Background: Endothelial heterogeneity represents the structural and functional ability of the endothelium to cater for the diverse needs of the underlying tissues in the vascular tree 1. TNF-α is an inflammatory cytokine which has been associated with endothelial dysfunction (ED) and hence cardiovascular disease 2.Objectives: To compare the in vitro TNF-α induced responses of two cell lines from distinct vascular sites, viz. cardiac microvascular endothelial cells (CMECs) and aortic endothelial cells (AECs).Methods: Adult rat (Rattus Norvegicus) CMECs and AECs were treated with 0.5, 5 and 20ng/ml TNF-α (24 and 48H). Nitric oxide (NO) and reactive oxygen species (ROS) were measured by DAF-2/DA and DHR fluorescence respectively. Cell viability was quantified by PI/Annexin-V fluorescence. The following proteins were measured by Western blotting: eNOS, PkB/Akt, heat shock protein 90 (HSP90), caveolin-1, nitrotyrosine and IkBα in cells treated with 5 and 20 ng/ml TNF-α (24h). All data are expressed as % of control (control:100%).Results: After 24H, AECs treated with 5ng/ml TNF-α showed significantly lower NO-production vs. control (AECs: 54±7.7% vs. Control, p<0.05). At 48h, all TNF-α concentrations significantly reduced NO-production in AECs and CMECs vs. controls; however, there were no differences in AECs vs. CMECs. TNF-α decreased DHR sensitive ROS in both cell lines vs. control, with a significantly greater reduction in CMECs vs. AECs. There were no differences in cell viability in either cell line after 24h. At 48h, CMECs showed higher % apoptosis vs. AECS, and 20ng/ml TNF-α significantly increased necrosis in CMECs (8.30±1.3%) vs. AECs (1.0±0.3%), p<0.05. At 20ng/ml TNF-α, phospho/total eNOS ratios were significantly lower in CMECs (68±5%) vs. AECs (94±10%); p <0.05. Although total PKB/Akt expression was significantly lower in CMECs vs. AECS (5ng/ml and 20ng/ml TNF-α), a significant reduction in phospho/total PKB/Akt ratio was only observed at 20ng/ml TNF-α (AECs: 94±4% vs. CMECs: 55±4%, p<0.05). HSP90 was decreased in both cell lines after TNF-α treatment vs. controls; with no differences observed in CMECs vs. AECs. IkBα expression was reduced in CMECs (5ng/ml: 70±2%; 20ng/ml: 50±10% vs. controls; p<0.05) and vs. AECs (5ng/ml:100±0.2%; 20ng/ml:75±10%; p<0.05). Caveolin -1 and nitrotyrosine levels remained unchanged.Conclusion: The PKB/Akt-eNOS pathway is a principal source of NO production in ECs 2. TNF-α resulted in a downregulation of this pathway in AECs and CMECs, which was more pronounced in CMECs. The TNF-α-induced attenuation of the PKB/Akt-eNOS pathway suggested an early dysfunctional response in our cells. The reduced expression of IkBα (more pronounced in CMECs) is a reflection of NFkB-activation, a major pro-inflammatory pathway3. Overall, CMECs appeared to be more susceptible to the harmful effects of TNF-α than AECs.