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The importance of the vascular endothelium in the regulation of cardiovascular homeostasis has become more and more apparent because of an increasing quantity of data. The endothelial activation und dysfunction plays an important role in the pathogenesis of numerous cardiovascular diseases. To fulfill their diverse functions endothelial cells express a multitude of membrane ion channels. The control of the calcium influx and the modulation of the membrane potential of the endothelial cell are therefore of particular importance. The alteration of the intracellular calcium concentration results from release of calcium from the intracellular space as well as via calcium influx from extracellular. The extracellular calcium influx is thereby, among others, mediated by non-selective cation channels. These include the 28 human members of the TRP family. The importance of TRP channels in human endothelial cells is not yet completely understood.
The aim of the present study was to examine systematically the expression of all human TRP channels in human dermal microvascular endothelial cells (HMEC-1) using the Real-Time RT-PCR, a
well-established method. GAPDH was used as a house-keeping gene, the expression of the endothelial marker vWF was likewise studied. To verify the results of every carried out RT-PCR, a gel electrophoresis was performed for every RT-PCR. In addition the quality of every primer was validated by applying linearity analyzes. The expression of the mRNA of TRPC1, TRPC4, TRPC6, TRPC7, TRPV1, TRPV2 and TRPV4, TRPM4, TRPM5, TRPM6, TRPM7, TRPM8 as well as TRPA1, TRPML2, TRPP1 and PKD2like1 could be demonstrated. The notably high expression of TRPC1, TRPC4, TRPC6, TRPM6, TRPM7, TRPM8 and TRPP1 was noteworthy.
Little is known about activation and modulation of TRP channels in human endothelial cells. Therefore, using RT-PCR, HMEC-1 were subsequently stimulated with different vasoactive (bradykinin, histamine, endothelin-1) and proangiogenic factors (VEGF, erythropoetin) to investigate whether these modulate the expression of TRP channels in HMEC-1. A gel-electrophoresis confirmed the results of the RT-PCR. A significant higher expression of TRPM8 after stimulation of HMEC-1 with VEGF as well as a significant higher expression of TRPM4, TRPM5 and TRPM8 after stimulation with bradykinin was able to be demonstrated.
In conclusion the results of this underlying study confirm that the human microvascular endothelial cells HMEC-1 express specific TRP channels. In addition it was able to be shown that some of the
TRP channels (TRPM4, TRPM5, TRPM8) have a significantly higher expression after endothelial stimulation. Hence, on the basis of endothelial stimulation it was demonstrated that vasoactive and proangiogenic factors can modulate the expression of TRP channels in the endothelium. In contrast to all expectations an extraordinary expression of certain TRP channels after stimulating despite statistical significance HMEC-1 with diverse substances was not shown.
The deeper understanding of the physiological function of the endothelium and of the vascular system in general provides the key to understanding pathophysiological processes and discloses new therapeutically perspectives. In the future, because of their complex functions in the cardiovascular system, TRP channels may gain importance for medical drug development.