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Final Report Summary - RTSAPA (The role of Toll-Like Receptors Signaling in Angiogenesis and its Potential Applications in Promoting Vascularization in Regenerative Medicine)


Publishable summary

The creation of adequate tissue constructs and other therapeutic products in tissue engineering and regenerative medicine is a highly challenging task. Adequate vascularization of tissue engineering constructs is an important prerequisite and might be promoted by allowing stem cells or progenitor cells to reach the site of injury/replacement for tissue repair. The development of new approaches leading to fast and successful vascularization is therefore one of the most intensively studied subjects in tissue engineering and regenerative medicine. Our previous work has demonstrated that endothelial progenitor cells from peripheral blood or cord blood are a potential autologous cell source for cellular therapies aiming to enhance the neovascularization of tissue engineering constructs. However, in these in vitro angiogenesis studies, the effect of one important system present in vivo, the immune system, has not been taken into account. In this project, we intend to identify the critical immune pathways involved in angiogenesis and to investigate the way that they affect angiogenesis and tissue regeneration in vitro and in vivo.

The objectives of first period are to investigate the role of TLR activation by exogenous and endogenous ligands in regulating/promoting angiogenesis by using our coculture system of OECs and POBs. I summarize my results in three parts. (A) I have characterized the expression of TLR2, TLR3 and TLR4 on OECs/POBs and studied their expression patterns after stimulation with different ligands: (B) I have developed a system approaches in quantitative analysis of angiogenesis in our coculture system of OECs and POB. The results have been written in the form of manuscript and submitted to Micron in last October; (C) I have demonstrated the essential role of TLR4 activation in promoting angiogenesis by using our coculture system of OECs and POBs. The results have been written in the form of manuscript and will be submitted to an international journal of high quality for publication soon.

Adequate vascularization of tissue engineering constructs and other therapeutic products is an important prerequisite for clinical translation and might be promoted by allowing stem cells or progenitor cells to reach the site of injury/replacement for tissue repair. However, quantitation of newly formed vessels/vessel-like structures in these constructs is a challenging task because of the complexity of the co-culture system and the lack of an efficient approach for quantitative analysis of these structures. To overcome these problems, we have established a systematic approach for the quantitative analysis of angiogenesis in co-cultures of outgrowth endothelial cells and osteoblasts. In this approach, we have utilized manual tracing to segment the vessels on fluorescent and confocal image print-outs. Our method is indispensable in the quantitative analysis of angiogenesis in our coculture system. The efficiency of this approach has been demonstrated by studying the effect of Toll-like receptor 3-ligand stimulation on the angiogenesis in co-cultures of outgrowth endothelial cells and osteoblasts. After stimulation with a TLR3-ligand, polyinosinic: polycytidylic acid, microvessel formation in the co-cultures increased significantly, which might indicate a potential role of TLR3 in promoting vascularization in co-cultures of outgrowth endothelial cells and osteoblasts. Our approach provides a convenient, robust, and reliable method for angiogenesis analysis and has a broad potential with regard to in vitro and in vivo angiogenesis studies, yielding accurate quantitative data on vascularization of tissue engineering constructs and other therapeutic products.

The development of new approaches leading to fast and successful vascularization tissue-engineering constructs is one of the most intensively studied subjects in tissue engineering and regenerative medicine. Our previous studies have demonstrated that endothelial progenitor cells from peripheral blood or cord blood are a potential autologous cell source for cellular therapies aimed at enhancing the neovascularization of tissue-engineering constructs. However, in these in vitro angiogenesis studies, the effect of one important system present in vivo, namely the immune system, has not been taken into account. Recently it has reported that TLR4 activation and LPS stimulation of endothelial cells might promote angiogenesis in a variety of settings. In this study we have demonstrated that TLR4 activation by Ultrapure LPS E. coli 0111:B4 (LPS-EB) enhanced significantly microvessel formation in our coculture system consisting outgrowth endothelial cells (OECs) and primary human osteoblasts (POBs). In addition, the precise modes of TLR4 action on the angiogenesis have been investigated. In monocultures of OEC and POBs we found that TLR4 activation through LPS-EB upregulated the expression level of TLR4/MYD88 and enhanced the angiogenesis and osteogenesis, respectively. Furthermore, by using the ELISA and qRT-PCR, we found that the level of two adhesion molecules (ICAM-1, E-selectin), two cytokines (IL-6 and IL-8) and growth factors (VEGF, PDGF-BB) related to angiogenesis increased significantly after LPS-EB treatment. The knowledge obtained will be very helpful to understand the role of TLR4 in the angiogenesis in different settings related to tissue repair and tissue engineering. In addition, since LPS and TLR4 agonists could improve angiogenesis and osteogenesis, TLR4 agonists (endogenous or synthetic) could be used for intervention of angiogenesis in vivo and therefore might be tested for their potential clinical applications in promoting angiogenesis in bone tissue engineering.


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UNIVERSITAETSMEDIZIN DER JOHANNES GUTENBERG-UNIVERSITAET MAINZ
Germany
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