阻断血管内皮生长因子C促进角膜移植后新生淋巴管和血管的“分离生长”

doi:10.3969/j.issn.2095-4344.2016.33.011

中国组织工程研究 ›› 2016, Vol. 20 ›› Issue (33): 4940-4948.doi: 10.3969/j.issn.2095-4344.2016.33.011

• 角膜组织构建 corneal tissue construction • 上一篇下一篇

阻断血管内皮生长因子C促进角膜移植后新生淋巴管和血管的“分离生长”

叶辉1，严浩2，钟蕾1，王涛1，邓娟1，凌士奇1

1中山大学附属第三医院眼科，广东省广州市 510630；2广东医学院附属南山医院眼科，广东省深圳市 510080

收稿日期:2016-05-15 出版日期:2016-08-12 发布日期:2016-08-12
通讯作者: 凌士奇，博士，中山大学附属第三医院眼科，广东省广州市 510630
作者简介:叶辉，男，1968年生，广东省人，1998年中山大学毕业，硕士，主治医师，主要从事角膜方面的研究。并列第一作者：严浩，男，1968年生，湖北省人，2004年华中科技大学毕业，博士，主任医师，主要从事角膜方面的研究。
基金资助:
国家自然科学基金项目(81070711)，广东省自然科学基金项目(S2013010016324)；广东省科技计划项目(2014A020212393)；深圳市科技计划项目(JCYJ20150402152130186)

Lymphatic vessels growing apart from blood vessels in transplanted corneas after the blockade of vascular endothelial growth factor C

Ye Hui1, Yan Hao2, Zhong Lei1, Wang Tao1, Deng Juan1, Ling Shi-qi1

1 Department of Ophthalmology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, Guangdong Province, China
2 Department of Ophthalmology, Nanshan Hospital of Guangdong Medical College, Shenzhen 510080, Guangdong Province, China

Received:2016-05-15 Online:2016-08-12 Published:2016-08-12
Contact: Ling Shi-qi, M.D., Department of Ophthalmology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, Guangdong Province, China
About author:Ye Hui, Master, Attending physician, Department of Ophthalmology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, Guangdong Province, China Yan Hao, M.D., Chief physician, Department of Ophthalmology, Nanshan Hospital of Guangdong Medical College, Shenzhen 510080, Guangdong Province, China Ye Hui and Yan Hao contributed equally to this work.
Supported by:
the National Natural Science Foundation of China, No. 81070711; the Natural Science Foundation of Guangdong, China, No. S2013010016324; the Science and Technology Project of Guangdong, China, No. 2014A020212393; and the Science and Technology Project of Shenzhen, China, No. JCYJ20150402152130186

摘要/Abstract

摘要：

文章快速阅读：

文题释义：
角膜新生淋巴管：正常角膜无淋巴管，但在炎症、烧伤、移植后均可出现角膜新生淋巴管。角膜新生淋巴管属于毛细微淋巴管，无色透明，临床上难以辨认。淋巴管可以回收组织间液，避免组织水肿。除此之外，角膜淋巴管可以加速角膜抗原呈递的速度，在角膜免疫中起着重要的作用。
血管内皮生长因子C：是一种与血管内皮生长因子同源的内皮细胞生长因子，其受体为血管内皮生长因子受体3和血管内皮生长因子受体2。血管内皮生长因子C介导了淋巴管的发生、生长和增生，是目前公认的淋巴管诱生因子。

摘要
背景：角膜淋巴管有利于角膜抗原的运输，从而加速了抗原提呈的进程，在角膜免疫中起着重要的作用。然而，由于角膜移植后角膜新生血管和淋巴管呈“平行”生长，因而很难对淋巴管在移植免疫中所起的具体作用大小作出准确的评估。
目的：通过阻断血管内皮生长因子C的表达，探索角膜移植后角膜新生淋巴管和血管的生长变化。
方法：建立同种异体角膜移植的大鼠模型130只，将受体大鼠随机等分为血管内皮生长因子C拮抗组和对照组。血管内皮生长因子C拮抗组大鼠在移植后第2天起腹腔注射单克隆抗体，隔天1次，连续2周，阻断角膜移植后血管内皮生长因子C的表达。对照组大鼠注射生理盐水。
结果与结论：①血管内皮生长因子C拮抗组大鼠角膜中血管内皮生长因子C的表达水平显著下降。在对照组中，角膜新生淋巴管和血管呈“平行”生长；②在血管内皮生长因子C拮抗组中，血管面积轻度减少，而淋巴管面积呈显著性减少(P < 0.05)；③在对照组中大鼠角膜排斥指数与血管面积、淋巴管面积均呈显著性正相关，而在血管内皮生长因子C拮抗组大鼠角膜中虽然排斥指数与血管面积显著性相关，但排斥指数与淋巴管面积间无相关性；④血管内皮生长因子C拮抗组中植片平均存活时间明显大于对照组；⑤说明拮抗血管内皮生长因子C后，角膜新生淋巴管和血管间出现了“分离生长”；而拮抗血管内皮生长因子C可以有效抑制角膜移植后的角膜新生淋巴管，从而提高植片的存活率。

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
ORCID: 0000-0002-4948-9236(Ling Shi-qi)

关键词: 组织构建, 组织工程, 角膜, 移植, 新生淋巴管, 新生血管, 植片排斥反应, 血管内皮生长因子C, 国家自然科学基金

Abstract:

BACKGROUND: Corneal lymphangiogenesis is beneficial to the transport of corneal antigenic materials, and accelerates the process of antigen presentation, thereby playing an important role in corneal immunity. However, due to the parallel outgrowth of corneal blood and lymphatic vessels in transplanted corneas, it is often difficult to accurately evaluate the role of corneal lymphatic vessels in allograft rejection.
OBJECTIVE: To explore the development of corneal lymphangiogenesis and angiogenesis in transplanted rat corneas after the blockade of vascular endothelial growth factor C (VEGF-C).
METHODS: 130 rats used to establish corneal allogenic transplantation models were equally randomized into two groups: the anti-VEGF-C group and the control group. VEGF-C was blocked in the anti-VEGF-C group by intraperitoneal injection of neutralizing monoclonal anti-VEGF-C antibody every other day for 2 consecutive weeks. Meanwhile, rats in control groups received intraperitoneal injections of saline. Corneal angiogenesis and lymphangiogenesis were characterized using whole mount immunofluorescence, and the immune rejection of the grafts was evaluated by scoring the rejection index (RI). In addition, the expression of VEGF-C was examined by real-time PCR. The relationship of corneal lymphangiogenesis and angiogenesis to RI in transplanted corneas was also characterized.
RESULTS AND CONCLUSION: VEGF-C expression was markedly downregulated after VEGF-C blockade. Corneal lymphangiogenesis developed in parallel with corneal angiogenesis in the control group. While there was a mild reduction in blood vessel area (BVA) and a significant decrease in lymphatic vessel area (LVA) in the anti-VEGF-C group (P < 0.05). In addition, RI was positively correlated with BVA (P < 0.05) and LVA (P < 0.05) in the control group. However, although RI was significantly correlated with BVA (P < 0.05) in the anti-VEGF-C group, the correlation between RI and LVA was not statistically significant (P > 0.05). the graft survival time in the anti-VEGF-C group was significantly increased compared with that in the control group (P < 0.05). Our results show that the outgrowth of lymphatic vessels is separated from that of blood vessels in transplanted corneas by blocking VEGF-C. The blockade of VEGF-C has a significant role in preventing corneal lymphangiogenesis in corneal beds, which results in higher allograft survival rates.

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

Key words: Cornea, Vascular Endothelial Growth Factor C, Tissue Engineering

中图分类号:

R318

叶辉，严浩2，钟蕾，王涛，邓娟，凌士奇. 阻断血管内皮生长因子C促进角膜移植后新生淋巴管和血管的“分离生长”[J]. 中国组织工程研究, 2016, 20(33): 4940-4948.

Ye Hui, Yan Hao, Zhong Lei, Wang Tao, Deng Juan, Ling Shi-qi. Lymphatic vessels growing apart from blood vessels in transplanted corneas after the blockade of vascular endothelial growth factor C[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(33): 4940-4948.

图/表（结果） 3

RESULTS

Quantitative analysis of experimental animals

During the experiment, four SD rats died of anaesthesia in the course of corneal transplantation and additional four SD rats were supplemented. Therefore, the number of SD rats keeps 140 in this experiment.

Corneal lymphangiogenesis and angiogenesis

To visualize corneal lymphatic and blood vessels, we labelled blood vessels green by intravenous injection of fluorescein isothiocyanate-conjugated lectin and labelled all vessels red by incubating the whole cornea with PE-conjugated anti-CD31 antibody. Upon merging the green and red fluorescent images, blood vessels appeared yellow and lymphatic vessels appeared red. With this method, we found that both corneal lymphangiogenesis and angiogenesis occurred and developed after keratoplasty. In the control group, the growth of lymphatic vessels was almost the same as the growth of blood vessels, gradually extending from the limbus to the graft. Both corneal lymphangiogenesis and angiogenesis reached a maximum 21 days after transplantation. However, in the anti-VEGF-C group, the growth of corneal lymphatic vessels was much slower than that of blood vessels. There were a large number of new blood vessels, but only a few lymphatic vessels extended to the cornea 21 days after the transplantation (Figure 1).

The expression of VEGF-C mRNA after keratoplasty

To investigate whether intraperitoneal injection of a neutralizing monoclonal antibody inhibited VEGF-C mRNA expression, we examined the expression of VEGF-C mRNA by real-time PCR at different time points after keratoplasty. Real-time PCR revealed that VEGF-C mRNA expression increased on day 3, decreased dramatically on day 7, and then increased conversely until day 14 in both the control and anti-VEGF-C groups. However, compared with the control group, VEGF-C mRNA expression was significantly decreased at all time points except for day 7 after corneal transplantation in the anti-VEGF-C group. The expression of VEGF-C mRNA dramatically increased in the control group, but was almost at the same level in the anti-VEGF-C group from day 14 to day 21 after keratoplasty (Figure 2). The data showed that VEGF-C blockade resulted in a significant inhibition of VEGF-C mRNA expression.

The growth of corneal lymphatic vessels after VEGF-C blockade

To characterize corneal lymphangiogenesis and angiogenesis, we examined LVA and BVA at different time points after keratoplasty, and compared the differences in them between the anti-VEGFC and control groups, respectively. In contrast with a mild reduction in BVA in the anti VEGF-C group, LVA was dramatically decreased. The difference in LVA between the 2 groups was statistically significant at all time points after keratoplasty (Figure 3) (all P < 0.05). Subsequently, we analyzed the correlation of BVA and LVA with RI in each group, and found that RI was strongly and positively correlated with both BVA (r² = 0.537 1; P < 0.05) and LVA (r²=0.451 8; P < 0.05) in the control group. However, although RI was significantly correlated with BVA (r² =0.710 6; P < 0.05) in the anti-VEGF-C group, the correlation between RI and LVA was not statistically significant (r² =0.046 5; P > 0.05) (Figure 4). These results suggested that corneal angiogenesis but not lymphangiogenesis contributed to allograft rejection after the blockade of VEGF-C.

Effect of VEGF-C blockade on corneal allograft survival

Compared with the graft survival time for the control group, the graft survival time for the anti-VEGF-C group was significantly increased (P < 0.05, Table 1). In addtion, the scores of RI were much higher in the control group in comparison with that in the anti-VEGF-C group (Table 2), suggesting that intraperitoneal injection of a monoclonal anti-VEGF-C antibody was effective in preventing corneal allograft rejection.

参考文献

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引言

INTRODUCTION

The cornea is normally devoid of blood vessels and lymphatic vessels, implying that it is a relatively immune privileged site that is likely to have a more favorable prognosis after corneal transplantation

compared with other solid organ transplantations. However, recent evidence from both human and animal transplanted corneas has reported that corneal angiogenesis and lymphangiogenesis could occur after keratoplasty, which implies that in normal corneal transplantation, the graft has a chance to connect to both the blood and lymphatic systems. While blood vessels provide an entrance for immune effector cells (e.g., CD4 alloreactive T lymphocytes and memory T lymphocytes), corneal lymphangiogenesis provides an exit from the graft to regional lymph nodes, which has been shown to be essential in promoting alloimmunization and subsequent graft rejection^[1-2]. By studying the time course of angiogenesis and lymphangiogenesis in transplanted corneas, an understanding of their roles in allograft rejection can be obtained, which could have therapeutic implications.

In previous studies, Yamagami et al.^[3] successfully inhibited allograft rejection after obstruction of corneal lymph drainage by bilateral cervical lymphadenectomy. Their study later showed that the survival rate of corneal grafts placed into the vascularized recipient beds was as high as 92%, suggesting that afferent corneal lymphatics may be equally or even more important than efferent corneal blood vessels in allograft rejection^[4]. Recently, we^[5] and Dietrich et al.^[6] at approximately the same time reported that corneal lymphangiogenesis played a crucial role in immune rejection after transplantation.

Anti-lymphangiogenesis therapies could increase the survival rate of corneal transplantations. However, our previous and other studies also showed that there was a parallel outgrowth of corneal blood and lymphatic vessels in transplanted corneas^[7-8]. The degree of corneal lymphangiogenesis was significantly associated with the degree of corneal angiogenesis after keratoplasty. Therefore, the respective roles of lymphatic or blood vessels in allograft immunity need to be elucidated in the induction of corneal lymphangiogenesis versus angiogenesis.

The present study targeted corneal lymphatic vessels by the blockade of vascular endothelial growth factor C (VEGF-C), and investigated whether anti-VEGF-C therapy had a selective inhibitory role in corneal lymphangiogenesis and in the inhibition of the parallel growth of blood vessels and lymphatic vessels after corneal transplantation. Together, these findings will broaden our understanding of the mechanisms important for corneal transplant rejection.

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

材料方法

MATERIALS AND METHODS

Design

A comparative observational study.

Time and setting

The experiment was performed in the animal center of Zhongshan Ophthalmic Center of Sun Yat-Sen University from January 2014 to June 2015.

Materials

A total of 140 Sprague-Dawley (SD) rats were included in this experiment, of them 130 were used as recipients, and the remaining 10 rats were used as normal controls. In addition, 65 male Wistar rats aged 8-10 weeks, weighing 180-200 g, and provided by the animal center of Southern Medical University (license No. SCXK (Yue) 2011-0015) were used as donors for corneal transplantation. The experimental conditions conformed to good laboratory practices (National Research Council of the USA, 1996) and all animals involved in the study were handled in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Prior to treatment, we confirmed that all rats were free from ocular diseases.

Methods

Orthotopic corneal transplantation

The procedure for corneal transplantation was adapted from a method described previously^[9]. In brief, rats were anesthetized with chloral hydrate (300 mg/kg) through the abdominal cavity, and 1% atropine was used 20 minutes before the operation. Donor corneas (3.5 mm in diameter) were removed and placed in Optisol solution. After the recipient’s right cornea was marked with a trephine of 0.30 mm diameter and excised under the operating microscope, the donor graft was sutured into the recipient’s bed with eight interrupted sutures (10-0 nylon). The anterior chamber of the eye was reformed by injection of balanced salt solution (BSS). The graft sutures were removed 2 weeks after the operation.

VEGF-C blockade

After corneal transplantation, the recipient rats were equally and randomly divided into two groups (n=65 rats in each group): anti-VEGF-C and control groups, respectively. To block VEGF-C, 20 mg/kg (0.1 mL) neutralizing goat anti-rat VEGF-C monoclonal antibody (Boshide Bio-Project Co., Wuhan, China) was injected intraperitoneally on the day after surgery and on alternate days up to 2 weeks. Rats in the control group intraperitoneally received 0.1 mL of normal saline.

In the anti-VEGF-C or control group, five corneas (the right cornea, including both parts of donor and recipient) were examined by whole mount immunofluorescence and real-time PCR at 3, 7, 10, 14, 18, and 21 days after transplantation. Five rats were used for slit-lamp observation to evaluate the state of corneal graft rejection, and the rejection index (RI) was recorded. In addition, 10 right corneas from normal SD rats were used as normal controls for immunofluorescence (5 corneas) or real-time PCR (5 corneas).

Immunofluorescence of whole mount corneas

The method was modified as previously described^[10-11]. Briefly, 10 mL of lectin-FITC from Lycopersicon esculentum (20 μg/mL; Sigma-Aldrich, St. Louis, Missouri, USA) was injected intracardially and allowed to perfuse for 2 minutes. The cornea was incubated in phycoerythrin-labeled anti-rat PECAM1 (CD31-PECAM) (1:50; BD Biosciences, San Diego, CA, USA) in TNB blocking buffer (TSA Biotin System; NEN Life Science, Boston, Massachusetts, USA) overnight at 4 ℃, after being dissected and fixed in acetone at -20 ℃ for 15 minutes. Corneas were photographed using a digital camera connected to a confocal fluorescent microscope (LSM 510 Meta; Carl Zeiss, Jena, Germany). The total areas covered by lectin⁻/PECAM⁺ were measured as lymphatic vessel areas (LVA) and the total areas covered by lectin⁺/PECAM⁺ were measured as blood vessel areas (BVA). The images were further analyzed under low magnification (×2), and LVA and BVA were quantified using Axiovision 4.7.2 digital image analysis software (Carl Zeiss).

Slit-lamp microscopy

The corneal grafts were examined by a masked observer every day after the transplantation. The clinical appearance of each graft was scored according to the controls established by Holland et al. ^[12]. Briefly, a RI of 0-12 was generated based on the sum of the three graft components: clarity, edema, and neovascularization (NV) (0-4 for each component). The following clarity scoring system was used: 0, clear cornea; 1, slight haze; 2, increased haze in the anterior chamber structures, but still clear; 3, advanced haze with a difficult view of the anterior chamber; and 4, an opaque cornea with no view of the anterior chamber. The following edema scoring system was used: 0, no stromal or epithelial edema; 1, slight stromal thickness; 2, diffuse stromal edema; 3, diffuse stromal edema with microcystic edema of the epithelium; and 4, bullous keratopathy. The scoring for NV was as follows: 0, no NV; 1, NV of the peripheral cornea; 2, NV of the corneal wound; 3, NV of the peripheral graft; and 4, NV of the entire graft. According to Zhang et al.^[13], a clarity score of ≥ 3.0 and a total score > 6 were recorded as rejected.

Real-time RT-PCR

Total RNA was isolated from the samples using TRIzol^® Reagent (Gibco-BRL Life Technologies, Gaithersburg, MD, USA). RNA was prepared following the protocol from the manufacturer. The RNA pellets were washed with 75% ethanol, centrifuged, and dried. The residual DNA was removed by treatment with DNase I. Pellets were resuspended in 30 μL of DEPC-treated water followed by the addition of 50 mmol/L Tris, pH 7.5, 10 mmol/L MgCl₂, 20 U of RNase-free DNase I, and 20 U of RNas in a total volume of 60 μL. Samples were incubated at 37 ℃ for 25 minutes. Then the RNA was cleaned using RNeasy Mini Kits (Qiagen, Valencia, CA, USA) following the manufacturer’s protocol. The RNA concentration and purity were determined by measuring optical density at 260 nm and 280 nm using a spectrophotometer (Metash Instru-ments Co., Ltd., Shanghai, China). The cDNA was generated from total RNA samples by using the Taqman Reverse Transcription Reagents kit (Applied Biosystems, Foster City, CA, USA). To prepare the cDNA, the total RNA from each sample was first incubated at 25 ℃ for 10 minutes, and then reverse transcribed at 48 ℃ for 30 minutes. Real-time RT-PCR was performed using a SYBR^® green dye (Applied Biosystems) with the ABI PRISM 7900HT (Applied Biosystems). The primers for VEGF-C were 5’-GTA AAA CTT GCT GCT GCA CAT T-3’ (sense) and 5’-GAA CGT CTA ATA ATT GAA TGA ACT TGT CT-3’(antisense) (GenBank accession no. AY032729). The DNA polymerase was first activated at 95 ℃ for 10 minutes, denatured at 95 ℃ for 15 seconds, and annealed/extended at 60 ℃ for 1 minute, for 40 cycles according to the manufacturer’s protocol. The products were sequenced to confirm that the correct gene sequence was being amplified. All PCR reactions were performed in triplicate. Relative quantitation of gene expression was performed using the standard curve method (User Bulletin number 2, ABI PRISM 7700 Sequence Detection System; Applied Biosystems). For a comparison of the transcript levels between samples, standard curves were prepared for both the target gene and the endogenous reference (18S ribosomal RNA). For each experimental sample, the amounts of target and endogenous reference were determined from the appropriate standard curves. Then, the target amount was divided by the endogenous reference amount to obtain a normalized target value. Each of the experimental normalized sample values was divided by the normalized control sample value to generate the relative expression levels.

Main outcome measures

Corneal blood and lymphatic vessels were examined by whole mount immunofluorescence under a confocal fluorescent microscope, the expression of VEGF-C mRNA was examined by Real-time PCR, and corneal graft rejection was observed and socred by a slit-lamp.

Statistical analysis

Analysis of the significance of differences between

two groups was performed using paired Student’s

t-test (SPSS 16.0 statistical software, SPSS Inc., Chicago, IL, USA). Pearson’s analysis was used to determine correlations among BVA, LVA, and RI. Values are presented as the mean ± SD. All reported P-values are two-tailed, and statistical significance was defined at α=0.05 level.

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

讨论

DISCUSSION

The lymphatic system plays an important role in maintaining tissue fluid homeostasis. It collects and transports protein-rich interstitial fluid via lymph nodes, large collecting lymphatic vessels, and lymphatic trunks (including the thoracic duct), and then facilitates passage back to the blood vascular circulation. The lymphatic system also plays an essential role in the immune response to infectious agents. Recent studies reported that new corneal lymphatic vessels, together with blood vessels, occur in inflammatory, burned, and transplanted corneas^[14-16]. Compared with blood vessels, lymphangiogenesis plays a crucial role in corneal immunity and may be more important in allograft rejection of normal and high risk corneal transplants^[4-6]. However, due to the unknown features of corneal lymphatics, it is difficult for ophthalmologists to identify the state of corneal lymphangiogenesis in order to assess the effect of drugs targeting lymphangiogenesis after keratoplasty (if available), or to choose a better time for regrafting. Therefore, the role of corneal lymphangiogenesis in allograft rejection still needs to be elucidated.

We previously examined the development of corneal blood and lymphatic vessels after alkaline burns, then divided the recipients according to the state of corneal angiogenesis and lymphangiogenesis, and performed keratoplasty to compare their respective roles in allograft rejection. We found that corneal lymphatic vessels played a crucial role in allograft rejection^{[5, 16]}. However, alkali-burned corneas were thought to be high risk recipient beds, not only because of vascularization, but also because of serious inflammation. In addition, inflammation was considered to be the common trait in the postnatal events that overlapped with lymphatic vessel growth, although the actual mechanism of lymphangiogenesis was unknown based on available reports^[17]. In another previous study, we found a significant relationship between corneal lymphangiogenesis and inflammation index in alkali-burned rat corneas^[18]. Because of the interference of inflammation, it was difficult to accurately evaluate the role of corneal lymphatic vessels in allograft failure in alkali-burned corneal beds.

Recently, we reported that VEGF-C blocking therapy could inhibit corneal lymphangiogenesis and promote allograft survival^[19]. Although some other unknown factors may be involved in the establishment and maintenance of the lymphatic vasculature, VEGF-C is thought to be the predominant lymphangiogenic factor^[20-21]. Direct evidence of the role of VEGF-C in promoting lymphangiogenesis comes from studies of transgenic mice overexpressing VEGF-C under the control of the keratin 14 (k14) promoter. These mice displayed a pronounced hyperplasia of cutaneous lymphatic vessels, whereas the growth of blood vessels was not affected^[22]. Conversely, lymphatic vessels transiently regressed in the skin in the inner organ of keratin 14-controlled VEGFR3-Ig mice^[23]. In a previous study, we examined transplanted rat corneas and found that VEGF-C expression dramatically increased, and there was a significant correlation between corneal lymphangiogenesis and VEGF-C mRNA expression after keratoplasty^[24]. Therefore, if the blockade of VEGF-C could selectively inhibit corneal lymphangiogenesis, which resulted in lymphatic vessels growing apart from blood vessels in tranplanted corneas, we could more easily evaluate the respective roles of lymphatic and blood vessels in allograft rejection.

In the current study, we compared VEGF-C mRNA, BVA, and LVA between the anti-VEGF-C group and control group. We found that VEGF-C mRNA levels were dramatically decreased, especially from day 10 to 21 after keratoplasty, in the anti-VEGF-C group, suggesting that the expression of VEGF-C was successfully inhibited following injection of the neutralizing monoclonal antibody. We also found both BVA and LVA were gradually increased after keratoplasty in the control group. However, in the anti-VEGF-C group, while BVA was increased to some extent, LVA was gradually decreased from 14-21 days after keratoplasty, which results in an increasing difference between BVA and LVA. Taken together with the known role VEGF-C plays in lymphangiogenesis, the downregulation of VEGF-C by VEGF-C blocking may have resulted from the outgrowth of corneal lymphatic vessels being separate from the outgrowth of blood vessels.

Our results also showed that the relationship between LVA and RI was not significant in the anti-VEGF-C group, suggesting that corneal lymphatic vessels were not associated closely with allograft immunity after the blockade of VEGF-C. The decrease of corneal lymphatic vessels in transplanted corneas induced by VEGF-C blocking resulted in corneal lymphangiogenesis having minimal effects on corneal immunity, which might be responsible for the lack of correlation of LVA and RI. However, there was a significant relationship between BVA and RI in the anti VEGF-C group, which suggested an outgrowth of corneal blood vessels being associated closely with allograft rejection after VEGF-C blockade. In additin, comparing the mean survival times of the grafts in the two groups, it was no more than 8 days longer after the anti-VEGF-C treatment. It suggested when there were no lymph vessels, rejection is delayed, but not stopped. Therefore, both lymph and blood vessels are important in allograft rejection.

Notably, we also found that BVA as decreased to some extent after VEGF-C blockade. In addition to inducing lymphangiogenesis via the VEGFR3 pathway, VEGF-C has also been reported to act by binding VEGFR2^[25-26]. Whereas the 31 kDa form of VEGF-C only bound to VEGFR3 and was therefore exclusively lymphangiogenic, the 21 kDa form also induced angiogenesis by binding to VEGFR2^[27-28]. However, studies showed the main function of VEGF-C involved binding to VEGFR3, but not to VEGFR2. Compared with the reduction in LVA, the reduction in BVA was much smaller. Therefore, we hypothesize that the majority of VEGF-C interacted with VEGFR3 to induce lymphangiogenesis, and that VEGF-C blockade had a crucial role in inhibiting the outgrowth of lymphatic vessels, but not blood vessels, in transplanted corneas.

In summary, our study examined the development of corneal lymphangiogenesis and angiogenesis in transplanted rat corneas after VEGF-C blockade. By targeting VEGF-C, we found that the outgrowth of corneal lymphatic vessels was separate from the outgrowth of blood vessels after keratoplasty. These findings are particularly valuable in view of the key roles of corneal lymphangiogenesis and/or angiogenesis in allograft rejection. In future studies, these results could provide the basis for novel treatments that target the lymphatic system after corneal transplantation.

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

阻断血管内皮生长因子C促进角膜移植后新生淋巴管和血管的“分离生长”

Lymphatic vessels growing apart from blood vessels in transplanted corneas after the blockade of vascular endothelial growth factor C

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