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The role of connective tissue growth factor, transforming growth factor β1 and Smad signaling pathway in cornea wound healing

2006; Lippincott Williams & Wilkins; Volume: 119; Issue: 1 Linguagem: Inglês

10.1097/00029330-200601010-00010

2542-5641

Xinyi Wu, Yongmei Yang, Hui Guo, Yuan Chang,

Connective Tissue Growth Factor Research

The cornea is a highly specialized and unique organ in the human body. Its main function is to project light from the external environment onto the retina, and it has a specific transparency to perform its function properly. The transparency and integrity of the cornea is of vital importance. The corneal wound, especially laceration deep to Bowman's membrane and stroma, which will inevitably cause scar formation, may cause the degeneration or even loss of sight. Injury can activate many biological factors in cornea as a strong stimulating signal. Transforming growth factors (TGF) and connective tissue growth factors (CTGF) are thought to be related to scar formation after injury. TGF can stimulate stroma cells of cornea and promote synthesis of matrix. Over expression of TGF causes scar formation.1,2 CTGF is a 38 kD cysteine-rich protein molecule and belongs to CCN family (CTGF/Fisp12, Cyr 61/CEF-10, Nov). In 1991, CTGF was firstly found in endothelial cells of human umbilical vein cultured in vitro3,4 CTGF acts as an important molecule that intermediates the processes of fibrosis, scarring, wound repairing, angiogenesis and embryonic development in many cell types. CTGF plays a unique role in proliferation, differentiation and adhesion of fibroblast cells, which in turn produces large amounts of collagen and other extracellular matrix (ECM) proteins.5-8 CTGF is upregulated in fibrotic diseases, including lung-, skin-, pancreas-, liver-and kidney fibrosis.9,10 This study reports the expressions and interactions of TGF-β1 and CTGF in corneal wound in vivo. This study aimed at determining the expressions and interactions of CTGF and TGF-β1 in Smad signaling pathway during the period when corneal wound was healing. METHODS Fifty-two New Zealand white rabbits were obtained from the Animal Supplier Center of Shandong University. Animal care and treatment in this investigation were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Epithelial defect in rabbit cornea The rabbits were anesthetized by intramuscular injection of xylazine (2 mg/kg) and topical application with 1% butyn. A central corneal trephine cut (3 mm in diameter) was created with a depth of 0.05 mm of the anterior lamellar stroma blade as a cornea wound model. Then, immediately after the surgery, 0.5% erythromycin ophthalmic ointment was applied to the wounded eyes. The contralateral eyes served as unwounded controls. Subjects The subjects were divided into 4 groups: ® control group: Four unwounded rabbits served as control group. © simple corneal injury group: The cornea wound was performed as previously reported. Then, the corneal wound healing was determined at 2 hours, 6 hours, 1 day, 3 days, 7 days and 21 days after corneal wound. There were 4 eyes at each time point. ©TGF- β1 antibody group:The cornea wound was produced as previously reported. In order to determine the interaction between TGF- β1 and CTGF, the wounded eyes were injected with 15.5 μg TGF-β1 antibody subconjunctival at the same time and then the corneal wound healing was determined at 3 days, 7 days and 21 days after TGF-β1 antibody injection. There were randomly 4 eyes at each time point. ©Smad 4 antibody injection group: The cornea wound was performed as previously reported. In order to determine whether TGF- β1 up regulated the expression of CTGF by Smad signaling pathway in the corneal wound healing, the wounded eyes were injected with 20 μ g Smad 4 antibody subconjunctivaly and then, the corneal wound healing was determined at 3 days, 7 days and 21 days after injection. There were randomly 4 eyes at each time point. Observations after corneal wound We observed the process of corneal wound healing everyday by slit lamp with fluorescein staining. The infected eyes were not included in the experiments. Histological analysis All surgical procedures were followed by mRNAse protection. A 4 mm-diameter piece of cornea tissue containing the wound area was scissored. The specimen was then fixed in 4% polyformaldehyde containing 0.1% DEPC, dehydrated in 30 minutes and embedded in paraffin. Deparaffinized sections were processed for histology and immunostaining. Immunohistochemistry analysis and mRNAin situhybridization of CTGF and TGF-β1 The slides were processed according to the manufacturer's instruction. The reagents in this study were included as follows: immunohistochemistry, reagent kit (Boster Corporation), CTGF,TGF- β1 and FN rabbit anti-rabbit antibody (Biovendor Corporation), CTGF in situ hybridization reagent Kit (MBL.CO.JP) and Type I collagen in situ hybridization reagent Kit (MBL.CO.JP). CTGF and TGF- β1 required antigen-repairing by microwave and FN required digesting by proteinase. RESULTS The corneal epithelial wound healing It took about (3.64±0.43) days, (4.89±0.32) days and (4.89 ± 0.32) days for corneal epithelium resurfacing in the simple injury group, the anti-TGF-β1 antibody group and the Smad 4 antibody group, respectively. The regenerated epithelium cells were found to be unstable and desquamated with fluorescence staining and they were not steady until 20 days after corneal wound. In HE staining we found that epithelial cells in the early stage after wound were flat and those in the edge of wound proliferated locally multipliers with irregular shapes and order. On the 21st day after corneal wound, the epithelial layer in the wound area was still thicker than that in peripheral, but with regular shapes and order. The wound healing of corneal stroma The stromal cells were activated in 6 hours after wound and its cell number increased. The flattened ovoid nuclei of strama cells in resting state disappeared and were replaced by round nuclei. These cells possessed the activity of fibroblast and excreted collagen, covering the wound by depositing beneath the regenerated cells. The newly produced collagen arranged in a clutter with edema. They did not become orderly and most stromal cells did not come back to resting states until the 21st day. Immunohistochemistry The expression of TGF- β1 The staining of TGF-β1 was not found in the cornea stroma and less expression in epithelium in control group at any time point. After corneal injury, TGF- β1 expression in epithelium increased markedly. However, it increased gradually in stroma, and reached the peak on the third day after wound (Fig. 1A). TGFβ1positive cells were also found in the endothelial side of stroma around the wound area. But less expression of TGF-β1 was detected in stroma after subconjunctival injection of Smad 4 antibody (Fig. 1B). On the 7th day after injury, TGF-β1 expression was only found in epithelium but almost negligible in the stroma. TGF-β1 antibody inhibited TGF-β1 expression in the epithelium.Fig. 1.: The expression of TGF- β1 in immunohistochemical analysis. A: the marked expression of TGFβ1 protein both in the epithelium and stroma on the third day after ablation. The arrows show positive granules (original magnification×100); B: the dramatic expression of TGFβ1 protein in the epithelium while no expression in the stroma on the third day after subconjunctival injection of Smad 4 antibody. The arrows show positive granules (original magnification×100).The expression of CTGF protein There was no CTGF expression in normal cornea (Fig. 2A). The CTGF-positive cells firstly appeared in epithelium in 1 day after corneal injury and the expression in the epithelium and Str- oma reached the peak on the third day. Moreover, obvious expression of CTGF appeared in the deeper layer of stroma around the wound area. There was no change of CTGF expression after subconjunctival injection of Smad 4 antibody (Fig. 2B). However, the CTGF expression in stroma decreased after subconjunctival injection of TGF-β1 antibody (Fig. 2C).Fig. 2.: The expression of CTGF in immunohistochemical analysis. A: There is no expression of CTGF in the normal cornea. The arrow shows the epithelial side (original magnification×200); B: The dramatic expression of CTGF protein both in the epithelium and stroma on the 3rd day after subconjunctival injection of Smad 4 antibody. The arrows show positive brown granules in cytoplasm (original magnification×100); C: The remarked expression of CTGF protein in the epithelium while less in the stroma on the 7th day after subconjunctival injection of TGFβ1 antibody (original magnification ×100).The expression of FN Fibronectin (FN) is the main component of cell matrix and basement membrane. The positive brown thread-like substances are mainly located in corneal stroma, basement membrane and intercellular space. FN existed in normal cornea (Fig. 3A). After injury, more expression of FN was found in intra-epithelial cells and stroma. TGF-β1 antibody inhibited the expression of FN in the matrix (Fig. 3B).Fig. 3.: The expression of FN. A: Dramatic expression of FN in the basement membrane of endothelium in the control group. The arrows show positive expression (original magnification×40). B: On the 7th day after subconjunctival injection of TG- β1 antibody, the expression of FN in the inter-epithelial cells and in the stroma decreases. However, FN is found beneath the basement membrane of epithelium (original magnification ×200).In situhybridization The expression of CTGF mRNA The expression of CTGF mRNA was not detected in normal cornea. The stromal cells were firstly activated in 6 hours after injury and the expression of CTGF mRNA was found to reach a peak on the third day, especially in anterior stroma cells around the wound area (Fig. 4A). There was no change of CTGF mRNA expression after giving Smad 4 antibody subconjunctively (Fig. 4B). On the 7th day after injury, the expression of CTGF mRNA was primarily detected in the epithelium and almost disappeared in the stroma. But the CTGF mRNA expression in the epithelium decreased after giving the TGF- β1 antibody subconjunctively. Less expression was detected in the epithelium on the seventh day after subconjunctival injection of TGF-β1 antibody (Fig. 4C), and they were not detected in the epithelium or stroma on the 21st days.Fig. 4.: The expression of CTGF mRNA. A: The expression of CTGF mRNA both in the epithelium and the stroma and more remarked expression are found in the stroma adjacent to the epithelium on the third day after wound. The arrows show positive brown granules in cytoplasm (original magnification× 100). B: Dramatic expression of CTGF mRNA in the stroma on the third day after subconjunctival injection of Smad 4 antibody (original magnification×100). C: Less expression of CTGF mRNA in the epithelium on the 7th day after subconjunctival injection of TGF-β1 antibody (original magnification×100).The expression of type I collagen mRNA The expression of Type I collagen was upregulated after wound with a peak on the third day. There was no effect for Smad 4 antibody on the expression of type I collagen. The expression was inhibited by TGF-β1 antibody and went back to basal level on the 21st day after wound. DISCUSSION The cornea wound healing is a complex process. The response of corneal tissue to a laceration has been well studied. Immediately after the laceration of the cornea, a fibrin plug forms at the wound edge, and stromal edema begins. Approximately 30 minutes after injury, neutrophils migrate to the injury site and release enzymes and chemotaxis factors. Epithelial wound healing starts approximately 1 hour after injury, with flattening and sliding of wing layer cells adjacent to the wound, covering the defect within 24 to 96 hours. Epithelial replication begins in basal cells adjacent to the injury site to restore the epithelium to its full thickness. The stromal fibroblastic proliferation begins in approximately 24 hours. The keratocyte and monocytes near the wound are activated and produce collagen and various glycosaminoglycans (GAGs) to form fibrous tissue. The epithelium wound healing is achieved by division, proliferation, differentiation and migration of epithelial cells, and then, the functional healing from the remodeling of fibrous tissues restores its nearly physical structure by some stroma components. The key to understand corneal wound healing is to recognize the orchestrated interactions between growth factors, cytokines, and the extracellular matrix (ECM) proteins and their receptors.Injury, as a strong biological stimulating signal, can upregulate the expression of biological factors related to corneal wound healing.11,12 They are involved in corneal wound healing by different intracellular signaling pathways. One of the recognized TGF signaling pathways is Smad pathway. Smad proteins are homogenous protein family of fruit fly Mad protein and wireworm Smad protein. They can conduct TGF- β1signal into the nucleus directly by membranous kinase receptors, offering a short cut between transmembrane signals and nuclear transcription. There are at least 8 members in Smads family that are divided into three groups: The first group is those activated by receptors, including Smad 1, 2, 3, 5, 8 subtypes. After phosphorylation they combine with Smad 4 in dimmers and translocate to nuclei. Smad 2 and 3 intermediate TGF-β1 signal transduction. The second group is current Smad. Smad 4 is the unique member. The third group is inhibiting Samds, including Smad 6 and 7. The combination with their receptors13 may block the binding of the first group and their receptors. Smad 4 antibody can block the TGFβ-mediated Smad pathway and therefore change certain functions. In normal rabbit cornea, there is little expression of TGF-β1 protein in the epithelium and there is no expression in resting stromal cells. However, after injury stromal cells are activated to possess the activity of fibroblasts and the expression of TGF- β1 are upregulated dramatically in the epithelial cells and fibroblasts. Smad 4 antibody completely blocks TGF- β1 expression in the fiboblasts in stroma. These data indicate that TGF-β1 not only activates stromal cells but also stimulates these cells to produce larger amounts of TGF. However, this effect just lasts 3-4 days after injury, concomitant with the upregulation of CTGF. Local application of Smad 4 antibody has no effect on CTGF expression but TGF- β1 antibody affects that markedly. That is because Smad 4 antibody just selectively blocks one of TGF-mediated pathways-Smad pathway while TGF-β1neutralizing antibody may block all the biological functions of TGF-β1. Therefore, we hypothesize that: TGF-β1 can upregulate the expression of CTGF, and the effect may be intermediated by signaling pathways other than Smad and Smad pathway may be involved in the regulation of TGF-β1 autosecretion. FN is the main component of cell matrix and stroma, while type I collagen is the main component of stroma and scar tissue, so both expressions demonstrate the activation of corneal stroma and fibroblasts. Smad 4 blocks expression of TGF-β1 in stroma, with no effect on expression of CTGF, FN and type I collagen. Moreover, the upregulation of CTGF is corresponding to the expression of FN and typeI collagen, indicating that the expressions of FN and type I collagen are mainly regulated by CTGF. At the time, on the 7th day after injury, the expression of TGF-β1 disappears in stroma, while the expressions of CTGF, FN and type I collagen last up to the 21st day. These data support our hypothesis that TGF-β1 plays an important role in activation of stromal cells in early phase of wound and the production of collagen by activated fibroblasts is mainly accomplished by CTGF. The expression of CTGF after wound directly affects production of connective tissue and the following scar formation. We also found that on the third day after injury, CTGF expression was detected in endothelial side of corneal stroma around the wound area, while mRNA expression of the same specimen at the same location were found in epithelial side of stroma. So we hypothesize that stroma cells in the basal layer around the wound area are firstly activated,therefore, when the cells produce CTGF, the corresponding cells of superior layer are still at the phase of CTGF mRNA expression. This is different from the activation of stromal cells at the wound and this sequence of activation may be related to intracellular signal transduction. In the corneal wound healing, we found that TGF-β1 antibody delayed early healing of epithelium. It took about (3.64±0.43) days and (4.89±0.32) days for the covering of corneal wound in the simple injury group and the anti-TGFβ1 antibody group, respectively, with significant statistical difference. This is different from the research before which were carried out on base of cell culture in vitro. As we know, the living environment in vitro is relatively simple while more complex in vivo, In vivo the network of various growth factors and other biological factors may affect many functions of cells synergistically. So the results attained in vitro just serve as reference for that in vivo. Our study firstly investigated the expressions of CTGF and TGF-β1 with immunohistochemistry and in situ hybridization in different phase of corneal wound healing in vivo. Moreover, we firstly found that TGF-β1 could upregulate the expression of CTGF in rabbit corneal wound healing, but the signaling was not intermediated by Smad pathway in vivo. In corneal wound healing,CTGF,as a downstream factor of TGF-β1, mainly regulates the synthesis of collagen and determines the production of connective tissue and the following scar formation. Thus, the regulation of CTGF expression may become a new target for the treatment of scar formation in cornea wound.