(253d) "Aquaporin-1: Its Presence, Distribution and Quantification in Arterial Endothelium and How These Change with Chronic Hypertension – with Relevance to Early Atherogenesis"

Atherosclerosis is responsible for over 50% of all deaths in the US and in all Western countries. The earliest events of atherosclerosis occur when pressure-driven convection transports low-density lipoproteins (LDLs) from the blood into the subendothelial intimae (SI) of high-pressure, large and intermediate sized arteries. Lipid binding to SI extracellular matrix appears to begin the cascade of events that lead to atherosclerosis, where these vessels subsequently narrow and harden, whose consequences can include stroke and/or heart attack. Over short time scales (minutes), studies have shown that macromolecular tracers focally cross the endothelium of these vessels via transient leaks that are associated with the junctions around rare (~1 cell every few thousand) endothelial cells that are temporarily not tight (lifetime~1 hr); some of these leaks are associated with cells that are either dying or dividing. Thus the transmural pressure-driven water flow both advects these tracers into the SI, where they can bind to extracellular matrix and dilutes unbound lipid in the SI, thereby decreasing the kinetics of such binding, and sweeps unbound lipid from the SI deeper into the wall. Thus the nature of this overall convective trans-wall water flux, not just the part through the rare widened junctions, is of critical interest. It has generally been accepted that water crosses the endothelium paracellularly, that is, through both the tight and leaky junctions between endothelial cells. Since 1990, however, a family of ubiquitous transmembrane proteins called aquaporins (AQP), which very efficiently facilitate transmembrane water transport appear to do so with great specificity and little or no ATP cost, has been identified. It is therefore natural to ask whether AQPs are present in arterial endothelial cells and, if so, if their presence implies a transcellular contribution in transmural water transport. In that case, vessels might have the capacity to regulate their hydraulic conductivity, the ratio of transmural water flux to pressure difference, actively by control of their AQP expression levels. This would render trans-endothelial water transport not simply a passive process, that goes up and down with a changing transmural pressure, but the subject of active control that would, by implication, affect the further transport of LDL that had already crossed the SI paracellularly through leaky junctions and in its kinetics of binding to SI ECM. We previously reported our use of immunohistochemistry to identify and show the existence of Aquaporin-1 (AQP1) on both the luminal and abluminal membranes of whole vessel rat aortic endothelial cells. We also showed evidence that either blocking aquaporins chemically or knocking down their expression reduces endothelial hydraulic conductivity, both in cultured monolayers and in whole vessels ex vivo. A known risk factor for atherosclerosis is hypertension and we postulated that one external condition that may influence a vessel's AQP expression is its transmural pressure. We then confirmed this hypothesis in both normotensive Wistar Kyoto rats and their genetically modified hypertensive cousins, the Spontaneously Hypertensive rats (SHR), i.e., by showing that endothelial cells from the chronically hypertensive rats appear to express far more AQP than their normotensive analogues. Non-genetically modified normotensive (Sprague-Dawley rats (no operation), Sprague-Dawleys that have undergone a sham operation and hypertensive (Sprague-Dawleys due to having undergone the Goldblatts procedure that induces the rennin-angiotensin pathway to hypertension) showed qualitatively similar results. This evidence, in aggregate, supports the hypothesis that aortic endothelial cells may be able to actively regulate their aquaporin expression in response to chronic hypertensive conditions so as to control their transmural transport processes.

In this present study, we present simple kinetic models based on the Law of Mass Action to describe molecular transcription and translation processes that describe our observed transmural pressure-induced differential AQP1 expression. This mechanism assumes that changes in transmural pressure impact the transcription factors that serve as a regulatory point for the control of gene expression. We first examine the model's steady states for different transmural pressures and compare with data from our lab. From these data we extract certain products of model kinetic parameters and find ranges of the individual parameters themselves. We then extend our findings by numerically solving (using Matlab® 7.3.0) the dynamical version of our model. In particular, we predict the time dependence of the concentration of transcription factors, mRNA and AQP1 in response to step changes in transmural pressure. We also postulate that siRNA against AQP1 mRNA destroys or degrades these mRNAs with second order kinetics, and in the process themselves are consumed. This loss of mRNA subsequently affects gene expression until the siRNA concentration is severely depleted and the mRNA and its protein can recover. We solve for the corresponding dynamics of AQP1 reduction upon with siRNA introduction and its subsequent recovery with siRNA depletion.

Improved understanding of AQP1 regulation by, e.g., transmural pressures, may lead to novel therapies, not just in the case of atherosclerosis, but also for a wide variety of human diseases.