Cutaneous wound remodeling by re-differentiating keratinocytes expressing CXCR3 l

通过表达CXCR3 l的角质形成细胞的再分化来重塑皮肤伤口

基本信息

批准号：
8644267
负责人：
Arthur Huen
金额：
$ 12.03万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-04-01 至 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8644267
关键词：
Adherens Junction Adhesions Adhesiveness Animal Model Animals Apoptosis Appearance Area Atrophic Basic Science CXCL10 gene CXCL11 gene CXCR3 gene Calcium Cell Culture Techniques Cell Density Cells Cellular Morphology Chairperson Chronic Cicatrix Clinical Coculture Techniques Commit Communication Communities Complex Contact Inhibition Controlled Study Cutaneous Depressed mood Dermal Dermatologist Dermatology Dermis Desmosomes Development Distal Doctor of Medicine Doctor of Philosophy Endothelial Cells Engineering Environment Epithelial Event Failure Fibroblasts Floor Foundations Funding Genetically Engineered Mouse Gills Goals Granulation Tissue Housing Human Human Engineering Immunofluorescence Immunologic Impaired wound healing In Vitro Intercellular Junctions Intermediate Filaments Intervention Investigation Knockout Mice Label Laboratories Laboratory Study Lead Ligands Literature Medical Medical center Medicine Mentorship Microscopy Modeling Molecular Mus Natural regeneration Organ Organ Culture Techniques Pathologic Processes Pathology Phase Phosphorylation Physician Executives Physiological Processes Principal Investigator Process Protein Kinase C Proteins Research Research Personnel Research Project Grants Research Proposals Research Training Reverse Transcriptase Polymerase Chain Reaction Role Scientist Signal Transduction Site Skin Staging Structure System Techniques Testing Therapeutic Intervention Time Tissues Tongue Training Translations Trauma Tumor Cell Invasion Universities Western Blotting Work Wound Healing career cell motility chemokine chemokine receptor cytokine design desmoplakin experience extracellular in vivo innovation keratinocyte keratinocyte differentiation medical schools meetings migration monolayer mouse model novel paracrine premature professor psychologic public health relevance regenerative repaired response symposium tissue culture tool validation studies wound

项目摘要

DESCRIPTION (provided by applicant): Arthur C. Huen M.D., Ph.D, is currently completing training as a dermatologist committed to pursuing basic research investigations into wound healing. Intrigued by research from the beginning, he had pursued training in basic science research during medical school, resulting in doctorate training focusing on the role of intercellular adhesion, specifically the role of desmoplakin, a desmosomal plaque protein, in providing a strong state of intercellular adhesion via its connection to intermediate filaments. This work was completed at Northwestern University under the mentorship of Kathleen J. Green Ph.D (Mayberry Professor of Pathology, Professor of Dermatology, Associate Chairman of Pathology, Northwestern University). His current clinical training is being pursued at the University of Pittsburgh Medical Center (UPMC) Department of Dermatology and his research training is being conducted in the laboratory of Alan Wells M.D., D.M.S (Vice Chairman, Department of Pathology, Thomas J Gill III Professor of Pathology and Medical Director of Section of Laboratory Medicine at the University of Pittsburgh Medical Center) and under the supervisory and career mentorship of Louis Falo (Professor and Chairman, Department of Dermatology, UPMC). Both Departments are fully committed to Dr. Huen's development and have designed a transitional training situation that focuses on investigative experience, combined with some classroom and seminar/short course training in targeted areas. The main thrust will be to introduce Dr. Huen to the research community as a junior investigator. The Well's laboratory studies molecular mechanisms regulating cellular migration in both physiologic and pathologic processes such as wound healing and tumor invasion, respectively. The focus is on developing new hypotheses derived from discovery approaches in the literature and laboratory to solve complex questions involving cell-tissue communications. The major techniques utilized in the laboratory, and in which Dr. Huen is being trained, include tissue culture and organotypic culture as well as microscopy. Much of the in vitro work is verified using mouse models, with animals being housed at the nearby Pittsburgh VA Medical Center. A unique aspect of this work is the focus on ex vivo organotypic models that are specifically engineered to allow the molecular manipulations of in vitro in an model approaching the organ complexity of in vivo. In addition, there are several other laboratories on the same floor and interactions are frequent via combined weekly lab meetings as well as semimonthly formal conferences. Training within this environment and funding of the currently proposed research plan will provide a transition for Arthur Huen to become an independent medical scientist. The currently proposed project focuses on the role of chemokine receptor CXCR3 ligands, including IP9 (CXCL11, ITAC) and IP10 (CXCL10), in coordinating the transition of cutaneous wound healing from the proliferative phase to the remodeling phase. In vitro studies have demonstrated that stimulation with IP9 results in increased migration by keratinocytes whereas stimulation in fibroblasts results in decreased migration. The goal of this research proposal is to determine the signals responsible for initiating the cascade of CXCR3 ligands that starts the remodeling phase of cutaneous wound healing. It is hypothesized that keratinocytes near, but distal to the wound edge that are undergoing re-differentiation during re-epithelialization are responsible for expression of CXCR3 ligands IP9 and, to a lesser extent, IP10. Involvement of these re- differentiating keratinocytes would best fit the spatio-temporal expression of IP9 and IP10. To test this hypothesis, three specific aims are proposed. The first aim will examine the role of cellular confluence, which induces keratinocyte differentiation, on expression of chemokines IP9 and IP10. In the second aim, the hypothesis that re-establishment of intercellular junctions by keratinocytes is a trigger for chemokine expression will be examined. Lastly, the third aim will examine the effect of exogenous IP9 and IP10 stimulation on the rate of re-epithelialization in organotypic skin culture and mouse skin. These studies will be accomplished not only in routine tissue culture, but also in animals using unique genetically engineered mouse models, and in novel ex vivo organotypic cultures systems. Part of this proposal will involve establishing and using an all-human engineered skin organ culture in which the different components can be individually altered at the molecular level. This will then be a unique and highly useful tool for launching the initial independent projects of Dr. Huen. In addition, successful completion of these studies will establish an innovative model of wound healing for further explorations some of which would allow for translational development. PUBLIC HEALTH RELEVANCE: The proposed research project will study control mechanisms for signals released by cells near the edge of skin wounds that initiate the later stages of wound healing. Errors in the timing or the amount of signal released by these cells may result abnormal wound healing; for example, lack of signaling seems to slow the rate of skin wounds to close while too much signaling is expected to close wounds quickly but form weaker, depressed scars. Understanding the control of these signals may lead to better ways to treat chronic wounds and to more rapidly restore skin function and appearance after trauma.

描述（由申请人提供）：Arthur C. Huen M.D.博士目前正在完成培训，作为皮肤科医生，致力于对伤口愈合进行基础研究调查。从一开始就引起了研究，他就在医学院期间接受了基础科学研究的培训，从而导致博士培训的重点是细胞间粘附的作用，特别是脱莫布拉金（一种脱骨斑块蛋白）的作用，在脱骨体斑块蛋白（通过与中间纤维中的内膜纤维之间的连接）提供了强烈的细胞间粘附状态。这项工作是在凯瑟琳·J·格林（Kathleen J.他目前的临床培训正在匹兹堡大学医学中心（UPMC）皮肤病学系进行，他的研究培训正在D.M.S（D.M.S）的艾伦·威尔斯（Alan Wells M.D. （UPMC皮肤科教授兼董事长）。这两个部门都完全致力于Huen博士的发展，并设计了一个过渡性培训状况，重点是调查经验，并结合了针对性领域的某些课堂和研讨会/短期课程培训。主要的推力是将Huen博士介绍给研究社区，作为初级研究员。 Well的实验室研究分别在生理和病理学过程（例如伤口愈合和肿瘤侵袭）中调节细胞迁移的分子机制。重点是开发从文献和实验室中发现方法得出的新假设，以解决涉及细胞组织通信的复杂问题。在实验室中使用的主要技术，正在接受Huen博士的训练，包括组织培养和器官培养以及显微镜。大部分体外工作都使用小鼠模型进行了验证，动物被安置在附近的匹兹堡弗吉尼亚州医疗中心。这项工作的一个独特方面是专门设计的外体器官模型，这些模型允许在接近体内器官复杂性的模型中对体外进行分子操作。此外，同一地板上还有其他几个实验室，并且通过每周的实验室会议以及半月份的正式会议进行频繁进行互动。在这种环境中的培训和当前建议的研究计划的资金将为亚瑟·休恩（Arthur Huen）成为独立的医学科学家提供过渡。目前提出的项目着重于趋化因子受体CXCR3配体的作用，包括IP9（CXCL11，ITAC）和IP10（CXCL10），在协调皮肤伤口从增殖阶段到重塑阶段的过渡。体外研究表明，使用IP9的刺激导致角质形成细胞的迁移增加，而成纤维细胞的刺激导致迁移减少。该研究建议的目的是确定负责启动CXCR3配体级联的信号，该配体开始了皮肤伤口愈合的重塑阶段。假设角质形成细胞附近，但是在重新上皮期间正在进行重新分化的伤口边缘远端是CXCR3配体IP9的表达，并且在较小程度上是IP10。这些重大分化的角质形成细胞的参与最适合IP9和IP10的时空表达。为了检验这一假设，提出了三个具体目标。第一个目的将研究诱导角质形成细胞分化的细胞汇合的作用，在趋化因子IP9和IP10的表达中。在第二个目的中，将研究趋化因子表达的诱因，即通过角质形成细胞重新建立细胞间接头的假说。最后，第三个目标将检查外源性IP9和IP10刺激对器官皮肤培养和小鼠皮肤中重新上皮化速率的影响。这些研究将不仅在常规组织培养中，而且还将在使用独特的基因工程小鼠模型以及新型的离体器官型培养系统中完成。该提案的一部分将涉及建立和使用全人类工程的皮肤器官培养物，其中可以在分子水平上单独改变不同的成分。然后，这将成为启动Huen博士最初独立项目的独特且非常有用的工具。此外，这些研究的成功完成将建立一种创新的伤口愈合模型，以进一步探索其中一些允许转化发展。公共卫生相关性：拟议的研究项目将研究由皮肤伤口边缘附近的细胞释放的信号的控制机制，这些信号启动了伤口愈合的后期。这些细胞释放的时间或信号量的错误可能导致伤口愈合异常；例如，缺乏信号传导似乎会减慢皮肤伤口关闭的速度，而预计过多的信号会迅速关闭伤口，但会较弱，但肌肉疲软。了解这些信号的控制可能会导致更好的方法治疗慢性伤口，并在创伤后更快地恢复皮肤功能和外观。