MACROPHAGE-ENDOTHELIAL CO-CULTURE SYSTEM AS AN IN VITRO DERMAL ANGIOGENESIS MODE

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 糖尿病慢性足溃疡是一个主要的医学、社会和经济问题。它是导致下肢截肢的主要原因。糖尿病患者的非愈合性溃疡的主要特征是持续性炎症和受损的血管再生（血管生成）。血管生成是一个复杂的生理过程，需要正常的功能和适当协调的巨噬细胞和内皮细胞（EC）之间的相互作用。在糖尿病慢性溃疡中，这些细胞活性和功能受损。其功能障碍的生化和细胞机制以及可能改变的巨噬细胞-内皮细胞（巨噬细胞-EC）细胞相互作用在受损的血管生成知之甚少，主要是由于缺乏体外伤口愈合血管生成模型。因此，缺乏对慢性伤口愈合病理学（如糖尿病溃疡）的机制的了解阻碍了开发新疗法和相应的干预分子靶点的努力。 巨噬细胞通过分泌多种细胞因子和生长因子（包括缺氧诱导因子1 α（HIF-1a））在伤口血管生成中起关键作用。HIF-1a通过诱导多种血管生成因子（包括VEGF、Flk 1和NOS）的表达来启动血管生成。 我们假设HIF-1是糖尿病溃疡中巨噬细胞反应和糖尿病伤口愈合中血管生成的关键调节因子。为了验证我们的假设，我们这个夏季项目的目标是建立一个巨噬细胞-EC共培养系统作为体外皮肤伤口血管生成模型。 具体目标1：开发和表征巨噬细胞-EC共培养系统。我们将使用Transwell系统，通过在同一Transwell系统的上室的可渗透支持物上生长真皮内皮细胞和在底部孔中生长巨噬细胞来建立巨噬细胞-EC共培养物。在这个系统中，巨噬细胞和内皮细胞之间没有直接的接触，但它们的通讯是由这些细胞分泌的生长因子或细胞因子促进的  模拟这两种细胞类型之间没有直接接触的体内环境。我们将评估细胞共培养对细胞增殖和细胞因子表达（包括HIF-1a和VEGF）的影响。 具体目的2：评价巨噬细胞-EC共培养系统作为体外真皮血管生成模型的可行性。我们将通过诱导炎症和缺氧状态来模拟伤口微环境，从而挑战这种共培养系统。我们将评估这些条件下的细胞活性（增殖，存活）和细胞因子表达（HIF-1a和VEGF）。 INBRE暑期项目将使申请人和她的导师之间的富有成效的合作，使一个高度需要的体外皮肤伤口血管生成模型的发展。这种皮肤伤口血管生成模型将提供一个强大的系统来进行机制研究，如研究糖尿病伤口的微环境对巨噬细胞和内皮细胞之间的HIF-1-VEGF信号通路的影响。从这项研究中获得的初步数据将用于向NIH提出的拨款建议，以及从她的家乡机构获得研究支持。UCA是一所本科院校，王博士在过去的3年里从事了8名本科生的研究。因此，通过这种奖学金机制提供的支持也可能会增加本科生的研究机会。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Diabetic chronic foot ulceration represents a major medical, social, and economic problem. It is the leading cause of lower extremity amputations. Key features of the non-healing ulcer in diabetic patients are persistent inflammation and impaired blood vessel regeneration (angiogenesis). Angiogenesis is a complex physiological process that requires normal functions and properly orchestrated interaction between macrophages and endothelial cells (ECs). In diabetic chronic ulcer, those cellular activities and functions are impaired. The biochemical and cellular mechanisms underlying their dysfunction and the possible altered macrophage-endothelial (macrophage-EC) cell interactions in impaired angiogenesis are poorly understood, primarily due to the lack of an in vitro wound-healing angiogenesis model. Consequently, the lack of knowledge of the mechanisms responsible for the pathologies of chronic wound healing such as diabetic ulcer hampers the efforts to develop new therapies and the corresponding molecular targets for intervention. Macrophages play critical roles in wound angiogenesis by secreting multiple cytokines and growth factors including hypoxia inducible factor 1 alpha (HIF-1a). HIF-1a is essential for initiating angiogenesis by inducing the expression of multiple angiogenic factors including VEGF, Flk 1, and NOS. We hypothesize that HIF-1 is a critical regulator of the response of macrophages in diabetic ulceration and for angiogenesis in diabetic wound healing. In order to test our hypothesis, our objective for this summer project is to develop a macrophage-EC co-culture system as an in vitro dermal wound angiogenesis model. Specific Aim 1: Develop and characterize a macrophage-EC co-culture system. We will use a Transwell system to establish a macrophage-EC co-culture by growing dermal endothelial cells on the permeable support of the upper chamber and macrophages in the bottom well of the same Transwell system. In such system, there is no direct contact between macrophages and ECs, but their communications are facilitated by growth factors or cytokines secreted by those cells  mimicking the in vivo environment where there is no direct contact between these two cell types. We will evaluate the effect of cell co-culture on cell proliferation and cytokine expression (including HIF-1a and VEGF). Specific Aim 2: Evaluate the feasibility of using the macrophage-EC co-culture system as an in vitro dermal angiogenesis model. We will challenge this co-culture system by inducing an inflammatory as well as a hypoxic state to mimic the wound microenvironment. We will evaluate the cell activities (proliferation, survival) and cytokine expression (HIF-1a and VEGF) under these conditions. The INBRE summer program will enable a productive collaboration between the applicant and her mentor to allow development of a highly needed in vitro dermal wound angiogenesis model. This dermal wound angiogenesis model will provide a robust system to conduct mechanistic studies such as to investigate the effect of the microenvironment of a diabetic wound on the HIF-1-VEGF signaling pathway between macrophages and endothelial cells. The preliminary data obtained from this research will be used for a grant proposal to NIH as well as to obtain research support from her home institution. UCA is an undergraduate institution and Dr. Wang has engaged eight undergraduate students in her research during past 3 years. Therefore, support through this fellowship mechanism will likely also lead to enhancing research opportunities for undergraduate students.