the 14-day period post-surgery. Bleb morphology and subepithelial fibrosis at the operating area were evaluated with slit lamp and confocal microscopic examinations and with histologic examinations. The results showed that, in cell culture studies, FDI-6 suppressed the proliferation, migration, collagen gel contraction and the expression levels of fibronectin (FN) and α-smooth muscle actin (α-SMA) in RTFs with TGF-ß treatment by down-regulating the TGF-ß1/Smad2/3 signaling pathway. In animal studies, the IOPs of the FDI-6-treated group were significantly lower than those of the saline-treated group after trabeculectomy. The FDI-6-treated eyes showed a better bleb appearance with fewer blood vessels compared to the saline-treated eyes. The analysis of confocal microscopy in vivo and histopathology revealed that subconjunctival fibrosis after trabeculectomy was significantly attenuated in the FDI-6-treated group compared to the controls. In conclusion, our studies indicate that FDI-6 exerts an inhibitory effect on subconjunctival fibrosis caused by trabeculectomy, holding potentials as a new antiproliferative agent used in anti-glaucoma filtration surgeries in the future.

Lentiviral vector-mediated expression of C3 transferase attenuates retinal ischemia and reperfusion injury in rats.

AIMS: Our previous study showed that intravitreal delivery of self-complementary AAV2 (scAAV2)-mediated exoenzyme C3 transferase (C3) can attenuate retinal ischemia/reperfusion (I/R) injury. The current study investigated the neuroprotective effects of lentivirus (LV)-mediated C3 transgene expression on rat retinal I/R injury. MAIN METHODS: The LV encoding C3 and green fluorescent protein (GFP) together (LV-C3-GFP) or GFP only (LV-GFP) was intravitreally injected to SPRAGUE-DAWLEY rats. On day 5 post-intravitreal injection, eyes were evaluated by slit-lamp examination. The GFP expression on retina was confirmed by in vivo and ex vivo assessments. RhoA GTPase expression in retina was examined by western blot. Retinal I/R injury was generated by transiently increasing intraocular pressure (110 mmHg, 90 min). Eyes were then enucleated, and retinas processed for morphological analysis and TdT-dUTP terminal nick-end labeling (TUNEL) assay. KEY FINDINGS: No obvious inflammatory reactions or surgical complications were observed after intravitreal injection of LV vectors. There was a significant decrease of total RhoA GTPase level in the retina treated with LV-C3-GFP. Compared to the blank control group, LV-C3-GFP and LV-GFP did not affect the retinal thickness, cell density in ganglion cell layer (GCL), or numbers of apoptotic cells in retinal flat-mounts. In the LV-GFP-treated retinas, I/R decreased the retinal thickness and GCL cell density and increased apoptotic retinal cell numbers. LV-C3-GFP significantly protected against all these degenerative effects of I/R. SIGNIFICANCE: This study indicated that LV-mediated C3 transgene expression exhibits neuroprotective effects on the retinal I/R injury and holds potential as a novel neuroprotective approach targeting certain retinopathies.

scAAV2-Mediated C3 Transferase Gene Therapy in a Rat Model with Retinal Ischemia/Reperfusion Injuries.

Glaucoma is characterized by retinal ganglion cell (RGC) death and axonal loss. Therefore, neuroprotection is important in treating glaucoma. In this study, we explored whether exoenzyme C3 transferase (C3)-based gene therapy could protect retinas in an ischemia/reperfusion (I/R) injury rat model. Self-complementary adeno-associated virus 2 (scAAV2) vectors encoding either C3 protein (scAAV2-C3) or enhanced green fluorescence protein (scAAV2-EGFP) were intravitreally delivered into both eyes of rats, and I/R models (acute ocular hypertension) were made in one eye of each rat at day 7 after the injection. The rats were divided into six groups: scAAV2-C3, scAAV2-C3 with I/R, scAAV2-EGFP, scAAV2-EGFP with I/R, blank control, and blank control with I/R. TUNEL (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling), immunohistochemistry of cleaved caspase-3, NeuN and Brn-3a, and H&E staining were used to detect apoptotic cells and other changes in the retina. The results showed that scAAV2-C3 significantly reduced the number of apoptotic RGCs and decreased cell loss in the ganglion cell layer after I/R injury, and the I/R-injured retinas treated with scAAV2-C3 were the thickest in all I/R groups. These results suggest that scAAV2-mediated C3 gene therapy is able to protect the rat retina from I/R injury and has potential in the treatment of glaucoma in the future.

Deletion of the Impg2 gene causes the degeneration of rod and cone cells in mice.

Variants in interphotoreceptor matrix proteoglycans (IMPG2) have been reported in retinitis pigmentosa (RP) and vitelliform macular dystrophy (VMD) patients. However, the underlying molecular mechanisms remain elusive due to a lack of suitable disease models. We developed two independent Impg2 knockout (KO) mouse models using the CRISPR/Cas9 technique to assess the in vivo functions of Impg2 in the retina. Impg2 ablation in mice recapitulated the RP phenotypes of patients, including an attenuated electroretinogram (ERG) response and the progressive degeneration of photoreceptors. The histopathological examination of Impg2-KO mice revealed irregularly arranged rod cells and mislocalized rhodopsin protein in the inner segment at 6 months of age. In addition to the pathological changes in rod cells, cone cells were also affected in KO retinas. KO retinas exhibited progressive cone cell death and impaired cone cell elongation. Further immunoblotting analysis revealed increased levels of endoplasmic reticulum (ER) stress-related proteins, including C/EBP homologous protein (CHOP), immunoglobulin heavy-chain-binding protein (BIP) and protein disulfide isomerase (PDI), in Impg2-KO mouse retinas. Increased gliosis and apoptotic cell death were also observed in the KO retinas. As autophagy is closely associated with ER stress, we then checked whether autophagy was disturbed in Impg2-KO mouse retinas. The results showed that autophagy was impaired in KO retinas, as revealed by the increased accumulation of SQSTM1 and other proteins involved in autophagy. Our results demonstrate the essential roles of Impg2 in the retina, and this study provides novel models for mechanistic investigations and development of therapies for RP caused by IMPG2 mutations.

C3 Transferase-Expressing scAAV2 Transduces Ocular Anterior Segment Tissues and Lowers Intraocular Pressure in Mouse and Monkey.

Glaucoma is a lifelong disease with elevated intraocular pressure (IOP) as the main risk factor, and reduction of IOP remains the major treatment for this disease. However, current IOP-lowering therapies are far from being satisfactory. We have demonstrated that the lentivirus-mediated exoenzyme C3 transferase (C3) expression in rat and monkey eyes induced relatively long-term IOP reduction. We now show that intracameral injection of self-complementary AAV2 containing a C3 gene into mouse and monkey eyes resulted in morphological changes in trabecular meshwork and IOP reduction. The vector-transduced corneal endothelium and the C3 transgene expression, not vector itself, induced corneal edema as a result of actin-associated endothelial barrier disruption. There was a positive (quadratic) correlation between measured IOP and grade of corneal edema. This is the first report of using an AAV to transduce the trabecular meshwork of monkeys with a gene capable of altering cellular structure and physiology, indicating a potential gene therapy for glaucoma.

Is "normal tension glaucoma" glaucoma?

Primary open angle glaucoma (POAG) represents a distinct disease entity with elevated intraocular pressure (IOP) as the main risk factor, even though the reasons for why the IOP is elevated remains to be elucidated. It is considered that normal tension glaucoma (NTG) is a subtype of POAG, comprising a special form of glaucomatous neurodegeneration or glaucomatous optic neuropathy (GON) almost exactly the same as that seen in POAG, but the IOP, as named, remains in the statistically normal range. Actually the disease entity of NTG has been a profound confusion and it is difficult to be accurately conceptualized. One of the reasons is that the IOP is closely linked to the occurrence of GON in POAG but not in NTG, and for the latter, it seems that GON is secondary to a number of local or systemic disorders. In recent years, increasing evidences suggest that NTG or IOP independent GON is a non-glaucomatous disease with different disease entities from POAG and with more diverse and complex etiologies. Here we hypothesized that NTG, at least for those with recognizable primary diseases, is not a glaucomatous disease; instead, it represents a group of disorders with GON as a characteristic clinical feature or phenotype.

Lentiviral Vector-Mediated Expression of Exoenzyme C3 Transferase Lowers Intraocular Pressure in Monkeys.

Primary open-angle glaucoma (POAG) is considered a lifelong disease characterized by optic nerve deterioration and visual field damage. Although the disease progression can usually be controlled by lowering the intraocular pressure (IOP), therapeutic effects of current approaches do not last long. Gene therapy could be a promising method for persistent treatment of the disease. Our previous study demonstrated that gene transfer of exoenzyme C3 transferase (C3) to the trabecular meshwork (TM) to inhibit Rho GTPase (Rho), the upstream signal molecule of Rho-associated kinase (ROCK), resulted in lowered IOP in normal rodent eyes. In the present study, we show that the lentiviral vector (LV)-mediated C3 expression inactivates RhoA in human TM cells by ADP ribosylation, resulting in disruption of the actin cytoskeleton and altered cell morphology. In addition, intracameral delivery of the C3 vector to monkey eyes leads to persistently lowered IOP without obvious signs of inflammation. This is the first report of using a vector to transduce the TM of an alive non-human primate with a gene that alters cellular machinery and physiology. Our results in non-human primates support that LV-mediated C3 expression in the TM may have therapeutic potential for glaucoma, the leading cause of irreversible blindness in humans.

Effects of Lentivirus-Mediated C3 Expression on Trabecular Meshwork Cells and Intraocular Pressure.

Purpose: We evaluated the effects of lentivirus-mediated exoenzyme C3 transferase (C3) expression on cultured primary human trabecular meshwork (HTM) cells in vitro, and on rat intraocular pressure (IOP). Methods: HTM cells were cultured and treated with lentivirus vectors expressing either green fluorescent protein (GFP) only (LV-GFP) or GFP and C3 together (LV-C3-GFP). Changes in cell morphology and actin stress fibers were assessed. The vectors were also injected into the anterior chamber of rats, and GFP expression was visualized by a Micron III Retinal Imaging Microscope in vivo and a fluorescence microscope ex vivo. Changes in rat IOP were monitored by using a rebound tonometer and the eyes were evaluated by slit lamp. Results: LV-mediated C3 expression induced morphologic changes in HTM cells. The cells became retracted and rounded. GFP expression in the anterior chamber angle of rats was observed in vivo from 8 days to 48 days after injection of LV-C3-GFP or LV-GFP. IOP was significantly decreased in the LV-C3-GFP group starting 3 days post injection, and lasting for at least 40 days, when compared to either the contralateral control eyes (the LV-GFP group) or the ipsilateral baseline before injection (P < 0.05). No obvious inflammatory signs were observed in either the LV-C3-GFP or LV-GFP groups. Conclusions: LV-mediated C3 expression induced changes in morphology of cultured HTM cells. Intracameral injection of LV-C3-GFP lowered rat IOP for at least 40 days. No significant inflammatory reactions were observed in either the LV-C3-GFP or LV-GFP groups. This study supports the possible use of C3 gene therapy for the treatment of glaucoma.

Ohmic transition at contacts key to maximizing fill factor and performance of organic solar cells.

While thermodynamic detailed balance limits the maximum power conversion efficiency of a solar cell, the quality of its contacts can further limit the actual efficiency. The criteria for good contacts to organic semiconductors, however, are not well understood. Here, by tuning the work function of poly(3,4-ethylenedioxythiophene) hole collection layers in fine steps across the Fermi-level pinning threshold of the model photoactive layer, poly(3-hexylthiophene):phenyl-C61-butyrate methyl ester, in organic solar cells, we obtain direct evidence for a non-ohmic to ohmic transition at the hole contact that lies 0.3 eV beyond its Fermi-level pinning transition. This second transition corresponds to reduction of the photocurrent extraction resistance below the bulk resistance of the cell. Current detailed balance analysis reveals that this extraction resistance is the counterpart of injection resistance, and the measured characteristics are manifestations of charge carrier hopping across the interface. Achieving ohmic transition at both contacts is key to maximizing fill factor without compromising open-circuit voltage nor short-circuit current of the solar cell.

Doped polymer semiconductors with ultrahigh and ultralow work functions for ohmic contacts.

To make high-performance semiconductor devices, a good ohmic contact between the electrode and the semiconductor layer is required to inject the maximum current density across the contact. Achieving ohmic contacts requires electrodes with high and low work functions to inject holes and electrons respectively, where the work function is the minimum energy required to remove an electron from the Fermi level of the electrode to the vacuum level. However, it is challenging to produce electrically conducting films with sufficiently high or low work functions, especially for solution-processed semiconductor devices. Hole-doped polymer organic semiconductors are available in a limited work-function range, but hole-doped materials with ultrahigh work functions and, especially, electron-doped materials with low to ultralow work functions are not yet available. The key challenges are stabilizing the thin films against de-doping and suppressing dopant migration. Here we report a general strategy to overcome these limitations and achieve solution-processed doped films over a wide range of work functions (3.0-5.8 electronvolts), by charge-doping of conjugated polyelectrolytes and then internal ion-exchange to give self-compensated heavily doped polymers. Mobile carriers on the polymer backbone in these materials are compensated by covalently bonded counter-ions. Although our self-compensated doped polymers superficially resemble self-doped polymers, they are generated by separate charge-carrier doping and compensation steps, which enables the use of strong dopants to access extreme work functions. We demonstrate solution-processed ohmic contacts for high-performance organic light-emitting diodes, solar cells, photodiodes and transistors, including ohmic injection of both carrier types into polyfluorene-the benchmark wide-bandgap blue-light-emitting polymer organic semiconductor. We also show that metal electrodes can be transformed into highly efficient hole- and electron-injection contacts via the self-assembly of these doped polyelectrolytes. This consequently allows ambipolar field-effect transistors to be transformed into high-performance p- and n-channel transistors. Our strategy provides a method for producing ohmic contacts not only for organic semiconductors, but potentially for other advanced semiconductors as well, including perovskites, quantum dots, nanotubes and two-dimensional materials.