Endothelial Cell Adhesion and Function on Smooth Muscle

内皮细胞对平滑肌的粘附和功能

• 批准号: 7387518
• 负责人: George A Truskey
• 金额: $ 26.96万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-01 至 2012-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7387518
• 关键词: Actins; Adhesions; Affect; Anti-Inflammatory Agents; Anti-inflammatory; Arteries; Atherosclerosis; Atomic Force Microscopy; Basement membrane; Blocking Antibodies; Blood Vessels; Cell Adhesion; Cell Adhesion Molecules; Cell Culture System; Cell Line; Cell physiology; Cell-Matrix Junction; Cells; Coculture Techniques; Complication; Condition; Data; Development; Elasticity; Endothelial Cells; Endothelium; Exposure to; Extracellular Matrix; Extracellular Matrix Proteins; Fibronectins; Focal Adhesions; Gene Expression; Gene Proteins; Hour; Human; Immunofluorescence Immunologic; In Vitro; Inflammation; Inflammatory; Integrins; Laminin; Leukocytes; Ligands; Long-Term Effects; Measures; Mechanical Stress; Modeling; Nuclear Translocation; Numbers; Permeability; Phenotype; Plastics; Play; Production; Property; Proteins; Regulation; Relative (related person); Research; Reverse Transcriptase Polymerase Chain Reaction; Role; Smooth Muscle; Smooth Muscle Myocytes; Source; Stem cells; Stress Fibers; Stretching; Surface; Testing; Thrombosis; Time; Tissue Engineering; Umbilical Cord Blood; Vascular Graft; Veins; Western Blotting; cell growth; cell type; cellular transduction; day; design; insight; monolayer; novel; occludin; polyacrylamide gels; pressure; protein expression; receptor; repaired; research study; response; shear stress; tensin; transcription factor; vascular endothelial cadherin-2

DESCRIPTION (provided by applicant): In order to establish a functional endothelium for tissue engineered arteries, we developed a novel co-culture model in which human endothelial cells (EC) form a confluent monolayer on quiescent smooth muscle cells (SMC). The co-culture model is a simplified representation of a blood vessel that permits rapid and efficient examination of a large number of experimental variables. Strong adhesion develops between EC and SMC, SMC are more differentiated in co-culture, and human co-cultures can be maintained for as long as 30 days. Integrins play an important role in endothelial force transduction. Our preliminary data suggest that EC cultured on extracellular matrix produced by SMC produce fibrillar adhesions rather than focal adhesions observed when ECs adhere to rigid substrates. Further, EC in co-culture show a reduced oxidative and inflammatory state relative to EC cultured on plastic suggesting a shift in the type of adhesion and integrins involved during co-culture may affect the function of endothelium. The shift in the type of adhesion can influence integrins involved in adhesion during co- culture and the subsequent function of ECs. To properly design tissue engineered vessels that produce appropriate EC function, it is necessary to understand the effect of EC adhesion to the matrix overlying SMC upon EC function following exposure to flow. Thus, we will test the hypotheses that (1) in co-culture, fibrillar adhesion formation and the elastic modulus of SMCs influence specific extracellular matrix proteins and integrins involved in EC adhesion; (2) fibrillar adhesions promote an anti-inflammatory and anti- thrombotic EC phenotype under static and flow conditions by regulating the level of the transcription factor KLF2, and (3) a combination of co-culture and pulsatile shear stresses lowers the permeability of endothelium by reducing the formation of actin stress fibers with fibrillar adhesions. Specific aims of the project are to: (1) identify integrins and adhesion molecules involved in fibrillar and focal adhesion formation between EC and SMC in co-culture; (2) determine the importance of SMC elasticity and fibrillar adhesions upon EC adhesion and function; (3) determine the effect of fibrillar adhesions upon the response of co-cultured EC to flow; and (4) determine the effect of long-term flow upon EC permeability in co-culture. These studies will provide important new information about EC and SMC function interactions that can influence the design of tissue-engineered blood vessels. Endothelial Cell Adhesion & Function on Smooth Muscle There is considerable need for new sources of blood vessels to repair or replace vessels damaged by atherosclerosis. Tissue engineering represents one such opportunity. The proposed research will examine the manner in which the cells that line arteries and veins (endothelial cells) attach and function on surfaces produced by smooth muscle cells. The studies will provide new insights into the manner in which these two cells of the vessel wall interact and provide a cell culture system to facilitate development of tissue- engineered arteries.

描述（由申请人提供）：为了建立组织工程动脉的功能内皮，我们开发了一种新的共培养模型，其中人类内皮细胞（EC）在静止平滑肌细胞（SMC）上形成融合单层。共培养模型是血管的简化表示，允许对大量实验变量进行快速有效的检查。EC与SMC之间有较强的附着力，SMC在共培养中分化更大，人类共培养可维持30天之久。整合素在内皮力转导中起重要作用。我们的初步数据表明，在SMC产生的细胞外基质上培养的EC产生纤维状粘连，而不是在刚性底物上观察到的局灶性粘连。此外，与塑料培养的内皮细胞相比，共培养的内皮细胞表现出更低的氧化和炎症状态，这表明共培养过程中粘附和整合素类型的改变可能会影响内皮细胞的功能。在共培养过程中，黏附类型的转变会影响参与黏附的整合素和ECs的后续功能。为了正确设计组织工程血管以产生适当的EC功能，有必要了解暴露于血流后EC与SMC上基质的粘附对EC功能的影响。因此，我们将验证以下假设：(1)在共培养中，纤维粘附形成和SMCs的弹性模量影响参与EC粘附的特异性细胞外基质蛋白和整合素；(2)纤维粘连通过调节转录因子KLF2的水平，在静态和流动条件下促进抗炎和抗血栓形成的EC表型；(3)共培养和脉冲剪切应力的结合，通过减少纤维粘连的肌动蛋白应激纤维的形成，降低内皮的通透性。该项目的具体目标是：(1)确定在共培养中EC和SMC之间纤维和焦点粘附形成的整合素和粘附分子；(2)确定SMC弹性和纤维粘连对EC粘连和功能的重要性；(3)确定纤维粘连对共培养EC对血流反应的影响；(4)确定长期流动对共培养EC渗透率的影响。这些研究将为EC和SMC功能相互作用提供重要的新信息，这些相互作用可以影响组织工程血管的设计。平滑肌上内皮细胞的粘附与功能由于动脉粥样硬化而受损的血管需要大量新的血管来源来修复或替代。组织工程就是这样一个机会。拟议的研究将检查排列在动脉和静脉上的细胞（内皮细胞）附着在平滑肌细胞产生的表面上并发挥作用的方式。这些研究将为这两种血管壁细胞相互作用的方式提供新的见解，并为促进组织工程动脉的发展提供细胞培养系统。