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: 7576823
• 负责人: George A Truskey
• 金额: $ 27.3万
• 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/7576823
• 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 Culture Techniques; Cell Line; Cell physiology; Cell-Matrix Junction; Cells; Coculture Techniques; Complication; Data; Development; Elasticity; Endothelial Cells; Endothelium; Exposure to; Extracellular Matrix; Extracellular Matrix Proteins; Fc Receptor; Fibronectins; Focal Adhesions; Gene Expression; Gene Proteins; Hour; Human; Immunofluorescence Immunologic; In Vitro; Inflammation; Inflammatory; Integrins; Laminin; Leukocytes; Ligands; Long-Term Effects; Measures; Mechanics; Modeling; Nuclear Translocation; 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; 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）：为了建立组织工程化动脉的功能性内皮，我们开发了一种新的共培养模型，其中人内皮细胞（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，其允许快速且有效地检查大量实验变量。EC和SMC之间形成强粘附，SMC在共培养中更分化，并且人类共培养物可以维持长达30天。整合素在内皮细胞力传递中起重要作用。我们的初步数据表明，EC上培养的细胞外基质SMC产生的纤维粘连，而不是局灶性粘连时观察到的EC坚持刚性基板。此外，相对于在塑料上培养的EC，在共培养中的EC显示出降低的氧化和炎症状态，这表明在共培养期间涉及的粘附和整合素的类型的转变可能影响内皮的功能。粘附类型的转变可以影响在共培养期间参与粘附的整合素和EC的后续功能。为了正确设计组织工程血管，产生适当的EC功能，有必要了解EC粘附到基质覆盖SMC后EC功能暴露于流动的影响。因此，我们将检验以下假设：（1）在共培养中，平滑肌细胞的纤维粘附形成和弹性模量影响参与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KLF 2的水平在静态和流动条件下促进抗炎和抗血栓形成EC表型，和（3）共培养和脉动剪切应力的组合通过减少具有纤维状粘附的肌动蛋白应力纤维的形成来降低内皮的渗透性。本课题的具体目标是：（1）鉴定与共培养EC和SMC之间纤维状和粘着斑形成有关的整合素和粘附分子;（2）确定SMC弹性和纤维状粘附对EC粘附和功能的重要性;（3）确定纤维状粘附对共培养EC对流动反应的影响;以及（4）确定在共培养中长期流动对EC渗透性的影响。这些研究将提供重要的新信息EC和SMC功能的相互作用，可以影响组织工程血管的设计。内皮细胞粘附与平滑肌的功能对于新的血管来源有相当大的需求，以修复或替换动脉粥样硬化损伤的血管。组织工程就是这样一个机会。这项拟议中的研究将检查动脉和静脉的细胞（内皮细胞）在平滑肌细胞产生的表面上附着和发挥作用的方式。这些研究将为血管壁的这两种细胞相互作用的方式提供新的见解，并提供一种细胞培养系统，以促进组织工程动脉的发展。