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.
