Methodological studies of eicosanoid enzyme gene expression in vascular endothelium exposed to biomechanical stress

Abstract: Prostaglandins are the major eicosanoids synthesized by vascular endothelial cells, and the vasculature is an important target organ for their bioactive metabolites. In vitro studies have shown that biomechanical forces are important regulators of vascular expression of prostanoid enzymes and release of metabolites. To extend the in vitro observations into the context of an intact blood vessel, we have used a new ex vivo perfusion model in which living conduit blood vessels can be studied under conditions of precisely defined biomechanical forces. However, gene expression studies are difficult to perform in such a model due to the limited amount of cells that can be retrieved from a small vessel preparation. In the present work, we have developed and validated two highly sensitive mRNA quantification methods based on assessment during the exponential phase of amplfication (capillary electrophoresis and real-time PCR). Furthermore, we evaluated the appropriateness of multiple reference genes to be used as endogenous controls in vascular gene expression studies.We have shown that shear stress and intraluminal pressure exert differential effect on prostacyclin synthesis-related enzymes. High shear induced the expression of cyclooxygenase-1 and cyclooxygenase-2 in a biphasic pattern with two peaks after 1.5 and 6 hours of stimulation. Elevated intraluminal pressure increased cyclooxygenase-2 expression, whereas cyclooxygenase-1 mRNA decreased by stimulation. Gene expressions of prostacyclin and thromboxane synthases were time-dependently induced by both stimuli. These effects were associated with changes in protein synthesis and metabolite formation. High shear stress increased formation of both prostacyclin and thromboxane A2, whereas elevated intraluminal pressure selectively suppressed prostacyclin release. Also, we found that shear and intraluminal pressure exerted distinct effects on immediate-early genes encoding proteins in the AP-1 transcription factor family, which could be involved in the differential transcriptional effects of biomechanical forces. Evaluation of multiple reference gene showed that several aspects must be considered in the selection of appropriate endogenous references, such as amplification efficiency, average expression levels, independence of experimental intervention, and systematic and error measurement variability. In conclusion, these findings show that mechanical forces have significant effects on expression of four key eicosanoid enzymes and formation of their metabolites in living human conduit blood vessels.