• AQP1
• atherosclerosis

Arterial endothelial aquaporin-1 expression and subendothelial intima thickness and its relevance to prelesion events leading to atherosclerosis
Teresa Liberto

Objective of the study

Motivated by the previous works around the atherosclerosis problem done by Prof. Rumschitzki team, this thesis attempts to experimentally explore the influence of increasing AQP1s on the intima thickness. To better understand the developments of this studies, following are detailed the most important results of these previous researches.
Huang et al. (A fiber matrix model for the filtration through fenestral pores in a compressible arterial intima, 1997) proposed a new hypothesis and develop a mathematical model to explain rationally the in vitro and in situ measured changes in the hydraulic conductivity of the artery wall of rabbit aorta with transmural pressure previously observed by Tedgui and Lever (Tedgui & Lever, 1984) and Baldwin and Wilson (Baldwin & Wilson, 1993).
Huang et al. claimed that the compaction due to pressure loading of the proteoglycans matrix in the arterial intima near fenestral pores of the internal elastic lamina (IEL) leads to a narrowing of the pore entrance area and a large decrease in local intrinsic Darcy permeability of the matrix. They proposed a local two-dimensional model to study filtration flow close to fenestral pores in a compressible intima.
This model provided several important predictions for the developing of the following work like that the hydraulic conductivity would decrease by one half if the thin intimal layer between the endothelium and the internal elastic lamina volume-compresses approximately fivefold. Hence the model supported the hypothesis that increase transmural pressure compacts the arterial intima near the IEL fenestral pores and causes the associated hydraulic conductivity changes. They also found a marked, nonlinear steepening of the radial intimal pressure profile near the fenestral pore when the intima significantly compacts. This suggested that the endothelium deforms near the fenestral pores at high luminal pressures and that the primary pressure drop occurs near the pore’s edge. The one-dimensional behavior exhibited in the intima for the intimal velocity and pressure distribution suggested that a one-dimensional approximation for these flow variables will be adequate in a more complex elastohydrodynamic model for a deformable intima to relate endothelial deformation to the local subendothelial pressure field.
Moreover a simple linear elastic behavior for intimal matrix compression is only suitable at low transmural pressures. This research of 1997 anticipated, on the basis of the experimental data, that the loose PG matrix first compresses at small transmural pressures ~75-100 mmHg. The matrix then exhibits a much stiffer behavior in which the collagen matrix predominantly carries the normal load. Huang et al (1997) Another fundamental work of Huang et al. (Structural changes in rat aortic intima due to transmural pressure, 1998) represented the first measurements of the effect of transmural pressure on intimal layer thickness and showed that the intimal matrix is, indeed, surprisingly compressible. Rat thoracic aortas were perfused in situ with 2 percent glutaraldehyde solution at 0, 50, 100, or 150 mm Hg lumen pressure and sectioned for light and electron microscopic observations. Electron micrographs showed a nonlinear decrease in average intimal thickness with the increase of the lumen pressure (0-150 mmHg). This study demonstrated that the arterial intima is indeed very compressible and that its percentage change in thickness is more than 20 times the relative change in medial thickness due to hoop tension assuming constant medial density. Even if the intimal layer comprising less than 1 percent of the vessel wall can have such a profound effect on the total wall's hydraulic conductivity. The combined effects of a large decrease in Darcy permeability due to intimal matrix compaction and a fivefold or greater narrowing of the entrance height to the fenestral pore openings far outweigh the small increase in fenestral pore area due to hoop tension and provide for an increased hydraulic resistance that is comparable to the entire media at pressures above the "critical pressure".
This entire effect is lost for a vessel denuded of its endothelium where the hydraulic conductivity Lp measurements in Tedgui and Lever (1984) and Baldwin and Wilson (1993) both show that Lp for a denuded vessel is independent of pressure. Recent studies of Prof. Rumschitzki group (Raval, 2012) (Shripad, 2012) have investigated the role of the plasma flow across the porous vessel wall. This flow is driven by the ΔP between inside and outside large arteries (Pi -Po ≈ 100 mmHg) and is composed mainly by water (the flow across the endothelium is detailed in the previous section). This studies has found the fundamental role of aquaporin (AQP) family in this water flow crossing and how the up-regulation of AQP1 expression with forskolin treatment also increases endothelial hydraulic conductivity (Lpe) in vessels and monolayers.
Shripad, in his work (A theory for how aquaporin-1 and trasmural pressure influence the mechanics of and the transport through the artery wall, 2012) extended Huang et al.’s (1997) local filtration model including the effects of transcellular water flow through AQP1 water channels.
This broad presupposition allows to appreciate the objective of this work.
The goal of this research is contribute at the previous results with an in vivo experiment with a direct measurement of the intima thickness at different pressure to investigated the effect of transmural blood pressure on intima compression in rat aorta. This study was performed on rat aorta pressurized at 50 and 100 mmHg perfused with a blank solution (DMSO) and a forskolin solution (DMSO with Forskolin). Forskolin, as said before, increased aquaporin. This research want to compare the compression of the intima and hence the hydraulic conductivity values, with and without functioning aquaporins.
Experimental steps
Briefly the goal of this thesis work is control the intima thickness at different pressure to investigated the effect of transmural blood pressure on intima compression in rat aortae. This study was performed on rat aortae pressurized at 50 and 100 mmHg perfused with a blank solution (DMSO) and a forskolin solution (DMSO with Forskolin). This perfusion differentiation was done to compare compression of the intima with and without functioning aquaporins at different transmural pressure.
In order to view the degree of intima compression, the following steps were taken. Ten different rat aortae were cultivated with the following pressures, three at 100 mmHg perfused with blank solution, two at 100 mmHg perfused with Forskolin solution, two at 50 mmHg perfused with blank solution, three at 50 mmHg perfused with Forskolin solution. The aortae were then placed in a glutaraldehyde solution to preserve the tissue. After the proper fixing, sectioning and staining protocol, cross sections of the aortae were viewed using a Transmission Electron Microscope, pictures were taken, and intima area was calculated and compared using a MATLAB program.