Conjunctival Fibroblasts Research Articles

Inhibition of mTOR differently modulates planar and subepithelial fibrogenesis in human conjunctival fibroblasts.

In the current investigation, the effects of the mTOR inhibitors, Rapa and Torin1 on the TGF-β2-induced conjunctival fibrogenesis were studied. Experimental research. 2D and 3D cultures of HconF were subjected to the following analyses; (1) planar proliferation evaluated by TEER (2D), (2) Seahorse metabolic analyses (2D), (3) subepithelial proliferation evaluated by the 3D spheroids' size and hardness, and (4) the mRNA expression of ECM proteins and their regulators (2D and 3D). Rapa or Torin1 both significantly increased planar proliferation in the non-TGF-β2-treated 2D HconF cells, but in the TGF-β2-treated cells, this proliferation was inhibited by Rapa and enhanced by Torin1. Although Rapa or Torin1 did not affect cellular metabolism in the non-TGF-β2-treated HconF cells, mTOR inhibitors significantly decreased and increased the mitochondrial respiration and the glycolytic capacity, respectively, under conditions of TGF-β2-induced fibrogenesis. Subepithelial proliferation, as evidenced by the hardness of the 3D spheroids, was markedly down-regulated by both Rapa and Torin1 independent of TGF-β2. The mRNA expressions of several ECM molecules and their regulators fluctuated in the cases of 2D vs 3D and TGF-β2 untreated vs treated cultures. The present findings indicate that mTOR inhibitors have the ability to increase and to reduce planar and subepithelial proliferation in HconF cells, depending on the inhibitor being used.

Lysophosphatidic Acid Modulates TGF-β2-Induced Biological Phenotype in Human Conjunctival Fibroblasts

Background: Although lysophosphatidic acid (LPA) is known to have multiple pathophysiological roles, its contributions to ocular tissues, especially conjunctival fibrogenesis, remain to be elucidated. Methods: To study this issue, the effects of LPA on transforming growth factor-β2 (TGF-β2)-induced fibrogenesis of two-dimensional (2D) and three-dimensional (3D) cultures of human conjunctival fibroblasts (HconF) were examined by the following analyses: (1) planar proliferation determined by transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran permeability measurements, (2) real-time metabolic analyses, (3) measurements of the size and stiffness of 3D spheroids, and (4) mRNA expression of extracellular matrix (ECM) molecules and their modulators. Results: LPA had no effect on TGF-β2-induced increase in the planar proliferation of HconF cells. LPA induced a more quiescent metabolic state in 2D HconF cells, but this metabolic suppression by LPA was partially blunted in the presence of TGF-β2. In contrast, LPA caused a substantial decrease in the hardness of 3D HconF spheroids independently of TGF-β2. In agreement with these different LPA-induced effects between 2D and 3D cultured HconF cells, mRNA expressions of ECM and their modulators were differently modulated. Conclusion: The findings that LPA induced the inhibition of both TGF-β2-related and -unrelated subepithelial proliferation of HconF cells may be clinically applicable.

TGFβ1-Induced Fibrotic Responses of Conjunctival Fibroblasts through the Wnt/β-Catenin/CRYAB Signaling Pathway

Conjunctival fibrosis (CF) is a common postoperative complication of glaucoma filtration surgery (GFS), resulting in uncontrolled intraocular pressure and surgery failure. Therefore, it’s urgent to understand the molecular mechanisms underlying CF and explore novel pharmacological anti-fibrosis therapies for GFS. Our 4D-DIA quantitative proteomic results, coupled with experimental data, revealed the activation of the Wnt/β-catenin pathway in TGFβ1-induced human conjunctival fibroblasts (HConFs). Treatment with ICG-001, a Wnt/β-catenin inhibitor, effectively inhibited cell proliferation and migration in TGFβ1-treated HConFs. ICG-001 treatment alleviated the increased generation of extracellular matrix (ECM) proteins induced by TGFβ1. In addition, ICG-001 reduced the expression level of alpha smooth muscle actin (α-SMA) and inhibited cell contractility in TGFβ1-treated HConFs. Proteomics data further suggested that CRYAB was a downstream target of the Wnt/β-catenin, which was upregulated by TGFβ1 and downregulated by ICG-001. Immunoblotting assay also indicated that ICG-001 reduced the expressions of ubiquitin and β-catenin in TGFβ1-treated HConFs, implying that CRYAB stabilized β-catenin by inhibiting its ubiquitination degradation. Exogenous CRYAB promoted cell viability, increased ECM proteins levels, and upregulated α-SMA expression of HConFs under TGFβ1 stimulation. Furthermore, CRYAB rescued TGFβ1-induced fibrotic responses that were suppressed by ICG-001. In conclusion, our study elucidates the regulatory mechanism of Wnt/β-catenin/CRYAB pathway in CF, offering promising therapeutic targets for mitigating bleb scarring after GFS.

Developing a synergistic rate-retarding polymeric implant for controlling monoclonal antibody delivery in minimally invasive glaucoma surgery

Monoclonal antibodies (mAbs) have garnered substantial attention within the field of ophthalmology and can be used to suppress scar formation after minimally invasive glaucoma surgeries. Here, by controlling mAb passive diffusion, we developed a polymeric, rate-controlling membrane reservoir loaded with poly(lactic-co-glycolic acid) microspheres to deliver mAb for several weeks. Different parameters were tested to ensure that the microspheres achieved a good quality characteristic, and our results showed that 1 %W/V emulsifier with 5 %W/V NaCl achieved mAb-loaded microspheres with the highest stability, encapsulation efficiency and minimal burst release. Then, we fabricated and compared 10 types of microporous films based on polylactic acid (PLA), polycaprolactone (PCL), and polyethylene glycol (PEG). Our results revealed distinct pore characteristics and degradation patterns in different films due to varying polymer properties, and all the polymeric film formulations showed good biocompatibility in both human trabecular meshwork cells and human conjunctival fibroblasts. Finally, the optimized microspheres were loaded into the reservoir-type polymeric implant assembled by microporous membranes with different surface coating modifications. The implant formulation, which was fabricated by 60 PCL: 40 PEG (3 %W/V) polymer with 0.1 %W/V poly(lactic-co-glycolic acid) barrier, exerted the best drug release profile that can sustained release mAb (83.6 %) for 4 weeks.

Biocompatibility of Nb2C MXene film for ophthalmic implants

The Nb2C MXene film was prepared by tetrafluoroboric acid (HBF4) and hydrofluoric acid (HF) etching and vacuum extraction. The surface structure, properties, and composition of Nb2C MXene were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and other methods. The biocompatibility of Nb2C MXene film was evaluated by in vivo and in vitro experiments. The results show the layered, micro–nano surface structure of Nb2C MXene with abundant hydroxyl groups (—OH) on the surface. Compared with the control group, the proliferation rate of conjunctival fibroblasts cultured with 1 mg/mL Nb2C MXene is 72.15% (p<0.05). The cytotoxicity of Nb2C MXene is Grade 0 (p>0.05), and the hemolysis rate is 0.38% (<5%). The oxidative stress and inflammatory responses in rabbit ocular tissues are inhibited by Nb2C MXene film, which promotes wound healing without inducing fibrosis, and its micro–nano structure promotes cell adhesion and proliferation, demonstrating good biocompatibility.

Endoplasmic Reticulum Stress and Unfolded Protein Response in Vernal Keratoconjunctivitis.

Vernal keratoconjunctivitis (VKC) is an ocular allergic disease characterized by a type 2 inflammation, tissue remodeling, and low quality of life for the affected patients. We investigated the involvement of endoplasmic reticulum (ER) stress and unfolded protein response in VKC. Conjunctival imprints from VKC patients and normal subjects (CTs) were collected, and RNA was isolated, reverse transcribed, and analyzed with the Affymetrix microarray. Differentially expressed genes between VKC patients and CTs were evaluated. Genes related to ER stress, apoptosis, and autophagy were further considered. VKC and CT conjunctival biopsies were analyzed by immunohistochemistry (IHC) with specific antibodies against unfolded protein response (UPR), apoptosis, and inflammation. Conjunctival fibroblast and epithelial cell cultures were exposed to the conditioned medium of activated U937 monocytes and analyzed by quantitative PCR for the expression of UPR, apoptosis, autophagy, and inflammatory markers. ER chaperones HSPA5 (GRP78/BiP) and HYOU1 (GRP170) were upregulated in VKC patients compared to CTs. Genes encoding for ER transmembrane proteins, PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), ER-associated degradation (ERAD), and autophagy were upregulated, but not those related to apoptosis. Increased positive reactivity of BiP and ATF6 and unchanged expression of apoptosis markers were confirmed by IHC. Cell cultures in stress conditions showed an overexpression of UPR, proinflammatory, apoptosis, and autophagy markers. A significant overexpression of genes encoding for ER stress, UPR, and pro-inflammatory pathway components was reported for VKC. Even though these pathways may lead to ER homeostasis, apoptosis, or inflammation, ER stress in VKC may predominantly contribute to promote inflammation.

Pharmacological Stimulation of Soluble Guanylate Cyclase Counteracts the Profibrotic Activation of Human Conjunctival Fibroblasts.

Conjunctival fibrosis is a serious clinical concern implicated in a wide spectrum of eye diseases, including outcomes of surgery for pterygium and glaucoma. It is mainly driven by chronic inflammation that stimulates conjunctival fibroblasts to differentiate into myofibroblasts over time, leading to abnormal wound healing and scar formation. Soluble guanylate cyclase (sGC) stimulation was found to suppress transforming growth factor β (TGFβ)-induced myofibroblastic differentiation in various stromal cells such as skin and pulmonary fibroblasts, as well as corneal keratocytes. Here, we evaluated the in vitro effects of stimulation of the sGC enzyme with the cell-permeable pyrazolopyridinylpyrimidine compound BAY 41-2272 in modulating the TGFβ1-mediated profibrotic activation of human conjunctival fibroblasts. Cells were pretreated with the sGC stimulator before challenging with recombinant human TGFβ1, and subsequently assayed for viability, proliferation, migration, invasiveness, myofibroblast marker expression, and contractile properties. Stimulation of sGC significantly counteracted TGFβ1-induced cell proliferation, migration, invasiveness, and acquisition of a myofibroblast-like phenotype, as shown by a significant downregulation of FAP, ACTA2, COL1A1, COL1A2, FN1, MMP2, TIMP1, and TIMP2 mRNA levels, as well as by a significant reduction in α-smooth muscle actin, N-cadherin, COL1A1, and FN-EDA protein expression. In addition, pretreatment with the sGC stimulator was capable of significantly dampening TGFβ1-induced acquisition of a contractile phenotype by conjunctival fibroblasts, as well as phosphorylation of Smad3 and release of the proinflammatory cytokines IL-1β and IL-6. Taken together, our findings are the first to demonstrate the effectiveness of pharmacological sGC stimulation in counteracting conjunctival fibroblast-to-myofibroblast transition, thus providing a promising scientific background to further explore the feasibility of sGC stimulators as potential new adjuvant therapeutic compounds to treat conjunctival fibrotic conditions.

Study of IL-17 and Intercellular Adhesion Molecule-1 in Conjunctivochalasis Using Correlation Analysis.

The aim of this study was to observe the expression of interleukin (IL)-17 and intercellular adhesion molecule (ICAM)-1 in conjunctivochalasis (CCH) and to analyze the correlations between cytokines and the severity of CCH. Serum samples were collected from 22 patients with CCH and 18 normal controls (NCs). The Ocular Surface Disease Index, tear film break-up time, Schirmer I test, and corneal fluorescein staining were used to evaluate the ocular surface signs and symptoms. The concentrations of IL-17, IL-23, and ICAM-1 in serum and cellular supernatants were measured by enzyme-linked immunosorbent assays, and the gene expression levels of cytokines were measured by a quantitative real-time polymerase chain reaction. The relationships between serum concentrations of IL-17, IL-23, and ICAM-1 with clinical ocular surface parameters in CCH were analyzed using the Spearman correlation analysis. The concentrations of IL-17 and ICAM-1 in serum and cellular supernatants of CCH were significantly higher than those of NCs (all P < 0.001). The concentrations of IL-23 in serum and cellular supernatants of CCH showed no significant difference from those of NCs ( P > 0.05). The mRNA expression levels of IL-17 and ICAM-1 in conjunctival fibroblasts of CCH were significantly higher than those of NCs (all P < 0.001). The mRNA expression of IL-23 in conjunctival fibroblasts of CCH was higher than that of NCs, without a significant difference ( P > 0.05). Furthermore, the serum concentrations of IL-17 and ICAM-1 were positively correlated with Ocular Surface Disease Index and fluorescein staining (all P < 0.05), and negatively correlated with break-up time and Schirmer I test of CCH (all P < 0.05). The expression levels of IL-17 and ICAM-1 were significantly increased in CCH serum and associated with the disease severity. We postulate that IL-17 and ICAM-1 may play a role in the pathogenesis of CCH. IL-17 and ICAM-1 antagonists may be a potential treatment option for CCH in the future.

A MEK inhibitor arrests the cell cycle of human conjunctival fibroblasts and improves the outcome of glaucoma filtration surgery

Better agents are needed to improve glaucoma filtration surgery outcomes compared to current ones. The purpose of this study is to determine whether mitogen-activated protein kinase kinase (MEK) inhibitors can effectively arrest the cell cycle of human conjunctival fibroblasts (HCFs) and inhibit the formation of fibrosis and scarring following glaucoma filtration surgery. A cell counting kit‑8 assay revealed that the MEK inhibitor PD0325901 exhibited concentration-dependent growth inhibition of HCFs. Quantitative PCR, immunocytochemistry, and western blotting demonstrated decreased expression of proliferating cell nuclear antigen (PCNA) and cyclin D1 and increased expression of p27 in HCFs treated with PD0325901. Flow cytometry indicated that PD0325901 arrested the cell cycle of HCFs in the G0/1 phase. The cell-migration assay showed that HCF migration rate was significantly suppressed by PD0325901 exposure. Rabbits were divided into PD0325901-treatment and control groups, and glaucoma filtration surgery was performed. Although intraocular pressure did not differ between PD0325901-treatment and control groups, bleb height was greater in the treatment group. Histopathological evaluation revealed that fibrotic changes were significantly attenuated in the PD0325901-treatment group compared to the control group. In conclusion, the MEK inhibitor impedes HCF proliferation via cell-cycle arrest and may be beneficial for glaucoma filtration surgery by reducing bleb scarring.

Evaluation of Autophagy in Conjunctival Fibroblasts.

Vernal keratoconjunctivitis (VKC) is a serious eye allergy characterized by poorly understood pathogenic mechanisms and a lack of effective treatments. Autophagy, a process involved in both triggering and suppressing immune and inflammatory responses, plays a role in VKC's pathophysiology. Understanding autophagy's involvement in VKC could lead to new treatment possibilities, such as utilizing specific topical substances to induce or inhibit autophagy and prevent severe complications of this eye condition. In our current protocol, we present a robust methodology established in our laboratory for studying autophagy in primary conjunctival fibroblasts. We assess autophagy through techniques like immunocytochemistry, immunoblotting, and qPCR.

Physical Properties and Cellular Metabolic Characteristics of 3D Spheroids Are Possible Definitive Indices for the Biological Nature of Cancer-Associated Fibroblasts.

The current study's objective was to elucidate some currently unknown biological indicators to evaluate the biological nature of cancer-associated fibroblasts (CAFs). For this purpose, four different CAFs, CAFS1, CAFS2, SCC17F and MO-1000, were established using surgical specimens from oral squamous cell carcinomas (OSCC) with different clinical malignant stages (CAFS1 and CAFS2, T2N0M0, stage II; SCC17F and MO-1000, T4aN2bM0, stage IVA). Fibroblasts unrelated to cancer (non-CAFs) were also prepared and used as controls. Initially, confirmation that these four fibroblasts were indeed CAFs was obtained by their mRNA expression using positive and negative markers for the CAF or fibroblasts. To elucidate possible unknown biological indicators, these fibroblasts were subjected to a cellular metabolic analysis by a Seahorse bioanalyzer, in conjugation with 3D spheroid cultures of the cells and co-cultures with a pancreas ductal carcinoma cell line, MIA PaCa-2. The mitochondrial and glycolytic functions of human orbital fibroblasts (HOF) were nearly identical to those of Graves'-disease-related HOF (GOF). In contrast, the characteristics of the metabolic functions of these four CAFs were different from those of human conjunctival fibroblasts (HconF), a representative non-CAF. It is particularly noteworthy that CAFS1 and CAFS2 showed markedly reduced ratios for the rate of oxygen consumption to the extracellular acidification rate, suggesting that glycolysis was enhanced compared to mitochondrial respiration. Similarly, the physical aspects, their appearance and stiffness, of their 3D spheroids and fibroblasts that were induced effects based on the cellular metabolic functions of MIA PaCa-2 were also different between CAFs and non-CAFs, and their levels for CAFS1 or SCC17F were similar to those for CAFS2 or MO-1000 cells, respectively. The findings reported herein indicate that cellular metabolic functions and the physical characteristics of these types of 3D spheroids may be valuable and useful indicators for estimating potential biological diversity among various CAFs.

Full thickness 3D in vitro conjunctiva model enables goblet cell differentiation

In vitro culture and generation of highly specialized goblet cells is still a major challenge in conjunctival 3D in vitro equivalents. A model comprising all physiological factors, including mucus-secreting goblet cells has the potential to act as a new platform for studies on conjunctival diseases. We isolated primary conjunctival epithelial cells and fibroblasts from human biopsies. 3D models were generated from either epithelial layers or a combination of those with a connective tissue equivalent. Epithelial models were investigated for marker expression and barrier function. Full-thickness models were analyzed for goblet cell morphology and marker expression via immunofluorescence and quantitative real-time PCR. Simple epithelial models cultured at the air–liquid interface showed stratified multi-layer epithelia with pathologic keratinization and without goblet cell formation. The combination with a connective tissue equivalent to generate a full-thickness model led to the formation of a non-keratinized stratified multi-layer epithelium and induced goblet cell differentiation. In our model, a high resemblance to natural conjunctiva was achieved by the combination of conjunctival epithelial cells with fibroblasts embedded in a collagen-hydrogel as connective tissue equivalent. In the future, our conjunctival in vitro equivalent enables the investigation of goblet cell differentiation, conjunctival pathologies as well as drug testing.

TGF-β Isoforms Affect the Planar and Subepithelial Fibrogenesis of Human Conjunctival Fibroblasts in Different Manners.

Three highly homologous isoforms of TGF-β, TGF-β-1~3, are involved in the regulation of various pathophysiological conditions such as wound healing processes in different manners, despite the fact that they bind to the same receptors during their activation. The purpose of the current investigation was to elucidate the contributions of TGF-β-1 ~3 to the pathology associated with conjunctiva. For this purpose, the biological effects of these TGF-β isoforms on the structural and functional properties of two-dimensional (2D) and three-dimensional (3D) cultured human conjunctival fibroblasts (HconF) were subjected to the following analyses: 1) transendothelial electrical resistance (TEER), a Seahorse cellular metabolic measurement (2D), size and stiffness measurements of the 3D HTM spheroids, and the qPCR gene expression analyses of extracellular matrix (ECM) components (2D and 3D). The TGF-β isoforms caused different effects on the proliferation of the HconF cell monolayer evaluated by TEER measurements. The differences included a significant increase in the presence of 5 ng/mL TGF-β-1 and -2 and a substantial decrease in the presence of 5 ng/mL TGF-β-3, although there were no significant differences in the response to the TGF-β isoforms for cellular metabolism among the three groups. Similar to planar proliferation, the TGF-β isoforms also induced diverse effects toward the mechanical aspects of 3D HconF spheroids, where TGF-β-1 increased stiffness, TGF-β-2 caused no significant effects, and TGF-β-3 caused the downsizing of the spheroids and stiffness enhancement. The mRNA expression of the ECMs were also modulated in diverse manners by the TGF-β isoforms as well as the culture conditions for the 2D vs. 3D isoforms. Many of these TGF-β-3 inducible effects were markedly different from those caused by TGF-β1 and TGF-β-2. The findings presented herein suggest that the three TGF-β isoforms induce diverse and distinctly different effects on cellular properties and the expressions of ECM molecules in HconF and that these changes are independent of cellular metabolism, thereby inducing different effects on the epithelial and subepithelial proliferation of human conjunctiva.

Role of the chloride channel blocker in the formation of filtering tract scars after glaucoma surgery in rats.

Filtration surgery is commonly performed for glaucoma treatment to reduce intraocular pressure (IOP); however, scarring of the filtering bleb is the main cause of failure. In this study, we evaluated the effects of the chloride channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) on scar formation in filtering blebs. A glaucoma filtering surgery model was generated using Sprague-Dawley rats, divided into the control and NPPB groups receiving injections of different NPPB concentrations. The IOP of all rats decreased 1-day post-surgery and gradually increased afterward. However, IOP in rats from the NPPB groups recovered more slowly than that of the control group rats. In addition, the area and survival times of filtering blebs in rats from the NPPB groups were substantially larger and longer than those in the control group. Twenty-eight days after surgery, the protein and mRNA expression of collagen I, fibronectin and α-smooth muscle actin in the filtering area of rats from the NPPB groups were significantly lower than that in the control group rats. Collectively, our study demonstrates that NPPB inhibits filtering bleb scar formation, maintains filtering bleb morphology and prolongs filtering bleb survival time by inhibiting the differentiation of conjunctival fibroblasts and extracellular matrix synthesis.

Prostanoid FP and EP2 Receptor Agonists Induce Epithelial and Subepithelial Fibrogenetic Changes in Human Conjunctival Fibroblasts in Different Manners.

Purpose: To examine the effects of prostanoid FP and EP2 receptor agonists, PGF2α and Omidenepag (OMD), respectively, on the transforming growth factor beta (TGF-β2) induced conjunctival fibrogenesis. Methods: Two-dimension (2D) and three-dimension (3D) cultures of these fibroblasts were subjected to following analyses: (1) planar proliferation evaluated by transendothelial electron resistance (TEER) measurements, (2) real-time metabolic analyses, (3) subepithelial proliferation evaluated by 3D spheroid' size and stiffness measurements, and (4) the mRNA expression of extracellular matrix (ECM) molecules and their modulators. Results: TGF-β2 induced increase in the planar proliferation was significantly decreased or enhanced by PGF2α or OMD, respectively. The proportion of oxygen consumption required to drive ATP synthesis compared with that driving proton leakage was increased by PGF2α and OMD independently with TGF-β2. In contrast, maximal mitochondrial respiration was decreased by PGF2α and OMD, and the OMD-induced effect was further enhanced by the presence of TGF-β2. In addition, the TGF-β2 dependent increase in the glycolytic capacity was cancelled by PGF2α and/or OMD. Alternatively, subepithelial proliferation, as evidenced by the stiffness of the 3D spheroids, was substantially increased by both PGF2α and OMD, and these were differently modulated by TGF-β2. The expression of several related factors as above fluctuated among the conditions for both 2D and 3D and TGF-β2 untreated or treated cultures. Conclusion: The present findings indicate that the prostanoid FP or the EP2 receptor agonist may solely and differently induce the planar and subepithelial proliferation of HconF cells and these were also modulated by TGF-β2.

Impairment of autophagy promotes human conjunctival fibrosis and pterygium occurrence via enhancing the SQSTM1-NF-κB signaling pathway.

Pterygium is a common ocular disease with a high recurrence rate, characterized by hyperplasia of subconjunctival fibrovascular tissue. Autophagy, an important process to maintain cellular homeostasis, participates in the pathogenic fibrosis of different organs. However, the exact role of autophagy in pterygium pathogenesis remains unknown. Here, we found that autophagic activity was decreased in human pterygium tissues compared with adjacent normal conjunctival tissues. The in vitro model of fibrosis was successfully established using human primary conjunctival fibroblasts (ConFB) treated with transforming growth factor-β1 (TGF-β1), evidenced by increased fibrotic level and strong proliferative and invasive capabilities. The autophagic activity was suppressed during TGF-β1- or ultraviolet-induced fibrosis of ConFB. Activating autophagy dramatically retarded the fibrotic progress of ConFB, while blocking autophagy exacerbated this process. Furthermore, SQSTM1, the main cargo receptor of selective autophagy, was found to significantly promote the fibrosis of ConFB through activating the PKCι-NF-κB signaling pathway. Knockdown of SQSTM1, PKCι, or p65 in ConFB delayed TGF-β1-induced fibrosis. Overexpression of SQSTM1 drastically abrogated the inhibitory effect of rapamycin or serum starvation on TGF-β1-induced fibrosis. Collectively, our data suggested that autophagy impairment of human ConFB facilitates fibrosis via activating the SQSTM1-PKCι-NF-κB signaling cascades. This work was contributory to elucidating the mechanism of autophagy underlying pterygium occurrence.

3D-printed long-acting 5-fluorouracil implant to prevent conjunctival fibrosis in glaucoma.

To develop a sustained release 5-fluorouracil (5-FU) implant by three-dimensional (3D) printing to effectively prevent conjunctival fibrosis after glaucoma surgery. 3D-printed implants composed of polycaprolactone (PCL) and chitosan (CS) were fabricated by heat extrusion technology and loaded with 1% 5-FU. Light microscopy and scanning electron microscopy were used to study the surface morphology. The 5-FU concentration released over 8 weeks was measured by ultraviolet visible spectroscopy. The effects on cell viability, fibroblast contractility and the expression of key fibrotic genes were assessed in human conjunctival fibroblasts. The PCL-CS-5-FU implant sustainably released 5-FU over 8 weeks and the peak concentration was over 6.1 μg/ml during weeks 1 and 2. The implant had a smooth surface and its total weight decreased by 3.5% after 8 weeks. The PCL-CS-5-FU implant did not affect cell viability in conjunctival fibroblasts and sustainably suppressed fibroblast contractility and key fibrotic genes for 8 weeks. The PCL-CS-5-FU implant was biocompatible and degradable with a significant effect in suppressing fibroblast contractility. The PCL-CS-5-FU implant could be used as a sustained release drug implant, replacing the need for repeated 5-FU injections in clinic, to prevent conjunctival fibrosis after glaucoma surgery.

Neuroprotective effect of mesenchymal stem cell-derived extracellular vesicles on optic nerve injury in chronic ocular hypertension.

Mesenchymal stem cells have neuroprotective effects that limit damage to the retina and photoreceptors, and which may be mediated by extracellular vesicles (or exosomes) released by mesenchymal stem cells. To investigate the neuroprotective effect of extracellular vesicles derived from umbilical cord mesenchymal stem cells on glaucoma, we established rat models of chronic ocular hypertension by injecting conjunctival fibroblasts into the anterior chamber to mimic optic nerve injury caused by glaucoma. One week after injury, extracellular vesicles derived from umbilical cord-derived mesenchymal stem cells were injected into the vitreous cavity. We found that extracellular vesicles derived from mesenchymal stem cells substantially reduced retinal damage, increased the number of retinal ganglion cells, and inhibited the activation of caspase-3. These findings suggest that mesenchymal stem cell-derived extracellular vesicles can help alleviate optic nerve injury caused by chronic ocular hypertension, and this effect is achieved by inhibiting cell apoptosis.

Role of Oncostatin M in the Pathogenesis of Vernal Keratoconjunctivitis: Focus on the Barrier Function of the Epithelium and Interleukin-33 Production by Fibroblasts.

Vernal keratoconjunctivitis (VKC) is a severe, recurrent allergic conjunctivitis. Previously, we found high concentrations of oncostatin M (OSM) in the tears of patients with VKC. Here, we investigated the role of OSM in VKC by focusing on epithelial barrier function and IL-33 production. To assess the effect of OSM on the barrier function of human conjunctival epithelial cells (HConEpiCs), we measured transepithelial electrical resistance and dextran permeability. We also assessed expression of tight junction-related proteins such as E-cadherin and ZO-1 in HConEpiCs by Western blotting and immunofluorescence. Then we used immunohistochemistry to evaluate expression of Ki-67, E-cadherin, epithelial-mesenchymal transition-related proteins, and IL-33 in giant papillae (GPs) from patients with VKC. In addition, we used Western blotting, microarray, quantitative real-time polymerase chain reaction, and enzyme-linked immunosorbent assay to examine whether OSM activates signal transducer and activator of transcription 1 (STAT1) or STAT3 and induces the expression of various genes in human conjunctival fibroblasts (HConFs). OSM reduced expression of E-cadherin and ZO-1 in HConEpiCs, indicating barrier dysfunction. In immunohistochemistry, Ki-67 expression was present in the lower epithelial layer of the GPs, and E-cadherin expression was reduced in the superficial and lower layers; double staining revealed that GPs had a high number of fibroblasts expressing IL-33. In addition, in HConFs, OSM phosphorylated both STAT1 and STAT3 and induced IL-33. OSM has important roles in severe, prolonged allergic inflammation by inducing epithelial barrier dysfunction and IL-33 production by conjunctival fibroblasts.

Amniotic membrane: Basic concepts, different kinds

AbstractAmniotic membrane (AM) use in ophthalmic surgery has provided an alternative for corneal and conjunctival reconstruction in many clinically challenging situations and it is currently part of our armamentarium to deal with ocular surface disease and corneal surgery. In 1910, AM was first utilized surgically as a skin graft substitute material. The first ophthalmic usage was in the 1940 s as a conjunctival substitute after removal of fibrotic tissue.The AM is a thin, avascular membrane. It is the innermost layer of the foetal membranes and is composed of three layers: the epithelium, a monolayer of cuboidal cells attached to the basal lamina by numerous hemidesmosomes; the basement membrane (BM), composed of collagen, fibronectin and laminin. and the stroma. Finally, the loose connective tissue of the stroma confers tensile strength.AMT is useful clinically due to its unique structure, biocompatible composition and subsequent biological functions. Studies on human AM have shown important biological properties: enhanced epithelialization by facilitating the migration of epithelial cells, reinforcing adhesion of basal epithelial cells, promoting epithelial differentiation maintaining the epithelial phenotype, and preventing apoptosis; inhibition of inflammation, neovascularization and scarring; and antimicrobial properties.Diverse Growth factors have been found, that may have an epithelial origin. The epithelium also provides cytokines that maintain the microenvironment of the stem cells (SCs) of the corneal epithelium. BM facilitates migration of epithelial cells, reinforces adhesion of basal epithelial cells, and prevents apoptosis. Finally, a component of the stroma suppresses signalling via TGF‐beta, which modulates the proliferation of normal corneal, conjunctival, and limbal fibroblasts, and reduces fibrosis. The stroma also contains antiinflammatory and antiangiogenic proteins and protease inhibitors that reduce stromal inflammation and ulceration.Different methods of AM preservation have been described, including Cryopreservation (the most common technique of preservation), lyophilisation and dehydration and low‐temperature vacuum evaporation.Preserved AM may be used as a graft, as a patch, or a combination of both. As a graft (epithelial‐side up) AM is used in cases of both epithelial and stromal defects. The AM replaces the missing stromal matrix and provides a BM for cells to grow on. As a patch (epithelial‐side down), the AM covers an epithelial defect without stromal loss, and is sutured to the episclera near the limbus and protects the ocular surface from external insults and provides substances that reduce inflammation and promote epithelialization. Combined transplantation utilizes both the inlay and onlay methodologies of transplantation, where the graft provides structural integrity, whilst the patch allows for the protection of the graft.Indications for AMT in Ocular Surface Diseases include (1) conjunctival reconstruction in areas with conjunctival tissue defects, (2) corneal surface reconstruction: AM as a graft provides a stroma and a BM on which normal corneal epithelium grows. AM heals corneal ulcers with stromal thinning of different aetiologies, the most common being neurotrophic corneal ulcers. Additionally, AMT can be used in cicatrizing disease associated with acute or chronic SC loss and acts as a substrate for culturing epithelial stem cells of the sclerocorneal limbus.