Expression, function and endogenous modulators of the transient receptor potential (TRP) channels TRPV1, TRPV4, TRPM8 und TRPA1 in human corneal endothelial cells

人的角膜内皮细胞(HCE)完成维持角膜透明度和正常视力所必需的各种功能。这些功能依赖于调节细胞内钙水平的细胞机制。瞬时受体电位通道(TRPs)在这一过程中起着重要作用。最近的研究表明，TRPV1和TRPM8的功能Trp通道亚型活性调节细胞凋亡(钙-细胞凋亡连接)。因此，有必要进一步研究这些Trp亚型的调节如何有助于HCE功能的支持。在最近的研究中，申请人还首次证明了HCE细胞表达包括TRPV4和TRPA1在内的其他Trp亚型。目前的研究证实，TRPM8和TRPA1在原代HCE细胞培养中也有功能表达。此外，我们的结果表明，这些TRPs在细胞内钙离子浓度的温度依赖性和渗透敏感性调节中发挥作用。本项目的目的是利用高灵敏度的方法(钙离子成像、膜片钳、定量聚合酶链式反应)，确定TRPs在调节角膜内皮相关生物学过程中的特性，并识别内源性Trp通道活性调节器。在已建立的体外HCEC-12细胞模型以及新鲜和培养的原代HCE细胞培养中，我们计划描述TRPs的分子、功能和药理学特性以及它们关联的信号通路，这些通路介导对组织生存至关重要的反应的控制。此外，我们还将确定TRPV1、TRPV4、TRPM8和TRPA1之间的功能串扰机制，以及HCE中与EGF和FGF-2相关的膜上同源受体。此外，长期接触眼科药物对色氨酸通道功能和调节的影响及其对角膜内皮功能的影响也将被研究。总体而言，这项研究的结果将有助于更好地了解HCE生物学，并有望开发新的策略来延长和改善角膜移植前后的角膜内皮细胞功能，并可能在长期接触临床证明有效的眼科药物期间也是如此。

The human corneal endothelium (HCE) fulfills various required functions mandatory for the maintenance of corneal transparency and normal vision. These functions are dependent on cellular mechanisms that mediate regulation of intracellular calcium levels. Transient receptor potential channels (TRPs) play a substantial role in this process. Recent studies showed that functional TRP channel subtype activity of TRPV1 and TRPM8 modulate apoptosis (calcium-apoptosis link). Therefore, further studies are warranted to determine how modulation of these TRP subtypes contributes to the support of HCE function. In recent studies, the applicants also demonstrated for the first time that HCE cells express other TRP subtypes that include TRPV4 as well as TRPA1. Current investigations confirmed functional expression of TRPM8 and TRPA1 also in primary HCE cell cultures. Furthermore, our results indicate that these TRPs play a role in the temperature-dependent and osmosensitive regulation of intracellular calcium concentration. The aim of this project is to determine the properties of TRPs in regulating corneal endothelial-relevant biological processes and to identify endogenous TRP channel activity modulators, using highly sensitive methods (Ca2+ imaging, patch-clamp, qPCR). In the established in vitro HCEC-12 cell model and fresh and cultured primary HCE cell cultures, we plan to delineate the molecular, functional and pharmacological properties of TRPs as well as their linked signaling pathways, which mediate control of responses that are essential for tissue viability. In addition, we will define mechanisms of functional cross-talk between TRPV1, TRPV4, TRPM8 and TRPA1 and the HCE-relevant membrane cognate receptors for EGF and FGF-2 in the HCE. Furthermore, the impact of long-term exposure to ophthalmic drugs on TRP channel function and regulation and its implication on corneal endothelial function will be investigated. Overall, the results of this study will contribute to a better understanding of HCE biology and hold promise to develop novel strategies for prolonging and improving corneal endothelial cell function before and after keratoplasty and possibly also during long-term exposure to clinically proven effective ophthalmological medications.