角膜基质细胞基质金属蛋白酶1，2活性与组织因子途径抑制物2的效应

doi:10.3969/j.issn.2095-4344.2014.02.015

中国组织工程研究 ›› 2014, Vol. 18 ›› Issue (2): 251-258.doi: 10.3969/j.issn.2095-4344.2014.02.015

• 组织构建与生物活性因子 tissue construction and bioactive factors • 上一篇下一篇

角膜基质细胞基质金属蛋白酶1，2活性与组织因子途径抑制物2的效应

俞建雄¹，袁静²，周炼红²

武汉大学人民医院，1外科，2眼科，湖北省武汉市 430060

收稿日期:2013-10-23 出版日期:2014-01-08 发布日期:2014-01-08
通讯作者: 袁静，博士，副主任医师，武汉大学人民医院眼科，湖北省武汉市430060
作者简介:俞建雄，男，汉族，1974年生，江西省婺源县人，2004年华中科技大学附属同济医学院毕业，博士，主治医师，主要从事基因工程与外科治疗研究。
基金资助:
国家自然科学基金资助项目(81100664)；武汉大学自主科研基金资助项目(111091)

Effect of tissue factor pathway inhibitor-2 on the expressions of matrix metalloproteinase 1 and 2 in keratocytes

Yu Jian-xiong¹, Yuan Jing², Zhou Lian-hong²

1 Department of Gastrointestinal Surgery, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China
2 Eye Center, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China

Received:2013-10-23 Online:2014-01-08 Published:2014-01-08
Contact: Yuan Jing, M.D., Associate chief physician, Eye Center, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China
About author:Yu Jian-xiong, M.D., Attending physician, Department of Gastrointestinal Surgery, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China
Supported by:
the National Natural Science Foundation of China, No. 81100664; Scientific Research Project of Wuhan University, No. 111091

摘要/Abstract

摘要：

背景：有研究表明，基质金属蛋白酶所参与的细胞外基质降解在角膜新生血管形成过程中起关键作用，组织因子途径抑制物2是新近发现的一种新型的丝氨酸蛋白酶抑制物，能有效抑制基质金属蛋白酶的活性。
目的：观察组织因子途径抑制物2对体外角膜基质细胞表达基质金属蛋白酶活性的关系。
方法：在体外对兔角膜基质细胞进行原代及传代培养，用脂质体介导的人类组织因子途径抑制物2真核表达载体转染兔角膜基质细胞，G418筛选阳性细胞。
结果与结论：RT-PCR，Western blot及明胶酶谱法分析结果显示，转染后角膜基质细胞组织因子途径抑制物2 mRNA和蛋白质的表达均上调(P < 0.05)，而基质金属蛋白酶1，2的活性下降(P < 0.05)。结果提示，组织因子途径抑制物2可明显抑制角膜基质细胞中基质金属蛋白酶1，2的活性。

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

关键词: 组织构建, 组织工程, 角膜基质细胞, 组织因子途径抑制物2, 基质金属蛋白酶, 细胞外基质, 角膜新生血管, 国家自然科学基金

Abstract:

BACKGROUND: The degradation of extracellular matrix, which is mediated by matrix metalloproteinases, plays a crucial role in the corneal neovascularization. Tissue factor pathway inhibitor 2, a new type serine proteinase inhibitor, can effectively inhibit the activity of matrix metalloproteinases.
OBJECTIVE: To elucidate the effect of tissue factor pathway inhibitor 2 on the expressions of matrix metalloproteinases in keratocytes in vitro.
METHODS: Rabbit keratocytes were primarily cultured and subcultured in vitro. Plasmid vector pBos-Cite-neo/TFPI-2 was transfected into keratocytes with Lipofectamine 2000. The positive cells were selected using G418.
RESULTS AND CONCLUSION: The results of reverse transcription-polymerase chain reaction, western blot analysis and gelatinase zymography analysis showed that, expression of mRNA and protein of tissue factor pathway inhibitor 2 was upregulated in the transfected keratocytes (P < 0.05), while activity of matrix metalloproteinase 1 and 2 was significantly decreased (P < 0.05). Experimental findings indicate that that tissue factor pathway inhibitor 2 strongly inhibits the activity of matrix metalloproteinase 1 and 2 in keratocytes.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: corneal stroma, metalloproteinase, extracellular matrix, cornea, culture medium

中图分类号:

R318

俞建雄，袁静，周炼红. 角膜基质细胞基质金属蛋白酶1，2活性与组织因子途径抑制物2的效应[J]. 中国组织工程研究, 2014, 18(2): 251-258.

Yu Jian-xiong, Yuan Jing, Zhou Lian-hong. Effect of tissue factor pathway inhibitor-2 on the expressions of matrix metalloproteinase 1 and 2 in keratocytes[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(2): 251-258.

图/表（结果） 2

Restriction enzymes were analyzed

After pBos-Cite-neo/TFPI-2 was digested by Xba I and Kpn I, two fragments of 5.05 kb and 440 bp were seen in 1% agarose gel electrophoresis, showing that there was 440 base pairs between polyclonal sites of Xba I and Kpn I (Figure 1). This evidence confirmed that the plasmid of pBos-Cite-neo/TFPI-2 contains human TFPI-2 gene. And the ratio of the absorbance at 260 and 280 nm was 1.80, showing that the purity of the plasmid was suitable to be transfect.

The morphology of keratocytes and gene transfection

After the explants were inoculated for 48 hours, some cells were derived from areas of corneal tissue. After 4- 5 days, cells grew around the tissues showing aristate formation, the cells were mainly fusiform with a small quantity of round or polygonal cells. After 2 weeks, the cells showed reticular forming. After 3 passage, the cells were fusiform approximately on the whole (Figure 1). Immunocytochemical staining of vimentin exhibited positive, which conformed that the cultured cells were keratocytes. Following transfection with lipofectamine 2000, cells were selected by G418, stable transfectants showed the same shape as non-transfected cells with the similar growth rate (data are not shown).

TFPI-2 mRNA expression in keratocytes as detected by reverse transcription-polymerase chain reaction

Semi-quantitative reverse transcription-polymerase chain reaction was performed to detect TFPI-2 mRNA expression (Figure 1). The expression level of TFPI-2 mRNA in K-P, K-V, K-TFPI-2 groups was represented by the IA ratio of TFPI-2 and β-actin (IA_TFPI-2/IA_β-actin). The results showed that TFPI-2 mRNA was expressed strongly in K-TFPI-2 group (P < 0.05), while there were slight expression in K-P and K-V groups. This finding suggested that keratocytes could acquire TFPI-2 mRNA strong expression after TFPI-2 transfection.

TFPI-2 protein expression in keratocytes as detected by western blot analysis

Western blot analysis was performed to detect TFPI-2 protein expression (Figure 1). The expression level of TFPI-2 protein in K-P, K-V, K-TFPI-2 groups was represented as the IA (IA_TFPI-2). The results showed that TFPI-2 protein was expressed strongly in K-TFPI-2 group (P < 0.05), while there were slight expression in K-P and K-V groups. This finding suggested that keratocytes could acquire TFPI-2 protein strong expression after TFPI-2 transfection.

The activity of MMP1 and MMP2 in keratocytes after transfection as detected by gelatin zymography

Gelatinolytic activities of MMPs were determined using prestained sodium dodecyl sulfate polyacrylamide gel electropheresis markers and MMP 1 and MMP 2 standards in zymography. Thus we could estimate the weights of the bands, approximately 54 and 72 kDa, respectively. The activity of MMP1 and MMP2 was represented as the IA of negatively stained bands (Table 1). The activity level of MMP1 (IA_{MMP-1)in K-P, K-V, K-TFPI-2 groups is shown in Figure 1. The results showed that, the activity of MMP1 and MMP2 was significantly decreased in K-TFPI-2 group (P < 0.05), and there were no significant differences in the level of MMP1 and MMP2 expressed between K-P and K-V groups.}

参考文献

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引言

Corneal neovascularization is a sight-threatening condition usually associated with inflammatory or infectious disorders of the ocular surface. It refers to the formation of new vascular structures in areas that are previously avascular. Following corneal injury, wound healing often proceeds without corneal neovascularization. However, corneal neovascularization may be induced during wound healing in several inflammatory, infectious, degenerative, and traumatic corneal disorders. Diseases associated with corneal neovascularization include inflammatory disorders, corneal graft rejection, infectious keratitis, contact lens-related hypoxia, alkali burns, stromal ulceration, aniridia, and limbal stem cell deficiency. In these conditions, the degradation of extracellular matrix is the key step for initiating the process of endothelial cell invasion into extracellular matrixand for recruiting endothelial progenitor cells to angiogenic site, which is accomplished by varieties of serine proteinases and matrix metalloproteinases (MMPs) derived from corneal cells^[1-6].

MMPs are zinc-dependent endopeptidases, which are collectively capable of degrading all constituents of extracellular matrix. Based on their structure and substrate specificity, MMPs can be divided into subgroups of collagenases, stromelysins, gelatinases, membrane-type MMPs and other MMPs. The cornea expresses eight out of the 24 identified MMPs including collagenase I (MMP-1 and MMP-13), gelatinases A and B (MMP-2 and MMP-9), stromelysin (MMP-3), matrilysin (MMP-7) and membrane type-MMP (MMP-14). Among these MMPs, MMP1 and MMP2 are two important members in the development of corneal neovascularization. MMP1 is located on 11q22 and is one of the widely expressed MMPs that can degrade type I, II and III collagens. MMP2 is able to degrade type IV collagen and some bioactive molecules. Therefore, to inhibit these proteinases could

be useful in maintaining the integrity of extracellular matrix and blocking angiogenic response^[7-10].

In previous works, most attention has focused on tissue inhibitors of metalloproteinase family and also implicated members of the serpin superfamily, i.e., α₂-macroglobulin and RECK, in the regulation of MMP activity. Tissue factor pathway inhibitor 2 (TFPI-2), also called placental protein 5, is known as a placenta-derived glycoprotein with serine proteinase inhibitory activity, which inhibits plasmin, trypsin, MMPs. It is a newly serine protease inhibitor with more broad inhibitory spectra compared with tissue inhibitors of metalloproteinase family, and plays a major role in keeping extracellular matrix from being degraded during angiogenesis^[11-17].

More interesting, there are a few introductions about TFPI-2 and cornea. Hence, in this experiment, we attempt to deliver the plasmid DNA coding for TFPI-2 into keratocytes and observe the activity of MMPs by keratocytes, basing for elucidating the relation between the expression level of TFPI-2 and the activity of MMP1, MMP2 and exploring a new gene-therapy aimed to corneal neovascularization in the laboratory and in the clinic.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

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材料方法

Design

A comparative observational experiment regarding cells.

Time and setting

This study was carried out in the laboratory for Eye Center, Renmin Hospital of Wuhan University, China from June 2011 to June 2012.

Materials

Animals

Six healthy purebred adult New Zealand rabbits of either gender, weighing 2.0-2.5 kg, were provided by the Animal Center at Renmin Hospital of Wuhan University, China (animal license No. SYXK (E) 2004-0027). All procedures were performed according to the Guidance Suggestions for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of China^[18].

Vector

TFPI-2 expression vector (pBos-Cite-neo/TFPI-2) was kindly gifted by Dr. Zhong Ren (Department of Hematology, Medical School Hospital of Qingdao University, China).

Methods

Cell culture and identification

Keratocytes were grown from explants using a modification of previously described method^[19]. In brief, eyeballs were isolated from healthy New Zealand White rabbits and sterilized corneas were obtained. Corneal tissues were rinsed with Hank’s balanced solution containing 100 U/mL penicillin and 100 ng/L streptomycin. After carefully removing the epithelia and endothelium by scraping, the remaining stroma was cut into several equal pieces (1 mm × 1 mm) and directly placed into six well-culture plate or into a 35-mm dish and dehydrated for 2-3 hours. The explants were then cultured in DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/mL) and streptomycin (100 ng/L), 2 mmol/L L-glutamine at 37℃ under 5% CO₂ and 95% humidity. The medium was renewed every 3-4 days. Cells were passaged at1:2 after confluence by trypsinization. For this study, cells of passages 3 or 4 were used. Keratocytes were identified by their characteristic fusiform appearance and by positive immunohistochemical staining with vimentin antibody by biomicroscope.

Reagents and instruments
Reagent and instrument Source
Dulbecco’s modified Eagle’s medium (DMEM), trypsin, D-Hank’s solution, vimentin antibody	Sigma, USA
Fetal bovine serum	HyClone, USA
Lipofectamine 2000, G418, 4-α-phorbol 12-myristate 13-acetate, Taq DNA	Promega, USA
Trizol reagent, murine leukemia virus reverse transcriptase	Gibco-BRL, USA
Anti-TFPI-2 rabbit polyclonal antibody, horseradish-peroxidase- conjugated anti-rabbit lgG, nitrocellulose membrane	Three Hawks Biotech, Wuhan, China
CCD camera and Quantity-one software, v4.4.0	Bio-Rad, USA
ECL western blot system	Pierce, USA
Image-Pro Discovery software	Media Cybernetics, USA
Phase contrast inverted microscope	Olympus, Japan
Densitometer	Personal Densitometer; Leica Q550iw; Germany

Preparation and purification of the plasmids

TFPI-2 expression vector (pBos-Cite-neo/TFPI-2) was kindly gifted from Dr. Zhong Ren (Department of Hematology, Medical School Hospital of Qingdao University). Plasmids were extracted by alkali disvolution and purified using the precipitation with lithium chloride and polyethylene glycol. The purity of nucleic acid was estimated by the absorbance at 260 and 280 nm. If the ratio of the absorbance at 260 and 280 nm was between 1.8-2.0, DNA had a high purity. Then plasmids were digested by Xba I and Kpn I and were separated on 1% agarose gel.

TFPI-2 gene transfection and selection of positive cells

The TFPI-2 expression vector was transfected into keratocytes with lipofectamine 2000 according to the manufacturer’s protocol. In brief, the keratocytes at passages 3 were seeded onto plates and transfected for 24 hours. Lipofectamine 2000 was diluted to 20 g/mL as solution A with serum-free DMEM medium and the plasmids of pBos-Cite-neo/TFPI-2 were diluted to 2.5 g/mL as solution B. Before transfection, solution A was mixed with solution B with equal volume. The mixture was incubated for 30 minutes at room temperature to allow the TFPI-2/Lipofectamine 2000 complexes to form. After transfection for 24 hours, the transfected cells were selected in medium containing 400 ng/L G418, and amplified in 200 ng/L G418 for 2 weeks. G418-resistant cells (K-TFPI-2) were used in subsequent experiments with the control of empty vector transfected cells (K-V) and non-transfected cells (K-P). Then, the expression levels of TFPI-2 were detected by reverse transcription-polymerase chain reaction and western blot analysis.

The expression of TFPI-2 mRNA as detected by reverse transcription-PCR

Total RNA was extracted from cells using Trizol reagent according to the manufacturer’s protocol. First-strand cDNA was synthesized and two micrograms of total RNA was reverse transcribed using murine leukemia virus reverse transcriptase. The resultant cDNA was used for PCR amplification with Taq DNA polymerase^[20] and 2 μL cDNA. Thirty-five cycles was performed, each consisted of 94 ℃ for 1 minute, 60 ℃ for 1 minute, and 72 ℃ for 1 minute. The size of predicted products is 440 bp. β-actin was amplified from the same cDNA sample as the internal control. These cycle numbers fell into the exponential range of polymerase chain reaction amplification. The amplified polymerase chain reaction products were then identified on 1.5% agarose gels. Images of ethidium bromide-stained agarose gels were acquired with a CCD camera, and semi-quantitative analysis involved use of the Quantity-one software, v4.4.0. Integral absorbance (IA) of band was used to assess the expression of the polymerase chain reaction products: IA=the mean absorbance×area. The ratio of IA_{TFPI-2and IA_{β-actin (IA_{TFPI-2/IA_{β-actin) was used to assess the expression of TFPI-2 mRNA.}}}}

Gene	Primers (5^’-3^’)	Product size (bp)
TFPI-2	Sense: GTC GAT TCT GCT GCT TTT CC Antisense: ATG GAA TTT TCT TTG GTG CG	`440`
β-actin	Sense: GAA ACT ACC TTC AAC TCC ATC Antisense: CGA GGC CAG GAT GGA GCC GCC	219

The expression of TFPI-2 protein as detected by western blot analysis

The protein of extracellular matrix was extracted as previously described^[21]. In short, extracellular matrix proteins were extracted from the three groups of cells. All the cell cultures were treated with 200 nmol of 4-α-phorbol 12-myristate 13-acetate per mL of the culture medium overnight. The cultures were washed with phosphate buffered saline (PBS) three times and then lysed with PBS containing 0.5% Triton X-100 (v/v) for 20 minutes at room temperature. After aspirating the Triton X-100 along with the lysed cells, the remaining extracellular matrix was washed three times with PBS and then twice with 20 mmol/L Tris-HCl (pH 7.4) containing 100 mmol/L NaCl and 0.1% (v/v) Tween-20. The culture dishes were then examined under a light microscope and ensured that no visible cells were present in the dishes. To collect extracellular matrix proteins, 200 μL of 1×sodium dodecyl sulfate polyacrylamide gel electrophoresis sample buffer was added to each dish and agitated for 20 minutes at room temperature. About 30 μL of these extracts were resolved on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and the proteins were subsequently transferred to a nitrocellulose membrane. The membranes were blocked with 10% nonfat dry milk and incubated overnight at 4 ℃ with anti-TFPI-2 rabbit polyclonal antibody diluted at 1:500. After three washes, the membranes were incubated with peroxidase-conjugated secondary antibody (1:5 000). TFPI-2 proteins were developed using an enhanced chemiluminescence method according to the manufacturer’s instructions. The western blots were quantified using Image-Pro Discovery software and IA of band was used to evaluate the percentage restoration of TFPI-2 protein levels in the three groups of keratocytes.

The activity of MMP1 and MMP2 as detected by gelatin zymography

Gelatinolytic activities of MMPs in samples were analyzed using substrate-gel electrophoresis (zymography). This is a sensitive and quantifiable method which allows visualization and quantification of proteolytic activity of enzymes, based on the molecular weights after gel electrophoresis. As the activation of the MMPs occurs after denaturation and renaturation processes, both forms of MMPs can be detected in the samples. In brief, the activity of MMP1 and MMP2 in conditioned medium of cultured keratocytes in three groups was analyzed by zymography using sodium dodecyl sulfate polyacrylamide gel electrophoresis (10%) containing 0.1% gelatin. Equal volumes of samples of conditioned cell culture medium were mixed with Laemmli-buffer under non-reducing conditions, loaded onto the gel and separated by electrophoresis. Thereafter, gels were washed three times for 3 hours at room temperature in buffer (50 mmol/L Tris-HCl, pH 8.0, 5 mmol/L CaCl₂, 0.02% NaN₃, and 2.5% Triton X-100) and incubated for 18 hours at 37℃ with same buffer except Triton X-100. Gels were stained with Coomasssie Brillant Blue R-2500 (0.1%) and destained in 5% methanol and 7% acetic acid. Gelatinolytic activity appeared as a clear band on a blue background. To quantitate the amount of gelatinase production, the stained zymograms were scanned on a densitometer and IA of band was used to evaluate the activity of MMP1 and MMP2 in the three groups of keratocytes.

Main outcome measures

Expression of mRNA and protein of TFPI-2 and the activity of MMPs in three groups of cells were observed.

Statistical analysis

Results are shown as mean ± SEM. Statistical analyses were performed using one-way analysis of variance test with SPSS 13.0 statistical software. A value of P < 0.05 was considered statistically significant.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

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讨论

There is no vessel in normal and health corneas. Corneal neovascularization is a sight-threatening condition usually associated with inflammatory or infectious disorders of the ocular surface and often accompanied by stromal edema, lipid deposits, keratitis, or scarring[22], which is a serious pathologic condition and can cause visual loss. It can also lead the anterior segment’s immune privileged, which plays a central role in the disequilibrium of ocular cytokine network and decreases reproductive system or tissue repair. Besides, pathological angiogenesis is thought to predispose to the reflection of corneal allografts by facilitating the exposure of antigens in the donor cornea to the immune system[23-26]. Statistics showed that in most progressing countries, corneal neovascularization was still the most common causes of blindness and disability. Conventional treatments such as corticosteroid, laser, surgical intervention could provide only symptomatic treatment of the disease without addressing the underlying cause, and there are also many limitations and complications associated with each of these treatment modalities. Till now, corneal neovascularization remains the most challenging one to treat, therefore, new modalities in the treatment are required urgently.

Recent studies suggest that the mechanism of corneal neovascularization is the result of an imbalance between angiogenic and anti-angiogenic factors and the predominance of angiogenic factors. Angiogenic factors implicated in the development of corneal neovascularization include vascular endothelial growth factor, basic fibroblast growth factor, MMPs, prostaglandins, interleukin-2 and -8, and platelet-derived growth factor. Several angiogenesis inhibitors have also been identified in the cornea, such as tissue inhibitor of metalloproteinases, thrombospondin-1, pigment epithelium-derived factor, prolactin, angiostatin, and endostatin. When the imbalance between angiogenic and anti-angiogenic factors happened, corneal neovascularization began to proceed physiologically involving the degradation of the capillary basement membrane, endothelial cell migration, capillary tube formation, and endothelial cell proliferation. Sprouting of new blood vessels from preexisting capillaries requires precise spatial and temporal proteolysis of the extracellular matrix, which is critical for initiating the process of endothelial cell invasion into extracellular matrix and for recruiting endothelial progenitor cells to angiogenic site[27-28]. Among these angiogenic factors, the MMPs can degrade most components of the extracellular matrix and play a critical role in the process of neovascularization[29-32]. MMPs are a group of zinc-binding proteolytic enzymes that engage in extracellular matrix remodeling and angiogenesis. The cornea expresses eight out of the 24 identified MMPs, including collagenase I (MMP-1 and MMP-13), gelatinases A and B (MMP-2 and MMP-9), stromelysin (MMP-3), matrilysin (MMP-7) and membrane type-MMP (MMP-14). In this study, we also confirmed that the activities of MMP1 and MMP2 were high in subcultured keratocytes, which suggested MMPs are the main proteinases in the degradation of extracellular matrix. Therefore, inhibition of MMPs would be a newly useful approach in gene-therapy for corneal neovascularization. In present reports, most attention has focused on the tissue inhibitor of metalloproteinases family[33-35]. However, some works from several groups also implicated members of the serpin superfamily, i.e., α2-macroglobulin and RECK, in the regulation of MMP activity[36-37]. The present study demonstrates inhibition of active MMPs by the serpin TFPI-2, probably by direct protein/protein interactions, employing domains distinct from those responsible for serine-proteinase inhibition. In view of these studies, we tried to make use of gene transfection techniques to enable a new inhibitor of MMPs, TFPI-2 gene into mediated cells (keratocytes, main target cells in corneal inflammation) and hoped to obtain the stable transfectants in our study, in an effort to search for a new gene therapy of corneal neovascularization and investigate the molecular mechanism and the secretory pathway of endogenous TFPI-2.

TFPI-2, is a 32-kDa novel serine proteinases inhibitor, whose gene mapped to chromosome 7q22. TFPI-2 belongs to superfamily of Kunitz inhibitors, consisting of three tandemly arranged Kunitz-type proteinase inhibitor domains flanked by a short acidic amino terminus and a highly basic C-terminal tail[11-17]. The first and third Kunitz-type domain consist of seven arginine residue, which involve in sites of interaction TFPI-2 and MMPs. Compared with conventional inhibitor, TFPI-2 has a serine proteinase inhibitory activity with broader spectra and stronger inhibitory activity. It acts against a wide range of serine proteases through their non-productive interaction with a P1 residue in its first Kunitz-type domain. TFPI-2 exhibits strong inhibitory activity towards a broad spectrum of proteinases, including trypsin, plasmin, chymotrypsin, cathepsin G, plasma kallikrein and the factor VIIa-tissue factor complex. In contrast, TFPI-2 exhibits little or no inhibitory activity towards urokinase plasminogen activator, tissue-type plasminogen activator and α-thrombin. TFPI-2 can be synthesized and secreted by keratinocytes, dermal fibroblasts, smooth muscle cells, synoviocytes and endothelial cells, transcripts are widely expressed in various adult human tissue, such as liver, skeletal muscle, heart, kidney and pancreas[12]. Three isoforms of TFPI-2 are synthesized by these cells and migrate with an apparent molecular weight of 33, 31 and 27 kDa due to differential glycosylation. Recent studies have shown that TFPI-2 expression plays a significant role in many physiological and pathological processes, such as the remodeling of the extracellular matrix, neovascularization, or the process of migration of tumor cell, it can inhibit tumor invasion and metastasis and progress of arthritis and atherosclerosis. However, little is known about the role of TFPI-2 in the expression of MMPs pathways in corneal keratocytes.

Here, we hypothesize that lack of TFPI-2 should contribute to enhanced matrix degradation in neovascularization and result in distinct microenvironments with corresponding variations in MMP/inhibitor ratios. Previous study also supported our hypothesis of that TFPI-2/MMP imbalance crucially determines extracellular matrix stability because of the deletion of gene or the mRNA instability of TFPI-2. In this study, we used suitable ratio of lipofectamine 2000/plasmids complex and successfully transfected pBos-Cite-neo/TFPI-2 by Lipofectamine 2000 into cultured keratocytes. The results showed that slight mRNA and protein of TFPI-2 were expressed in K-V and K-P groups, but obvious expression in K-TFPI-2 group. This evidence suggests that keratocytes only could synthesize and secrete extraordinary small quantity of TFPI-2, whose levels were not enough to protect the integrity of extracellular matrix and block angiogenic response, although we cannot conclude the cause as deletion of gene, or the mRNA instability. However, the expression of TFPI-2 could be significantly upregulated by gene transfection. In this study, we also compared the expression of TFPI-2 between the cells of K-P and K-V and the study delayed no significant different between them, which showed that the plasmids of pBos-Cite-neo/TFPI-2 itself has no effect on the expression of keratocytes and the transfection of the plasmids was safe method to acquire the higher expression of endogenous gene. Meanwhile, we also observed the cellular shape and grow rate after transfection without significant difference comparing with non-transfected cells and transfected pBos-Cite-neo cells. This showed that increased levels of TFPI-2 probably resulted from increased expression of TFPI-2 per keratocytes rather than more numbers of keratocytes, which was different with Shinode E’ report. They reported that the recombinant TFPI-2 stimulated DNA synthesis and cell proliferation in a dose-dependent manner and activated mitogen-activated protein kinase activity and stimulated early proto-oncogene c-fos mRNA expression in smooth muscle cells. The mitogenic function of recombinant TFPI-2 is considered to be mediated through mitogen-activated protein kinase pathway. Concerning the two difference results, we assumed reasons as follow: (1) vascular smooth muscle cells and keratocytes are different cell types and structurally similar TFPI-2 maybe have no a mitogenic activity for the keratocytes. (2) The origin of TFPI-2 was different between two reports. In Shinode E’s study, they use the recombinant TFPI-2 from the purified mitogenic substance from the conditioned medium of the cultured human umbilical vein endothelial cells, which is exogenous cytokine added to the smooth muscle cells. But in our study, we transfected the plasmids of pBos-Cite-neo/TFPI-2 to keratocytes and acquired endogenous TFPI-2 of cells. The different design of experiments may be bring about different experimental result. Certainly, the mechanism whether TFPI-2 is or not a mitogen for keratocytes need to investigate further.

MMP1 and MMP2 have several substrates, but their function is mainly attributed to their collagenase and gelatinase activities, which result in important roles in extracellular matrix turnover and cell migration. They are able to degrade the basement membrane proteins, such as type IV collagen and laminin, which are important to the invasion process of pathogens and of tumor cells. These enzymes also regulate the cell behavior during inflammation, contributing to the cell activation and migration. In our study, we investigated the activity of MMPs in subcultured keratocytes by zymography, although zymography results may not always accurately reflect the proteolytic activity of the enzymes in vivo, and could detect the expression of MMP1 and MMP2. The expression of MMP1 and MMP2 in the subcultured keratocytes suggested a role for these enzymes in the process of neovascularization. In the entire process of neovascularization, the local host tissue microenvironment can be an active participant. Besides the vascular, immune and inflammatory cells, the microenvironment of cornea consists mainly of keratocytes. These activated keratocytes in process of corneal neovascularization in vivo can be simulated by the subcultured keratocytes. Here, we show that the keratocytes can induce the expression of their MMP1 and MMP2, which suggest that the keratoctes synthesize MMPs via an autocrine mechanism.In addition, we observed that TFPI-2 limits the enzymatic activity of MMPs, which also suggested a mechanism by which TFPI-2 reduced matrix degradation by stable transfectants after TFPI-2 gene transfected into keratocytes. Thus, these findings indicate a biological role for TFPI-2 as an inhibitor of matrix degradation in corneal neovascularization, which is coincidence with the Herman’s study on atherosclerosis but different with Du’s study. We analyzed the cause probably resulted from the different cell type and different experimental design. Previous and our study indicated that TFPI-2 might participate in matrix-protective processes described inhibition of activation of MMP, however, the mechanism remained unclear. Considering the probable action pattern, we think there are two different mechanisms: one is to inhibit plasmin, trypsin, MMPs directly, the other is to prevent extracellular matrix from degradation by proteinases by combining it through heparin-like molecules. But in corneal tissue, whether both of the two mechanisms of TFPI-2 involved in the matrix-protective processes or a mechanism among them need to further study.

In summary, we observed expression of TFPI-2 in non-transfected cell and transfectants and analyzed the activity of MMPs. In accordance with the in vitro data, we supports the hypothesis that TFPI-2 limits extracellular matrix degradation and that the increase of TFPI-2 might promote extracellular matrix homeostasis in corneal neovascularization, which provide a experimental base for gene therapy to corneal neovascularization. However, whether TFPI-2 indeed crucially regulates MMP activity in vivo will require further study.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

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角膜基质细胞基质金属蛋白酶1，2活性与组织因子途径抑制物2的效应

Effect of tissue factor pathway inhibitor-2 on the expressions of matrix metalloproteinase 1 and 2 in keratocytes

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