In hypertension do smaller holes in arterial internal elastic lamina lead to fewer routes for myoendothelial junctions and hence less EDHF response?
PhD thesis, University of Glasgow.
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The work presented in this thesis describes the influence of the endothelium on smooth muscle cells, and how the structure of the internal elastic lamina (IEL) affects this relationship in mesenteric and saphenous arteries. This was enabled by the study of functional and confocal microscopy dye transfer experiments. Normotensive (WKY) and hypertensive (SHR) rats of 12 weeks and 6 months of age were used to assess the effect of hypertension and ageing on endothelial and smooth muscle cell communication.
The endothelium-derived hyperpolarising factor (EDHF) response in mesenteric arteries was investigated using wire myography, and the involvement of myoendothelial gap junctions (MEGJs) was assessed using the putative gap junction inhibitor carbenoxolone. Carbenoxolone attenuated the EDHF response in the WKY, suggestive of the involvement of myoendothelial gap junctions in EDHF. In the saphenous artery, incubation with L-NAME and indomethacin abolished the relaxation to ACh, indicating that there was no EDHF response in this artery.
Dye transfer experiments using luminally loaded calcein-AM and the gap junction blocking peptides (GJi) 37,43Gap27 and 40Gap27 in mesenteric arteries demonstrated that the dye moved from endothelial cells to smooth muscle cells by means of gap junctional transfer in the WKY. In the SHR, calcein staining in the smooth muscle showed a significant reduction to that observed in the WKY at both age points (with endothelial cell staining remaining constant between strains), and GJi incubation did not reduce smooth muscle cell staining from that of the control. Dye transfer was correlated with the EDHF function of the vessels, and was consistent with the increasing evidence supporting the role of MEGJS in the EDHF response. No conclusive evidence could be obtained from saphenous artery imaging of dye transfer or fenestrae area due to its unsuitability for this approach. However, functional experiments illustrated this artery (reported to have a lack of MEGJs) to be devoid of an EDHF response, thereby making it a potential control artery for the mesenteric artery.
Differences in the organisation of elastin in the IEL were assessed by measurement of fenestrations in the IEL, which are the only points through which endothelial and smooth muscle cells can connect. In WKY, the fenestrae appeared to fuse with age, causing a parallel increase in fenestrae area and decrease in fenestrae number which was not apparent in the SHR due to the inability of fenestrae to fuse in this strain. The smaller fenestrae area in the 6 month old SHR is thought contribute to the reduced EDHF response compared with WKY.
Collectively, the results of this study imply that there is a relationship between the EDHF response and myoendothelial coupling within an artery. EDHF response and smooth muscle cell staining following luminal calcein-AM application were reduced in the 6 month old SHR compared with aged-matched WKY. The smaller fenestrae in the IEL of the 6 month old SHR is a possible explanation as to why there is reduced cellular coupling, as this would restrict the passage of substances or charge between the endothelium and smooth muscle, be it by gap-junctional transfer or by diffusion.
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