Stable local haemodynamic microenvironments may determine the phenotype of endothelial cells (EC) in different regions of the circulation, but acute changes in flow might also modulate functional responses. We aim to understand how different levels or patterns of shear stress applied to endothelial cells regulate inflammatory responses, and in particular, leukocyte recruitment. For this purpose, we developed models in which human EC of various types (HUVEC from umbilical veins; HUAEC from umbilical arteries; HCAEC from coronary arteries) were cultured in glass capillaries coated with desired substrates. These constructs were conditioned by different levels of shear stress for different periods, or exposed to abrupt changes in shear. Conditioning could be combined with treatment with cytokines such as tumour necrosis factor-α (TNF) and interleukin-1β (IL-1), and adhesion and migration of flowing neutrophils analysed as an ‘inflammatory’ readout. Initial studies showed that conditioning of HUVEC for 24h at increasing shear stress acted to powerfully suppress responses to TNF, but not IL-1, judged by neutrophil recruitment (Sheikh et al., 2003; 2005). However, in subsequent studies, responses to both cytokines were suppressed by shear conditioning for HUAEC and HCAEC. Studies in which culture medium constituents, such as basic fibroblast growth factor, were swapped, indicated that this difference between the endothelial cells arose from culture conditions rather than from an in vivo imprinted phenotype. The fact that the original ‘static’ cultures of each cell type showed similar abilities to support adhesion and migration of neutrophils also indicated that the phenotypes of EC were plastic and could be re-set by conditioning in vitro. Taking this further, we analysed expression of selected genes in HUVEC immediately after digestion from veins, after standard culture in vitro and then after shear conditioning. Changes were induced by initial culture, which were reversed in part at least by the return to a shear environment. Thus it seems that endothelial phenotype is highly pliable, with enviromental factors, such as shear stress and growth factors, modifying responses in an inter-linked but reversible manner. We thus investigated whether the less responsive state induced in vitro by shear stress would change when flow was ceased. This might be relevant to ischaemic conditions in vivo (for instance linked to thrombo-embolism, surgical interventions or organ transplantation), where an inflammatory response typically follows reperfusion. We found that response of EC to TNF only increased slowly over 24-48h after cessation of flow, and that if a very low level of shear stress was retained, then the response remained suppressed (Matharu et al.,2008). In all of the above, functional changes could be linked to changes in expression of receptors such as E-selectin and the shear-sensitive transcription factor KLF-2, and in activation of NFkB. However, anomalies in the correlations between the different responses indicated that other modulatory events occurred outside of these well-described mediators. Nevertheless, most of the changes noted were over hours and linked to modulation of gene expression. In studies of flow reduction, however, we also noted an early pro-adhesive response. In the period around 60-120min after cessation of flow, neutrophils adhered to otherwise unstimulated HUVEC, when included in medium used to ‘reperfuse’ it (Matharu et al., 2008). Others have shown that an early oxidative response follows flow cessation (Manevich et al., 2001). Here, the transient neutrophil adhesion observed was attributable to oxidant-induced upregulation of expression of P-selection on the EC. Taken together, these studies suggest that local conditioning of endothelial cells contributes to vessel- and organ-specificity in inflammatory responses, and predisposition of certain sites to development of inflammation. At the same time, acute responses of EC to disruption of flow may contribute to outcome of ischaemia and reperfusion.