The culturing process rapidly erases differential arteriovenous gene expression. HUAECs and HUVECs lose their differential expression profile, including that of TFs, upon culture. (A) Hierarchical clustering analysis for freshly isolated or cultured HUAECs (red) or HUVECs (blue) revealing that for fresh samples replicates of each cell type tightly cluster together and both clusters are nicely separated. For cultured samples, incorrect clustering occurred. (B) Diagram representing the probe set intensity difference between HUVECs and HUAECs for each probe of the arteriovenous fresh profile (showing arterial probes [A] on the left and venous probes [V] on the right) for cultured (filled diamonds) or freshly isolated (open triangles) cells, revealing only minor differences in cultured cells. (C) Diagram representing the expression (±SEM; n = 4) determined by qRT-PCR for arterial TFs in freshly isolated cells (open triangles) or cultured cells (filled diamonds) relative to their expression in freshly isolated cells. *P < .05 vs freshly isolated cells. (D-E) Diagrams showing probe set intensity (±SEM; n = 4) for arterial (A) and venous (V) probes of the arteriovenous fresh profile for cultured (filled diamonds) or freshly isolated (open triangles) HUAECs (D; symbols in red) or HUVECs (E; symbols in blue). (F) Diagram showing that the average probe set intensity for all arterial probes (dashed lines) or venous probes (full lines) in freshly isolated HUAECs (left; red) or HUVEC (right; blue) “bleach” to a default average expression level (gray dashed line) upon culturing (middle; pink). *P < .05 vs fresh. NS, not significant vs fresh (n = 4-5).