Mimicking Vascular Endothelial Cell In Vivo Environment in Cell Culture

导论：实验血管生物学领域的科学家们一直面临着两个主要挑战：1)将多种机械力（如剪切应力、循环应变和静水压力）整合到使用体外细胞培养（如血管内皮细胞）的实验系统中；2)大多数细胞培养是在环境O2（即21% O2）下生长的，而不是在大多数器官/组织中看到的5% O2的生理水平。***就前者而言，相对于剪切应力和循环应变的研究，静水压力对EC响应的影响/机制的研究受到的关注要少得多。静水压力（例如静水压力的增加）曾经被认为是纯粹的“机械成分”，因此是一种相对良性的病理生理状况，最近已被认为是一种诱导机械信号传导的潜在刺激。有限的研究结果（包括我们自己的研究）表明，静水压力的增加引起复杂和高度敏感的细胞反应，因此可能在微循环调节中起关键作用。静水压力诱导血管内皮细胞生理/病理生理反应变化的机制方面仍然知之甚少。***细胞生物学家面临的另一个主要挑战是大多数细胞生长和用于体外实验的条件。通常的做法是在环境O2（即21% O2）下培养细胞。然而，身体的大多数细胞暴露在大约5%的O2中，从而使通常接受的细胞生长和实验条件（例如室内空气；21% O2）严重“高氧”和非生理性的。***研究方法：本研究计划的主要目标是研究血管内皮细胞对静水压力的反应（如分子信号传导和功能后果），并进一步开发新的实验模型，以研究生理氧水平下体外静水压力的作用/机制。***本研究将使用代表各种血管床的人血管内皮细胞原代培养物（例如，来自脑、肺、皮肤的微血管EC），这些细胞将在5% O2和静水压力（0-30 mmHg）的条件下培养并用于实验，采用最先进的微灌注和细胞共培养方法。将评估细胞功能/功能障碍的各个方面（例如氧化剂的产生，细胞因子，细胞骨架的重排，渗透性和亲粘附表型的变化等）。***期望：本研究项目将开发一种新的体外实验模型，密切模仿体内的生理/病理生理条件。此外，它将为至少三名研究生（硕士）的培训提供良好的基础。

Introduction: Scientists in the field of experimental vascular biology are constantly confronting two major challenges: 1) integration of multiple mechanical forces (e.g. shear stress, cyclic strain, and hydrostatic pressure) into the experimental system(s) that employ in vitro cell cultures (e.g. vascular endothelial cells) and 2) the fact that most cell cultures are grown at the ambient O2 (i.e. 21% O2) as opposed to physiological levels of 5% O2 seen in most organs/tissues. ***In regard to the former, studying the effects/mechanisms of hydrostatic pressure on EC responses have received much less attention relative to studies of shear stress and cyclic strain. Hydrostatic pressure (e.g. increase in hydrostatic pressure), that once was considered a purely “mechanical component” and hence, a relatively benign pathophysiological condition, recently has been recognized as a potential stimulus inducing mechanosignaling. Limited research findings (including our own) indicate that increase in hydrostatic pressure induces complex and highly sensitive cellular responses, therefore, may play a critical role in regulation of the microcirculation. The mechanistic aspects of hydrostatic pressure-induced changes in vascular endothelial cell physiological/pathophysiological responses remain poorly understood.***The other major challenge for cell biologists is the conditions at which most of cells are grown and used in the in vitro experiments. The common practice is to culture the cells at the ambient O2 (i.e. 21% O2). However, most of the body's cells are exposed to approximately 5% O2, thus making commonly accepted conditions of cell growth and experimentation (e.g. room air; 21% O2) severely “hyperoxic” and unphysiological. ***Research Approach: The main goal of this research program is to investigate vascular endothelial cell responses (e.g. molecular signaling and functional consequences) to hydrostatic pressure and to further develop a new experimental model allowing investigating the effects/mechanisms of hydrostatic pressure in vitro under physiological O2 levels.***This study will use primary human vascular endothelial cell cultures representing various vascular beds (e.g. microvascular EC derived from brain, lung, skin) which will be cultured and used in the experiments under conditions of 5% O2 and hydrostatic pressure (0-30 mmHg) employing state-of-the-art microperfusion and cell co-culture approaches. Various aspects of cell function/dysfunction (e.g. production of oxidants, cytokines, rearrangement of cytoskeleton, changes in permeability and pro-adhesive phenotype, etc.) will be assessed. ***Expectations: This research program will allow to develop a new experimental model in vitro closely mimicking physiological/pathophysiological conditions in vivo. In addition, it will provide excellent grounds for training of at least three graduate (MSc) students.