Macromolecular Structure of Arterial Walls

动脉壁和动脉瓣膜是复杂的大分子结构。这些结构的主要元素之一是支架，它提供了完成手头任务的力量和灵活性，要么保持血管中的血液对抗动脉压，要么通过冠状动脉瓣膜的功能维持压力。在过去的几年里，很明显，这些大分子的实际微观结构和组成可以影响不同疾病状态的进展，最明显的是动脉粥样硬化和瓣膜钙化。为了更好地了解这一过程，我们开始研究动脉血管床中大分子的精细结构，使用一种新的光学成像技术，该技术依靠胶原和弹性蛋白的非线性激发(NLE)在未固定的新鲜样品中提供其结构的亚微米图像，并使用相干反斯托克斯拉曼散射(CARS)直接监测水通过管壁的渗透。利用这些方法，我们做了以下观察：1)我们描述了用于监测生物组织中的水运动的CARS方法。我们基于键振动的光谱密度和脉冲激光的功率特性建立了物理模型，证明了为什么飞秒脉冲在检测CARS信号方面比传统的皮秒脉冲高出约300倍，而不会损失氘和氢之间的区别。我们还在实验上证实了这是一个模型和生物系统。这一进步使汽车能够监测各种生物系统中的水运动。2)使用氚作为示踪剂，我们用CARS显微镜确定了水渗透的主要屏障，也就是压力梯度最大的地方，在内皮细胞的基侧膜上。尽管内皮细胞被认为起到了水屏障的作用，但之前还没有确定具体的膜。我们还证明了水通道蛋白水通道蛋白AQP1集中在内皮细胞的顶膜上，这也与该膜与血管空间屏障接触时对水的高渗透性一致。我们认为，这种透水性的排列，在顶膜很高，在基侧膜很低，导致与心脏周期相关的压力波穿过内皮细胞体内，而对这个敏感细胞几乎没有压缩。然而，在内皮细胞基外膜与基底膜和内弹力板的弹性大分子的交界处，动脉压力波被传递给这些旨在承受这种压力的大分子。这一水屏障和跨越内皮细胞的相关压力梯度的变化可能在许多血管疾病状态中发挥重要作用。3)我们正在探索微流体模型系统，以评估CARS和其他简单荧光染料在亚微米级别的水通量定量中的使用，特别是评估这些系统中质子电导与大量水运动之间的差异，最终在生物系统中。

The arterial wall and arterial valves are complex macromolecular structures. One of the major elements of these structures is the scaffold that provides the strength and flexibility to perform the task in hand either retaining the blood in vessels against the arterial pressure or maintaining pressure via the function of coronary valves. In the last several years it has become apparent that the actual microstructure and composition of these macromolecules could influence the progress of different disease states most notably atherosclerosis and valve calcification. To gain a better understanding of this process, we have embarked on studies to understand the fine structure of the macromolecules in arterial vascular bed using a novel optical imaging technique that relies on the non-linear excitation (NLE) of collagen and elastin to provide sub-micron images of their structure in unfixed fresh samples together with direct monitoring of water permeation through the wall using Coherent anti-Stokes Raman Scattering(CARS). Using these approaches we have made the following observations: 1) We have characterized the CARS method of monitoring hydrogen or deuterium associated water for monitoring water motion in biological tissues. We have constructed physical models based on the spectral density of the bond vibrations and the power characteristics of pulsed lasers that document why femtosecond pulses are approximately 300 fold more effective in detecting the CARS signal than conventional picosecond pulses without a loss of discrimination between deuterium and hydrogen. We have also experimentally confirmed this is model and biological systems. This advance makes possible CARS monitoring of water motion in a variety of biological systems . 2) Using deuterium as a tracer we have established using CARS microscopy that the major barrier to water permeability, and thus where the largest pressure gradient is, is at the basolateral membrane of the endothelial cell. Thought the endothelial cell has been believed to play a role as the water barrier, the specific membrane had not previously been determined. We also demonstrated that the water channel protein aquaporin 1 is concentrated in the apical membrane of the endothelial cell, again consistent with this membrane in contact with the vascular space barrier being highly permeable to water. We suggest that this arrangement of the water permeability, very high in the apical membrane and very low at the basolateral membrane results in the pressure waves associated with the cardiac cycle passing through the body of the endothelial cell with little or no compression of this sensitive cell. However, at the interface of the endothelial cell basolateral membrane with the elastic macromolecules of the basement membrane and internal elastic lamina the arterial pressure wave is imparted on these macromolecules designed to withstand this pressure stress. Alterations in this water barrier and associated pressure gradients across the endothelial cell may play an important role in many vascular disease states.3) We are exploring microfluidic model systems to evaluate the use of CARS and other simple fluorescent dyes in the quantitation of water fluxes on the sub-micron scale, specifically evaluating the differences between proton conductance versus bulk water motion in these systems and then eventually in biological systems.