Aihua Hou

Rho GTPases and regulation of cell migration and polarization in human corneal epithelial cells.

Purpose Epithelial cell migration is required for regeneration of tissues and can be defective in a number of ocular surface diseases. This study aimed to determine the expression pattern of Rho family small G-proteins in human corneal epithelial cells to test their requirement in directional cell migration. Methods Rho family small G-protein expression was assessed by reverse transcription-polymerase chain reaction. Dominant-inhibitory constructs encoding Rho proteins or Rho protein targeting small interfering RNA were transfected into human corneal epithelial large T antigen cells, and wound closure rate were evaluated by scratch wounding assay, and a complementary non-traumatic cell migration assay. Immunofluorescence staining was performed to study cell polarization and to assess Cdc42 downstream effector. Results Cdc42, Chp, Rac1, RhoA, TC10 and TCL were expressed in human corneal epithelial cells. Among them, Cdc42 and TCL were found to significantly affect cell migration in monolayer scratch assays. These results were confirmed through the use of validated siRNAs directed to Cdc42 and TCL. Scramble siRNA transfected cells had high percentage of polarized cells than Cdc42 or TCL siRNA transfected cells at the wound edge. We showed that the Cdc42-specific effector p21-activated kinase 4 localized predominantly to cell-cell junctions in cell monolayers, but failed to translocate to the leading edge in Cdc42 siRNA transfected cells after monolayer wounding. Conclusion Rho proteins expressed in cultured human corneal epithelial cells, and Cdc42, TCL facilitate two-dimensional cell migration in-vitro. Although silencing of Cdc42 and TCL did not noticeably affect the appearance of cell adhesions at the leading edge, the slower migration of these cells indicates both GTP-binding proteins play important roles in promoting cell movement of human corneal epithelial cells.

Comparison of gene expression profiles in primary and immortalized human pterygium fibroblast cells

PURPOSE Pterygium is a fibrovascular growth on the ocular surface with corneal tissue destruction, matrix degradation and varying extents of chronic inflammation. To facilitate investigation of pterygium etiology, we immortalized pterygium fibroblast cells and profiled their global transcript levels compared to primary cultured cells. METHODS Fibroblast cells were cultured from surgically excised pterygium tissue using the explant method and propagated to passage number 2-4. We hypothesized that intervention with 3 critical molecular intermediates may be necessary to propage these cells. Primary fibroblast cells were immortalized sequentially by a retroviral construct containing the human telomerase reverse transcriptase gene and another retroviral expression vector expressing p53/p16 shRNAs. Primary and immortalized fibroblast cells were evaluated for differences in global gene transcript levels using an Agilent Genechip microarray. RESULTS Light microscopic morphology of immortalized cells was similar to primary pterygium fibroblast at passage 2-4. Telomerase reverse transcriptase was expressed, and p53 and p16 levels were reduced in immortalized pterygium fibroblast cells. There were 3308 significantly dysregulated genes showing at least 2 fold changes in transcript levels between immortalized and primary cultured cells (2005 genes were up-regulated and 1303 genes were down-regulated). Overall, 13.58% (95% CI: 13.08-14.10) of transcripts in immortalized cells were differentially expressed by at least 2 folds compared to primary cells. CONCLUSION Pterygium primary fibroblast cells were successfully immortalized to at least passage 11. Although a variety of genes are differentially expressed between immortalized and primary cells, only genes related to cell cycle are significantly changed, suggesting that the immortalized cells may be used as an in vitro model for pterygium pathology.

Evaluation of Global Differential Gene and Protein Expression in Primary Pterygium: S100A8 and S100A9 as Possible Drivers of a Signaling Network

Purpose Pterygium is a wing shaped fibrovascular growth on the ocular surface, characterized by fibrosis, angiogenesis, extracellular matrix remodeling, and inflammatory infiltrates. Epidemiologic studies have linked pterygium formation to various chronic inflammatory conditions, such as ultraviolet radiation, sawdust exposure, and dry eye disease. The purpose of this study is to identify proteins that are differentially expressed in primary pterygium by using a combination of gene microarray and proteomic platforms. Methods Paired pterygium and uninvolved conjunctiva tissues of four patients were evaluated for differences in global gene transcript levels using a genechip microarray. Proteins extracted from another four pairs of tissues were quantified by iTRAQ approach. Western blot and immunofluorescent staining on additional patients were used to validate dysregulated protein expression obtained from microarray and proteomics data. In addition, primary conjunctival fibroblasts were treated with recombinant S100A8, S100A9 or both. Transcript level changes of a panel of potential target genes were evaluated by real time-PCR. Results The following were up-regulated at both protein and transcript levels S100 A8 and A9, aldehyde dehydrogenase 3 family, member1 (ALDH3A1) and vimentin (VIM). Conversely, serpin peptidase inhibitor clade A member 1 (SERPINA1) and transferrin (TF) were down-regulated. Upon adding S100A8, S100A9 or both, the inflammatory chemokine CXCL1, matrix proteins vimentin, biglycan, and gelsolin, as well as annexin-A2, thymosin-β4, chymase (CMA1), member of Ras oncogene family RAB10 and SERPINA1 were found to be up-regulated. Conclusions We identified 3 up-regulated and 2 down-regulated proteins by using a stringent approach comparing microarray and proteomic data. On stimulating cells with S100A8/9, a repertoire of key genes found to be up-regulated in pterygium tissue, were induced in these cells. S100A8/9 may be an upstream trigger for inflammation and other disease pathways in pterygium.

Tissue resident memory T cells in the human conjunctiva and immune signatures in human dry eye disease

Non-recirculating resident memory (TRM) and recirculating T cells mount vigorous immune responses to both self and foreign antigens in barrier tissues like the skin, lung and gastrointestinal tract. Using impression cytology followed by flow cytometry we identified two TRM subsets and four recirculating T-subsets in the healthy human ocular surface. In dry eye disease, principal component analysis (PCA) revealed two clusters of patients with distinct T-cell signatures. Increased conjunctival central memory and naïve T cells characterized Cluster-1 patients, and increased CD8+ TRMs and CD4+ recirculating memory T cells characterized Cluster-2 patients. Interestingly these T-cell signatures are associated with different clinical features: the first signature correlated with increased ocular redness, and the second with reduced tear break up times. These findings open the door to immune-based characterization of dry eye disease and T-subset specific immunotherapies to suppress T-subsets involved in disease. They may also help with patient stratification during clinical trials of immunomodulators.

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

Conjunctivochalasis is the precursor to pterygium

Pterygium is a fibrovascular proliferative condition of the ocular surface with no known pathological mechanism. This condition affects vision due to dry eyes, astigmatism or physical occlusion of the visual axis for severe cases. The only definitive treatment for this condition is surgical excision. Interestingly, it is a lesion that may be related to UV radiation and elaboration of proteases. Conjunctivochalasis is a dry eye related condition that is exemplified by excessive conjunctiva or the mucous membrane of the front of the eye around the cornea. Both pterygium and conjunctivochalasis are associated with elaboration of matrix metalloproteinases as well as inflammatory cytokines. We propose that under specific conditions, conjunctivochalasis in the nasal part of the conjunctiva can progress to pterygium. The progression of conjunctivochalasis to pterygium may be related to special kinds of oxidative or inflammatory damage that affects only the part of the loose conjunctival tissue adjacent to the cornea. Protease expressed may then breakdown the conjunctival and corneal epithelium causing the head of pterygium to be very adherent to the cornea. This explains the fact that surgically excised pterygium tissue has stromal tissue enclosed by epithelia on both surfaces. In addition, it explains the existence of a surgical plane when an instrument is passed under the neck of the pterygium tissue but not at the apex. The implications of this hypothesis are first, treatment should be directed to the protection of conjunctivochalasis before it transforms to pterygium. This may be achieved by anti-inflammatory measures, anti-protease treatment, or preventing the triggering of the changes at the head of pterygium, such as avoidance of sunlight. Second, during resection of pterygium, it may not be necessary to resect the pterygium too extensively away from the cornea, since this effectively removes relatively normal conjunctiva.

A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable dry eye animal model to test novel therapeutics to treat autoimmune dry eye conditions. This protocol describes a chronic autoimmune dry eye rat model. Lewis rats were immunized with an emulsion containing lacrimal gland extract, ovalbumin, and complete Freunds adjuvant. A second immunization with the same antigens in incomplete Freunds adjuvant was administered two weeks later. These immunizations were administered subcutaneously at the base of the tail. To boost the immune response at the ocular surface and lacrimal glands, lacrimal gland extract and ovalbumin were injected into the forniceal subconjunctiva and lacrimal glands 6 weeks after the first immunization. The rats developed dry eye features, including reduced tear production, decreased tear stability, and increased corneal damage. Immune profiling by flow cytometry showed a preponderance of CD3+ effector memory T cells in the eyeball.

Mechanistic role of transglutaminase-2 in focal adhesions

Transglutaminase (TG)-2 interacts with matrix proteins and integrins, forming focal adhesions (FA) to initiate cell migration, thus playing a vital role in wound healing. Previously we showed that TG-2 influenced phosphorylation of paxillin and other FA proteins. Here, we aimed to investigate the molecular mechanism of TG-2 regulation of paxillin. Human corneal epithelial cells expressing shRNA against TG-2 (shTG) and scrambled sequence control (shRNA) were cultured. TG-2 was pulled down by anti-paxillin antibody, but not MAP3K12. Cell-free interaction assay with immobilized paxillin shows that TG-2 bind to paxillin directly. JNK was the strongest kinase for paxillin phosphorylation in the in-vitro kinase screen, but TG-2 could not phosphorylate paxillin directly. Increasing TG-2 concentrations did not increase the amount of JNK in the TG-2/paxillin complex. Immunofluoresent staining shows that TG-2 colocalises with vinculin and paxillin in FA of migrating cells. TG-2 binds to paxillin and JNK-containing FA but does not recruit JNK directly. Taken together with previous findings, TG-2 binds paxillin non-covalently, and JNK can phosphorylate paxillin, these processes critically regulate corneal epithelial adhesion and migration.

Altered expression level of inflammation-related genes and long-term changes in ocular surface after trabeculectomy, a prospective cohort study

PURPOSE We aim to evaluate changes in the ocular surface in a cohort of post-trabeculectomy patients and whether these were associated with conjunctival inflammatory gene expression. METHODS This is a single-arm interventional cohort performed in a tertiary referral center. These were assessed: dry eye symptom questionnaire, tear osmolarity, Schirmers test, non-invasive tear break up time (BUT), conjunctival redness and corneal fluorescein staining evaluation. Conjunctival impressions were performed using Eyeprim, and after RNA extraction, transcripts of 255 inflammatory genes were analysed using the Nanostring nCounter assay. RESULTS Thirty three patients were recruited with age 66.88 ± 9.76 at baseline, with a predominance of men. There was a significant decrease in inferior corneal staining at 6 months (p < 0.05) (n = 22) and significant decrease in tear osmolarity at 12 months (p < 0.01) (n = 27). No patient required glaucoma eyedrops post-surgery up to 3 years. At baseline 31/33 transcript profiles passed the quality control, and after normalization, 249 transcripts were subsequently analysed. Increased discomfort was associated with higher Protein Tyrosine Kinase-2 at the cross-sectional analysis at baseline. Lower baseline complement factor-D and higher levels of Mitogen associated kinase-8, MAP3K1 and MyD88, were associated with presence of corneal staining at 6 months. Nine genes, including the proinflammatory lipo-oxygenase (ALOX5) showed a significantly reduced level at 3 years (n = 5). CONCLUSIONS Glaucoma surgery may confer long term beneficial effect on the ocular surface, if anti-glaucoma eyedrops are no longer necessary. This may be due to reduced expression of conjunctival proinflammatory genes and immune-related genes.

Expression, Regulation, and Effects of Interleukin-17f in the Human Ocular Surface

ABSTRACT Purpose: To evaluate the basal and possibly stimulated expression of interleukin (IL)-17 in the context of the ocular surface and potential downstream effects. Methods: Western blot and immunofluorescent staining were used to evaluate IL-17F expression in human cornea/conjunctival tissues and cornea epithelial cell line. Cytokines and matrix metalloproteinase (MMP) transcripts were quantified by qPCR. IL-17F effects on NF-κB were investigated by the secretary alkaline phosphatase assay. Results: IL-17F was expressed in the cytoplasm of human corneal and conjunctival epithelial tissues. In the corneal cell line, exogenous IL-17F did not increase the NF-κB activity, but Pam3CSK4 increased IL-17F transcripts. IL-17F stimulated MMP-9 activity, promoted IL-6, IL-8, tumor necrosis factor-α transcripts levels, and depressed monocyte chemoattractant protein-1 expression, but did not affect transforming growth factor beta-1 transcript levels. Conclusions: Normal corneal/conjunctival epithelial cells express IL-17F. Microbial agents may stimulate IL-17F via the NF-κB pathway. Matrix dissolution stimulated by IL-17F may have a role in the melting and necrosis of cornea in severe inflammation.