Quantitative Vasodilation Studies

描述（由申请人提供）：本研究计划描述了一系列旨在帮助确定循环系统中阻力血管（即小动脉和毛细血管）控制血管张力的确切机制的实验。具体来说，这一建议将有助于确定红细胞（RBC）在控制肺血管阻力中的作用。当红细胞穿过微血管床时，如在肺中，红细胞受到机械变形。先前的研究结果表明，红细胞是肺中一氧化氮（NO）合成所必需的，并且红细胞通过微米大小的孔隙或管道导致ATP的释放，ATP是内皮细胞NO合成的已知刺激物。在这篇论文中，我们描述了一系列的研究，旨在量化红细胞穿过由聚二甲基硅氧烷（PDMS）芯片制成的微孔通道时ATP释放的速率和持续时间，这些微孔通道的内径与完整循环中的阻力血管相当。具体来说，我们将研究内径和微孔通道长度的改变以及流速对兔红细胞ATP释放的影响。此外，我们还将监测一氧化氮在存在和不存在红细胞衍生ATP的情况下产生的量。因此，在本提案中，我们提出了以下假设：ATP是红细胞在机械变形时释放的，是内源性NO合成的刺激，因此是肺循环中血管阻力的重要决定因素。在这里，我们打算1)证明通道直径的减小、通道长度和流速的增加刺激了合成微芯片中这些细胞释放ATP； 2)证明红细胞固有的某些特性，即细胞的可变形性和细胞年龄，可以影响红细胞释放ATP； 3)证明固定在微芯片通道管腔内的内皮细胞可以用来模拟体内真正的阻力血管的内皮。在红细胞来源的ATP刺激下，固定的内皮细胞产生和释放一氧化氮，可以用芯片上的电流法测量。这些研究的成功完成将使我们对控制肺循环血管阻力的机制有更全面的了解。这一信息将允许关于红细胞对健康和疾病中血管口径控制的贡献的新假设的发展。

DESCRIPTION (provided by applicant): This research proposal describes a series of experiments that are designed help to determine the exact mechanism by which resistance vessels in the circulatory system, namely arterioles and capillaries, control vascular tone. Specifically, this proposal will help define the role of the red blood cell (RBC) in the control of pulmonary vascular resistance. When traversing microvascular beds, such as in the lung, RBCs are subjected to mechanical deformation. Previous findings indicate the RBCs are required for nitric oxide (NO) synthesis in the lung, and that passage of RBCs through micrometer-sized pores or tubing results in the release of ATP, a known stimulus for endothelial cell NO synthesis. In this proposal, we describe a series of studies designed to quantify both the rate and duration of ATP release from RBCs as they traverse microbore channels fabricated in polydimethylsiloxane (PDMS) chips with internal diameters comparable to those of resistance vessels in the intact circulation. Specifically, we will examine the effect of alterations in the internal diameter and the length of the microbore channel, as well as the velocity of flow on ATP release from RBCs of rabbits. In addition, we will also monitor the amount of NO produced in the presence and absence of RBC derived ATP. Thus, in this proposal we address the hypothesis that: ATP, released from RBCs in response to mechanical deformation, is a stimulus for endogenous NO synthesis and, thereby, is an important determinant of vascular resistance in the pulmonary circulation. Here, we intend to 1) demonstrate that decreases in channel diameter and increases in channel length and flow velocity stimulate ATP release from these cells in a fabricated microchip, 2) demonstrate that certain properties intrinsic to the RBC, namely cell deformability and cell age, can affect ATP release from RBCs and 3) demonstrate that endothelial cells immobilized to the lumen of a microchip channel can be employed to mimic the endothelium of a real resistance vessel in vivo, and that the NO production and release from immobilized endothelial cells, stimulated by RBC-derived ATP, can be measured amperometrically on-chip. The successful completion of these studies will lead to a more comprehensive understanding of those mechanisms that are responsible for he control of vascular resistance in the pulmonary circulation. This information will permit the development of new hypotheses regarding the contribution of RBCs to the control of vascular caliber in health and disease.