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

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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.