De-Quan Li

Suppression of transforming growth factor-beta isoforms, TGF-beta receptor type II, and myofibroblast differentiation in cultured human corneal and limbal fibroblasts by amniotic membrane matrix.

Down‐regulation of the transforming growth factor‐beta (TGF‐β) signaling system is a strategy for preventing scarring during wound healing. Human corneal and limbal fibroblasts were cultured on the stromal matrix side of preserved human amniotic membrane. The levels of TGF‐β1, β2, and β3 and TGF‐β type II receptor transcripts and TGF‐β1 and β2 proteins were suppressed as early as 8 hr and more dramatically at 24 hr after contact with an amniotic membrane. This suppressive effect was accompanied by down‐regulation of α‐smooth muscle actin, EDA spliced form of fibronectin, and integrin α5. It persisted even when challenged by 10 ng/ml TGF‐β1. In contrast with their counterparts grown on plastic or in collagen gel, such suppression in amniotic membrane cultures remained complete after 1 week of culturing. Cells cultured on amniotic membrane showed significantly reduced [3H]‐thymidine incorporation compared to cells cultured on plastic and displayed no DNA fragmentation. These results reveal a novel mechanism by which the TGF‐β signaling system, DNA synthesis, and subsequent myofibroblast differentiation can be suppressed by an amnionic membrane matrix. This action explains in part the antiscarring results of amniotic membrane transplantation used for ocular surface reconstruction, a surgical technique applicable to other subspecialties. It may also explain in part why fetal wound healing is scarless. J. Cell. Physiol. 179:325–335, 1999.

Characterization of putative stem cell phenotype in human limbal epithelia.

This study evaluated proposed molecular markers related to stem cell (SC) properties with the intention of characterizing a putative SC phenotype in human limbal epithelia. Human corneal and limbal tissues were cut in the vertical and horizontal meridians for histology, transmission electron microscopy (TEM), and immunostaining. Semiquantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) and in situ hybridization were used to evaluate gene expression. TEM showed that the limbal basal cells were small primitive cells. Immunostaining disclosed that p63, ABCG2 and integrin α9 were primarily expressed by the basal epithelial cells of limbus. Antibodies against integrin β1, epidermal growth factor receptor (EGFR), K19, enolase‐α, and CD71 stained the basal cells of the limbus more brightly than the suprabasal epithelia. Integrin α6, nestin, E‐cadherin and connexin 43 did not stain the limbal basal cells, but the suprabasal epithelia of the cornea and limbus showed strong immunoreactivity. K3 and involucrin stained only corneal and limbal superficial cells. RT‐PCR showed higher levels of p63, ABCG2 and integrin α9 mRNA, but lower levels of K3, K12 and connexin 43 expressed in the limbal epithelia than the corneal epithelia. In situ hybridization showed that p63 transcripts were located in basal layer of the limbal epithelium. This work suggests that the basal epithelial cells of the limbus are p63, ABCG2 and integrin α9 positive, and nestin, E‐cadherin, connexin 43, involucrin, K3, and K12 negative, with relatively higher expression of integrin β1, EGFR, K19, and enolase‐α. This putative SC phenotype may facilitate the identification and isolation of limbal epithelial SCs.

ABCG2 Transporter Identifies a Population of Clonogenic Human Limbal Epithelial Cells

ABCG2, a member of the ATP binding cassette (ABC) transporters, has been identified as a molecular determinant for bone marrow stem cells and proposed as a universal marker for stem cells. This study investigates ABCG2 expression and its potential as a marker that identifies human limbal epithelial stem cells. ABCG2 expression was evaluated by immunofluorescent and immunohistochemical staining, laser scanning confocal microscopy, flow cytometry, and semiquantitative reverse transcription–polymerase chain reaction. Cells selected from primary limbal epithelial cultures by flow cytometry with ABCG2 monoclonal antibody (mAb) or Hoechst 33342 dye staining were evaluated for their gene expression and colony‐forming efficiency (CFE). ABCG2 protein was mainly located in the basal cells of limbal epithelia but not in the limbal suprabasal and corneal epithelia. ABCG2 staining was also observed in primary limbal epithelial cultures. Limbal epithelia express higher levels of ABCG2 and ΔNp63 mRNAs than corneal epithelia. Labeling with ABCG2 mAb yielded 2.5%–3.0% positive cells by flow cytometry. The ABCG2‐positive cells exhibited greater CFE on a 3T3 fibroblast feeder layer than ABCG2‐negative cells. A side population (SP) was detected by the Hoechst 33342 exclusion assay. SP cells displayed stronger expression of ABCG2 and ΔNp63 mRNA and greater CFE than the non‐SP cells. In conclusion, these findings demonstrate that ABCG2 transporter was exclusively expressed by limbal basal cells and that the ABCG2‐positive and SP cells possess enriched stem cell properties, suggesting for the first time that ABCG2 could serve as a marker to identify the putative limbal epithelial stem cells.

IL-17 disrupts corneal barrier following desiccating stress.

T helper (Th)-17 is a recently identified subtype of Th response that has been implicated in host defense and autoimmunity. We investigated whether there is evidence for a Th-17 response in human and experimental murine dry eye (DE). Gene expression in the human DE conjunctiva showed increased levels of the Th-17 inducers, interleukin (IL)-23, IL-17A, and interferon-gamma (IFN-γ). In the murine model, we found that desiccating stress increased matrix metalloproteinase-9, Th-17-associated genes (IL-6, IL-23, transforming growth factor-β1 and -2, IL-23R, IL-17R, IL-17A, retinoid-related orphan receptor-γt, and CC chemokine attractant ligand-20) and IFN-γ in cornea and conjunctiva. Furthermore, we found a significantly increased concentration of IL-17 in tears and number of IL-17-producing cells on the ocular surface. Antibody neutralization of IL-17 ameliorated experimental DE-induced corneal epithelial barrier dysfunction and decreased the expression of matrix metalloproteinases 3 and 9. Taken together, these findings suggest that IL-17 has a role in corneal epithelial barrier disruption in DE.

Hyperosmolar saline is a proinflammatory stress on the mouse ocular surface.

Purpose. To investigate whether hyperosmolar stress stimulates production of inflammatory mediators and activates the mitogen-activated protein kinase (MAPK) signaling pathways, c-jun n-terminal kinases (JNKs), extracellular-regulated kinases (ERKs), and p38 on the mouse ocular surface. Methods. 129SvEv/CD-1 mixed mice were treated with a balanced salt solution (BSS) (305 mOsM) or a hyperosmotic saline solution (HOSS) (500 mOsM). Untreated age-matched mice were used as controls. The concentrations of interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) were measured by enzyme-linked immunosorbent assay. Gelatinase activity was determined by in situ zymography. Corneal and conjunctival epithelia were lysed for Western blot with MAPK antibodies or used for semiquantitative reverse transcription and polymerase chain reaction and gene array. Results. Compared with age-matched controls and mice treated with BSS, the concentration of IL-1β in tear fluid washings and the concentrations of IL-1β and TNF-α and gelatinolytic activity in the corneal and conjunctival epithelia were significantly increased in mice treated with HOSS for 2 days. The expressions of IL-1β, TNF-α, and matrix metalloproteinase 9 (MMP-9) messenger RNA by the corneal and conjunctival epithelia were also notably stimulated in mice treated with HOSS. The levels of phosphorylated JNK1/2, ERK1/2, and p38 MAPKs in the corneal and conjunctival epithelia were slightly increased in mice treated with BSS, but markedly increased in mice treated with HOSS. Conclusions. These results show that the hyperosmolarity stimulates expression and production of IL-1β, TNF-α, and MMP-9 and activates JNK, ERK, and p38 MAPK signaling pathways on the mouse ocular surface. These findings suggest that hyperosmolar stress, as it may occur in dry eye, promotes ocular surface inflammation.

Matrix Metalloproteinase-9 Knockout Confers Resistance to Corneal Epithelial Barrier Disruption in Experimental Dry Eye

Altered corneal epithelial barrier function is the cause for ocular irritation and visual morbidity in dry eye disease. Increased matrix metalloproteinase (MMP)-9 activity has been observed in the tear fluid of dry eye patients. To determine the pathogenic role of MMP-9 in the corneal epithelial disease of dry eye, the effects of experimentally induced dry eye on corneal epithelial morphology and barrier function were compared in MMP-9 knockout mice and their wild-type littermates. Dry eye was created through cholinergic blockade and exposure to a desiccating environment. The tear fluid MMP-9 concentration increased in response to dryness in wild-type mice. Corneal epithelial permeability to three different-sized molecules increased in dry eye wild-type mice, but not in MMP-9 knockout mice. Topical administration of active MMP-9 to dry eye MMP-9 knockout mice significantly increased corneal epithelial permeability. Compared to MMP-9 knockout mice, wild-type mice showed greater desquamation of differentiated apical corneal epithelial cells that expressed the tight junction protein occludin in response to dryness. This was accompanied by an increase in lower sized (50 kd) occludin in the corneal epithelia of wild-type mice. These findings could be replicated in cultured human corneal epithelial cells that were treated with active MMP-9. These studies indicate that increased MMP-9 activity on the ocular surface in response to dryness disrupts corneal epithelial barrier function. This appears to be because of accelerated loss of tight junction bearing superficial corneal epithelial cells, perhaps by proteolytic cleavage of occludin.

Production and Activity of Matrix Metalloproteinase-9 on the Ocular Surface Increase in Dysfunctional Tear Syndrome

PURPOSE To evaluate production and activity of metalloproteinase (MMP)-9 on the ocular surface of patients with dysfunctional tear syndrome (DTS) and determine any correlation between MMP-9 activity and clinical parameters. METHODS Forty-six patients with newly diagnosed DTS and 18 control subjects were recruited. Complete ocular surface examinations were performed. Tear MMP-9 activity was assessed with an MMP-9 activity assay in 1 microL of unstimulated tear fluid. Using conjunctival epithelial cells from 19 patients with DTS and 16 controls, levels of MMP-9 and its regulating cytokine mRNA transcripts were evaluated by semiquantitative real-time PCR. RESULTS Each of four DTS severity-based groups had significantly higher mean MMP-9 activities than did the control group, which was 8.39 +/- 4.70 ng/mL. The DTS4 group had the highest MMP-9 activity (381.24 +/- 142.83 ng/mL), for which the mean was significantly higher than that of other DTS groups. In addition, patients with DTS had significantly higher levels of IL-1beta, IL-6, TNF-alpha, and TGF-beta1 mRNA transcripts in their conjunctival epithelia than did the control subjects. Tear MMP-9 activities showed significant correlation with symptom severity scores, decreased low-contrast visual acuity, fluorescein tear break-up time, corneal and conjunctival fluorescein staining, topographic surface regularity index (SRI), and percentage area of abnormal superficial corneal epithelia by confocal microscopy. CONCLUSIONS Tear MMP-9 activity was significantly higher in patients with DTS. This activity was associated with increased mRNA expression of MMP-9 and its regulating genes and correlated strongly with clinical parameters. MMP-9 appears to be a potentially useful biomarker for diagnosing, classifying, and monitoring DTS.

Cell Size Correlates with Phenotype and Proliferative Capacity in Human Corneal Epithelial Cells

This study investigated whether cell size correlates with phenotype and proliferative capacity in human corneal epithelial cells. Primary cultured human corneal epithelial cells were sorted by flow cytometry based on forward scatter profile in comparison with the profile of beads of known size. Four fractions (A, B, C, and D) of cells ranging in size from 10 to 16, 17 to 23, 24 to 30, and ≥31 μm in diameter, respectively, were collected to evaluate their 5‐bromo‐2‐deoxyuridine (BrdU) label retention properties, cell phenotype, and clonal growth capacity on a 3T3 fibroblast feeder layer. Among these four populations, cell size was shown to positively correlate with the expression of the differentiation markers keratin (K) 3, K12, and involucrin and inversely with the levels of stem cell–associated markers ΔNp63 and ABCG2 and with colony‐forming efficiency (CFE) and growth capacity. Population A with the smallest size, accounting for 11.0% ± 4.5% of the entire population, contained the greatest number of BrdU label‐retaining slow‐cycling cells, displayed the highest percentage of cells immunopositive to p63 and ABCG2 and negative to K3 and involucrin, expressed the highest levels of ΔNp63 and ABCG2 mRNA and the lowest levels of K3, K12, and involucrin, and possessed the highest CFE and growth capacity. These findings suggest that cell size correlates with cell differentiation phenotypes and proliferative capacity in human corneal epithelial cells. The smallest cells in population A seem to be enriched for putative stem cells, and small cell size may represent one of the important properties of adult corneal epithelial stem cells.

Hyperosmolarity-Induced Apoptosis in Human Corneal Epithelial Cells Is Mediated by Cytochrome c and MAPK Pathways

Purpose: To study whether hyperosmolarity induces apoptosis in human corneal epithelial cells through cytochrome c-mediated death pathways and by activation of mitogen-activated protein kinases (MAPKs). Methods: Primary human corneal epithelial cells cultured in normal osmolar media (312 mOsM) were switched to hyperosmolar media (450, 500, and 550 mOsM) by adding 70, 90, and 120 mM NaCl, respectively, with or without the c-jun N-terminal kinase (JNK) inhibitor SB202190 or the extracellular-regulated kinase (ERK) inhibitor PD98059. Apoptosis was assessed by the ApopTag In Situ Oligo Ligation (ISOL) assay. Confocal microscopy was used to detect cytochrome c and active caspase-3. Total RNA was extracted and subjected to reverse transcriptase-polymerase chain reaction for apoptosis-associated genes. Western blots were performed on cell extracts for the apoptogenic molecules cytochrome c and Smac/DIABLO, and phospho-JNK and ERK. Results: ISOL-positive apoptotic cells significantly increased from 3.3 ± 1.6% in control medium to 11.4 ± 5.8%, 18.9 ± 4.8%, and 43.9 ± 8.8% in 70, 90, and 120 mM NaCl added media, respectively. The 90 mM NaCl high saline medium notably increased release of cytochrome c and Smac/DIABLO from mitochondria; activated caspase-3, JNK and ERK; stimulated mRNA expression of interleukin-1-converting enzyme and Bax; and reduced Bcl2 expression. SB202190 and PD98059 significantly suppressed hyperosmolarity-induced JNK/ERK activation and ISOL-positive cells. In addition, PD98059 inhibited the release of cytochrome c and Smac/DIABLO from mitochondria. Conclusions: These findings show that hyperosmolarity induces apoptosis of human corneal epithelial cells through a cytochrome c-mediated death pathway, which may be mediated by JNK and ERK MAPK signaling pathways.

Differential regulation of keratinocyte growth factor and hepatocyte growth factor/scatter factor by different cytokines in human corneal and limbal fibroblasts

Corneal epithelial stem cells and transient amplifying cells are located in the limbal and corneal regions, respectively. In a serum‐free medium with or without different cytokines, limbal fibroblasts consistently produced greater levels of keratinocyte growth factor (KGF) transcript and protein than corneal fibroblasts, whereas corneal fibroblasts produced greater levels of hepatocyte growth factor/scatter factor (HGF/SF) transcript and protein than limbal fibroblasts. Expression of HGF/SF transcript and protein was up‐regulated mildly by epidermal growth factor (EGF), transforming growth factor‐α (TGF‐α), or platelet‐derived growth factor B (PDGF‐BB) but markedly by interleukin‐1β (IL‐1β) and was more pronounced in limbal than in corneal fibroblasts. Expression of KGF transcript was down‐regulated by EGF, TGF‐α, and PDGF‐BB, was markedly up‐regulated by IL‐1β, and was more pronounced in limbal than in corneal fibroblasts. Expression of KGF protein was up‐regulated markedly by IL‐1β and moderately by PDGF‐BB, especially in limbal fibroblasts. TGF‐β1 uniquely turned off transcript and protein expression of HGF/SF and KGF in corneal fibroblasts. Although its transcript levels were similarly down‐regulated in limbal fibroblasts, KGF protein levels were paradoxically up‐regulated by TGF‐β1 when added alone or with TGF‐α or IL‐1β. These data indicate that KGF and HGF/SF, two fibroblast‐derived epithelial mitogens, are expressed differentially by limbal and corneal fibroblasts and are modulated by cytokines activated during epithelial‐mesenchymal interactions, suggesting that they may play a different role in modulating corneal epithelial stem cells and transient amplifying cells. J. Cell. Physiol. 172:361–372, 1997.