In diabetics, abnormalities are seen in the retina, lens, lid, iris, ciliary body, and nerves.
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2 Diabetic corneal complications, such as abnormalities in the basement membrane of the corneal epithelium, barrier function impairment in the corneal epithelium, decreased corneal perception, abnormal collagen depositions in Descemet′s membrane, morphological changes in corneal endothelial cells, and pump function deterioration are also common.
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6 Hemidesmosomes and anchoring fibrils passing through the stroma in the adhesion complex are reduced, resulting in weakened hemidesmosome adhesion. As a result, preservation of the corneal epithelial layer (corneal epithelialization) is impaired. Furthermore, abnormalities in the corneal epithelial basement membrane may impair normal barrier function or induce edema in the corneal stromal area. Thus, depending on the healing of wound and inflammation there may be substantial impediments to tissues recovery.
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4 In addition, as the corneal endothelial cell pump function is damaged by diabetes, water is transported to the anterior corneal stromal tissues; therefore, particular cautions are required to reduce corneal complications such as keratitis.
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Furthermore, tissues react differently to cytokines in normal individuals, versus those with chronic diseases, such as diabetes, hypertension, or tumor lesions. These differences arise through differences in signal transduction pathway activation. We used microarrays and real-time polymerase chain reaction (real-time PCR) to measure the expression of genes or proteins involved in corneal stromal cell signal transduction. We compared normal cells in the spontaneously generated diabetes animal model (OLETF), with and without induction by pro-inflammatory cytokines.
Materials and Methods
1. Cell culture
Fifty-week-old genetically-affected type 2 diabetic (OLETF, Otsuka Long-Evans Tokushima Fattay, Japan) and normal rat eyes were purchased from Otsuka pharmaceutical company (Japan). The corneal epithelium was removed, the corneal stroma isolated, and washed several times in saline containing antibiotics. The corneal endothelial layer was removed, and the corneal stromal tissue was cut into small pieces, and cultured serially in culture medium. The culture medium was Dulbeco's Modified Eagle medium (DMEM, Gibco BRL, NY, USA) containing 10 % fetal bovine serum(FBS, Gibco BRL. NY, USA), 100 units/ml penicillin (Gibco BRL, NY, USA), and 100 mg/ml streptomycin (Gibco BRL. NY, USA). The culture medium was changed at 2-3 day intervals. When the cells became confluent, the culture medium was removed, the cells were washed once with Dulbeco's Phosphate-Buffered Saline (D-PBS, Gibco BRL, NY, USA) and dissociated with 0.25 % trypsin-0.02 % EDTA. For microarray experiments, cells between the 3rd and 4th generation of serial culture were collected and stored at -70℃.
2. Cytokine treatment
The cytokines IL-1α (R&D Systems, MN, USA) and TNF((R&D Systems, MN, USA) were used to treat the corneal stromal cells of diabetic and normal rats treated at a concentration of 20 ng/ml for 6 hours.
3. RNA extraction
Total cellular RNAs were extracted from the corneal stromal cells of diabetic and normal rats in primary culture using the RNeasy mini kit (QIAGEN inc, 74104, USA) for gene microarrays. First, denaturing solution was added to cell pellets on ice for 5 min, then phenol and chloroform were added to the samples, they were centrifuged, and the supernatant containing RNA was transferred to a new tube. The process was repeated, and subsequently, -20℃ isopropanol was added to precipitate the RNA, centrifuged, and the supernatant discarded. The pellet was washed with -20℃ 80 % ethanol and air-dried. The RNA was quantitated and confirmed by electrophoresis.
4. DNA gene microarrays
Sixty-mer oligonucleotides corresponding to each gene were synthesized, and subsequently placed on a slide using a robotic gene microarray. The robotic gene microarray places 0.25-1 nl DNA samples on a slide sample in spots averaging 100-150 nm. cDNA was synthesized from the isolated RNA samples using RT primers (Genisphere Inc, PA, USA) and SuperScript II reverse transcriptase (Invitrogen, NY, USA). The purified cDNA was hybridized to an Agilent Rat oligo 22K chip (Agilent tech, G4130A, USA). For hybridization, slides were placed in a dark hybridization chamber at 62℃ for 16 h. The slides were removed, washed 3 times, and dried using a centrifuge. A fluorescent dye was added to DNA capture reagents that bind RT primers, and a second hybridization was performed for 4 h. The slides were removed, washed, and dried using a centrifuge. Afterward, the fluorescence level was measured using an Axon laser fluorescence scanner (Axon ins, CA, USA). The microarray analyzer used in our study has 105 rows and 215 columns. A total of 22575 genes pertinent to the signal transduction were included.
5. Analysis
The hybridized slides were scanned by an Axon GenePix Laser Fluorescence Scanner and analyzed by GenePix Pro 5.1 (Axon, CA, USA) and GeneSpring 7.0 (Silicongenetics, CA, USA). Genes showing more than a two-fold difference in expression level between normal and diabetic rats were classified as significant. Genes showing similar patterns were distinguished by a Pearson correlation. The images show a gene as red if it is up-regulated in the diabetic rat, and green if it is down-regulated.
6. Real-time polymerase chain reaction
Real-time polymerase chain reaction (real time-PCR) was performed with SYBR Green I immunofluorescence dye and the HotStarTag DNA polymerase QuantiTect SYBR Green PCR Kit (QIAGEN GmbH, Germany). To quantitate the control group, a standard curve of genes to be tested and GAPDH was prepared. The reactions were carried out in an ABI PRISM 7900 Sequence Detection System version 1.6 software (Perkin-Elmer Biosystems, Foster City, CA, USA). Relative quantitation measurements were performed applying the relative standard curve method according to the manufacturer's instructions.
Discussion
In multicellular organisms, biological activities are controlled by cellular signals. Diverse signal transduction pathways regulate apoptosis, proliferation, migration, polarity, and other processes.
10 In corneal tissues, the interaction between the stromal and epithelial layers plays an important role in proliferation, migration, differentiation, morphogenesis, and other functions. Furthermore, it has been shown to contribute to homeostasis in the ocular surface and wound healing.
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Abnormal signal transduction between the stroma and epithelial layers may result in impaired corneal tissue development or delayed wound healing. In diabetics, reduced corneal perception, an abnormal corneal epithelial basement complex, and impaired corneal epithelium barrier function develop. Furthermore, spot-like corneal epithelial defects, delayed corneal epithelium regeneration after trauma, and neurotrophic keratitis are common as well.
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6 Therefore, studies on genes pertinent to corneal signal transduction pathways are important for understanding the mechanism of corneal diseases induced by diabetes.
In our study, the expression of genes associated with signal transduction in normal corneal stroma cells was compared to expression in diabetic corneal stromal cells. The purpose of this study is to provide a basis of gene therapy by identifying gene alterations induced by diabetes. In addition, we compared genes related to signal transduction following cytokine stimulation. Responses to inflammatory reactions were examined using IL-1α and TNF-α
Generally, when the corneal epithelial layer is damaged, the tissue secretes cytokines. While corneal stromal cells undergo apoptosis, the cytokines induce chemotaxis, proliferation of activated stromal cells, and excessive secretion of extracellular matrix substances such as collagen, eventually resulting in corneal wound recovery. IL-1α and TNF-α are representative of the cytokines involved in this process.
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14 Because the cytokines are primarily secreted from damaged corneal epithelial cells,
14 corneal stromal cells were exposed to IL-1α and TNF-α for approximately 6 hours. This time is sufficient to mimic the effect of cytokines released in response to corneal epithelial layer damage. Genes expressed at that time were classified as normal or diabetic and compared with each other.
The insulin, MAPK, calcium, and TGF-β pathways showed a large difference in expression between normal and diabetic corneal stromal cells. It has been shown that the insulin pathway improved the migration capacity of cells and thus accelerated wound healing instead of suppressing cell proliferation, following epithelial defect in corneal epithelial cells.15 In other words, the delayed wound healing that has been detected frequently in diabetes could be explained by abnormal insulin signaling. In our study, 5 of 6 insulin signal transduction genes were down-regulated.
The MAPK signal pathway has 3 subtypes, the c-jun N-terminal kinase (JNK) pathway, the extracellular signal-regulated kinase (ERK) pathway, and the p38 MAPK pathway. Upon activating the MAPK pathway, the tight junction in corneal epithelial cells is destroyed. Thus the pathway plays an important role in morphological control and corneal epithelial cell barrier function.
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17 All 7 genes involved in the MAPK signal pathway were up-regulated in diabetic corneal stromal cells.
Changes, such as cell migration and proliferation, are required for wound healing upon corneal epithelium damage requiring signal transduction among cells. Calcium-dependent signal transduction is required to convert extracellular stimulation to intercellular signal transduction. Calcium levels are increased in corneal epithelial cells in response to mechanical damage, transducing the signal to adjacent cells, thus facilitating wound healing.
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19 Therefore, our study suggests, that wound healing is delayed when corneal tissues are damaged in diabetics, because calcium signal transduction genes are down-regulated. It is thought that a deficiency of calcium-dependent signal transduction between adjacent cells may delay wound healing.
The TGF-β receptor is up-regulated after corneal wound, suggesting that the TGF-β signal pathway may play a role in repairing corneal epithelial cells.
20 In addition, TGF-β controls corneal stromal cell and myofibroblast differentiation. Thus it has a role in scar formation in the wound healing process.
21 Because the expression of pertinent TGF-β genes was decreased markedly, delayed wound healing in diabetes may be associated with the abnormal TGF-β signaling.
In the drosophila eye, Notch induced cell proliferation, but blocked cell differentiation in the primordial. The expression of Notch-related genes was up-regulated in diabetes.
22 Although the cell proliferation occurs to heal corneal tissue wounds, the cell differentiation is not overly active, as to impair the normal eye surface.
The Jak-STAT pathway mediates the action of growth factors such as epidermal growth factor. Prolactin, a Jak-STAT gene, expression was decreased.
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24 This may explain why cell proliferation at the corneal surface layer is not active in diabetes.
Genes associated with the TGF-β MAPK, insulin, phosphatidylinositol, Wnt, calcium, and Notch signal pathways reacted to IL-1α and TNF-α treatment. Among genes reactive to IL-1α Sp1 transcription factor (TGF-β) and peptidylprolyl isomerase D (calcium) were down-regulated, and secreted frizzled related protein 1 (Wnt) was up-regulated. All were unresponsive to TNF-α. Secreted frizzled related protein 1 had a distinct expression level. It has been shown to play a role in controlling axonal growth in retinal ganglion cells and impeding the wound healing process. Thus it is thought to be associated with delayed wound healing in diabetes.
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26 Thrombospondin 4 (TGF-β), cell division cycle 2 homolog A and angiotensin II type-I receptor (calcium), and CDP diacylglycerol synthase 1 (phosphatidylinositol) were down-regulated, and FBJ murine osteosarcoma viral oncogene homolog and Jun D proto-oncogene gene (MAPK) were up-regulated in response to TNF-α and unresponsive to IL-1α. The angiotensin II type-I receptor gene showed a marked reduction that is primarily associated with delayed wound healing after conjunctival injury. It has been reported that the expression is decreased in diabetes, thus delaying wound repair.
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Genes newly expressed after IL-1α treatment were associated with the Toll-like receptor, calcium, MAPK, Wnt, insulin, and the Jak-STAT signal pathways. Sphingosine kinase 1, and CD38 antigen (calcium), Neutrophic tyrosine kinase II, and Nuclear receptor subfamily 4 group A member 1 (MAPK), and axin 2, and Wnt inhibitory factor 1 (Wnt) were up-regulated. Reduced expression was associated with the Toll-like receptor, insulin, Jak-STAT pathways. Toll-like receptor 2 and mitogen activated protein kinase 6 were down-regulated.
The Toll-like receptor signal pathway was especially involved following cytokine treatment. This pathway has been shown to be involved in the host defense mechanism. In other words, it is involved in both innate and acquired immune responses.
29 According to our results, the expression of all genes associated with the Toll-like receptor was reduced in diabetes following inflammatory reaction. In diabetics the in vivo immune system reaction to cytokines is decreased; thus, in diabetes, the inflammatory response is weak. The host innate immune response is involved in infectious keratosis, and thus decreased noticeably in response to cytokines.
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In diabetes, genes associated with the calcium, MAPK, Notch, Toll-like receptor, insulin, and Wnt signal pathway were newly expressed after TNF-α treatment. The MAPK, Notch, and Wnt pathways were up-regulated, and the calcium, insulin, and Toll-like receptor pathways were down-regulated. Gene expression was decreased in all cases. Interferon β 1 and phospholamban showed marked down-regulation. Interferon β 1 has been reported to elevate anti-viral effects in the cornea. In diabetes with a concomitant inflammatory reaction, a reduced anti-viral effect can be detected due to immune system damage.
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Genes associated with the insulin, TGF-β, Toll-like receptor, and adipocytokine pathways were newly expressed in response to both IL-1α and TNF-α. The V-crk sarcoma virus CT 10 oncogene, suppressor of cytokine signaling 2, and liver glycogen phosphorylase (insulin), follistatin, TGF-β2, and BMP 2 (TGF-β, IL-6 and chemokine ligand 5 (Toll-like receptor), and Neuropeptide Y (adipocytokine) were down-regulated. Reduced Neuropeptide Y expression may delay angiogenesis and thus prolong wound healing.
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We further characterized several genes by real-time PCR, including Ppm 1A, which showed significant up-regulation after cytokine treatment. We also characterized TGF-β 2 and IL-6, which were down-regulated by cytokine treatment. The expression pattern was identical to the microarray analysis results.
By applying a microarray, we were able to observe differentially expressed genes pertinent to signal transduction pathways between normal and diabetic corneal stromal cells. By examining up- or down-regulated genes in diabetes, the signal transduction pathways involved in delayed wound healing and inflammatory response could be assessed. By looking at gene expression in response to interleukin or tumor necrosis factor, it is anticipated that genetic treatments for diabetic keratopathy and to prevent delayed corneal wound healing may be possible in the future.