Hyaluronan in the cornea: Regulation of limbal stem cell fate and lymphangiogenesis

项目摘要 角膜缘干细胞（LSC）是角膜损伤后重建角膜上皮所必需的。损伤 导致LSC或LSC龛（LSCN）实质性损伤的角膜可能导致LSC缺陷 LSCD是一种严重的医学病症，其导致角膜混浊、炎症、血管形成、严重的角膜炎性病变和严重的角膜炎性病变。 疼痛和结膜炎，并最终导致视力完全丧失。移植 LSC离体扩增到受损角膜上是一种常见的外科手术， 在全球范围内，总体成功率为60%-70%。离体培养LSC的主要障碍是， 它们容易分化成中央角膜上皮细胞，妨碍了它们用于治疗应用 并强调需要在体外再生LSCN微环境以用于LSC扩增。 有趣的是，我们最近发表的工作表明，LSCN是由透明质酸（HA） 丰富的基质，其是支持干细胞表型所需的。本提案的目标1将描述 LSCN中存在的复杂HA基质的精确组成。目标2将致力于复制 在培养条件下存在于LSCN中的基质用于LSC的离体扩增， 存在于LSCN中的HA基质如何在体内调节LSC表型。淋巴管 仅存在于利姆布斯（角膜的富含HA的区域）中，因此角膜是无血管的。眼部 损伤和炎症，例如LSCD，经常导致不可逆的角膜血管生成， 淋巴管生成，这降低了角膜透明度，导致视力丧失，并限制了成功的 LSC移植。淋巴管标志物Lyve-1（淋巴管内皮受体1）是一种免疫调节剂。 透明质酸粘附素（一种特异性结合HA的蛋白质），并在整个淋巴系统中表达。Lyve-1 能够结合可溶性和固定化HA;然而，HA/Lyve-1相互作用是否起作用， 在淋巴管生成中的作用仍然难以捉摸。有趣的是，我们的初步数据显示， 基因敲除小鼠在利姆布斯中仅显示残留的淋巴管，这表明HA支架可能是 角膜淋巴管生成所必需的。HA在整个角膜中的上调先于加重 淋巴管的延伸，将HA注射到角膜基质中导致淋巴管的延伸， 船舶.因此，我们推测HA在调节角膜淋巴管生成中起重要作用。 目的3提出确定角膜利姆布斯内HA调节角膜上皮细胞增殖的机制。 淋巴管的发育。

Project summary Limbal stem cells (LSCs) are required for reconstituting the corneal epithelium after injury. Injury to the cornea leading to substantial damage of LSCs or the LSC niche (LSCN) may lead to LSC deficiency (LSCD), a serious medical condition that causes corneal opacification, inflammation, vascularization, severe pain and conjunctivalization, and which can ultimately lead to complete loss of vision. The transplantation of LSCs expanded ex vivo onto the damaged cornea is a common surgical procedure that is carried out all around the world with an overall success rate of 60%- 70%. A major hurdle in culturing LSCs ex vivo is that they readily differentiate into central corneal epithelial cells, hampering their use for therapeutic applications and underscoring the need to regenerate the LSCN microenvironment in vitro for LSC expansion. Interestingly, our recently published work demonstrates that the LSCN is composed of an hyaluronan (HA) rich matrix which is required to support the stem cell phenotype. Aim 1 of this proposal will characterize the precise composition of the complex HA matrix present in the LSCN. Aim 2 will work towards reproducing the matrix present in the LSCN in culture conditions for ex vivo expansion of LSCs and also establishing how the HA matrix present in the LSCN regulates the LSC phenotype in vivo. Lymphatic vessels are present solely in the limbus (the HA rich region of the cornea) and the cornea is therefore avascular. Ocular injuries and inflammation, such as LSCD, often lead to irreversible corneal angiogenesis and lymphangiogenesis, which reduce corneal transparency, leading to vision loss and limiting the success of LSC transplantation. The lymphatic vessel marker Lyve-1 (LYmphatic Vessel Endothelial receptor 1) is a hyaladherin (a protein that specifically binds HA) and is expressed throughout the lymphatic system. Lyve-1 is capable of binding both soluble and immobilized HA; however, whether the HA/Lyve-1 interaction plays a role in lymphangiogenesis remains elusive. Interestingly, our preliminary data reveal that corneas of HA knock-out mice show only vestigial lymphatic vessels in the limbus, suggesting that an HA scaffold may be necessary for corneal lymphangiogenesis. HA up-regulation throughout the cornea precedes exacerbated extension of lymphatic vessels, and injecting HA into the corneal stroma leads to the extension of lymphatic vessels. Therefore, we postulate that HA plays an important role in regulating corneal lymphangiogenesis. Aim 3 proposes to identify the mechanism by which HA within the corneal limbus regulates the development of lymphatic vessels.