Abstract
Abstract: :
Purpose:Retinal blood flow is controlled locally by vasodilators such as nitric oxide, prostacyclin and the vasoconstrictor endothelin–1.There is considerable evidence in pathologies associated with Normal Tension Glaucoma of some endothelial dysfunction, whereby the balance of these vasoactive agents are not adequately regulated, thus leading to vasopasm in response to cold–stimuli.As hemodynamic forces have been shown to have a profound influence on endothelium–mediated events, we hypothesize that expression and function of these vasoactive agents are controlled by mechanical forces due to blood flow. Methods:Using a perfused transcapillary co–culture system (Cellmax), co–cultures of bovine retinal endothelial cells (BREC) with bovine retinal pericytes were exposed to low and high pulsatile flow for 24 hours, corresponding to a flow rate of 0.3 mls/min (6mmHg pulse pressure; 0.5 dyn/cm2 shear stress) and 24 mls/min (56mmHg pulse pressure; 23 dyn/cm2 shear stress) respectively. Results:Using an antibody specific to phospho–serine1179–eNOS,we found BRECs pp–eNOS increased 1.9 fold from low to high pulsatile flow when harvested from a co–culture (n=3).Correlated with this increase in enzyme activity, using an assay for nitrate, we found a 2.12 fold (n=4) increase in nitrate levels in the media from low to high pulsatile flow. Previous studies have shown COX–2 is regulated by laminar shear stress at the cAMP responsive element, and there is also some evidence suggesting COX–1 is mechanically sensitive. Levels of prostacyclin in media samples were increased 2.1 fold (n = 4) from low to high pulsatile flow. ET–1 is a 21 kilodalton peptide which causes relaxation via Endothelin–B receptors and constriction via the predominant ET–A receptor found on pericytes. We found a 2.0 fold increase in media ET–1 levels in co–culture. It is well known that mitogen–activated protein(MAP) kinases play important roles in the intracellular signaling of a variety of agonists. We have investigated the involvement of MAP kinases (p38 & p44/p42 MAP kinase) in response to pulsatile flow and found no difference between both total & phosphorylated protein levels of each after 24 hours pulsatile flow. Increases in phoshorylated levels of p38 & p44/p42 MAP kinase were however found after 2 & 8.5 hours high pulsatile flow. Conclusions: These results indicate that pulsatile flow within the Cellmax co–culture system can modulate the vasoactive agents released from the endothelial cell layer which should in turn have a profound effect upon the pericyte layer.
Keywords: blood supply • vascular cells • nitric oxide