Here we showed an over-view of the cellular process altered in cytokine-activated brain endothelium using mRNA array analysis. Previous studies have shown that microvascular and macrovascular human endothelium have several differences in response to inflammatory stimulus however some similarities are also shared [12, 14]. In our analysis, we observed that some of the early changes in the gene expression pattern induced by TNFα in HUVECs [11, 12, 14] were also seen during long-term exposure to TNFα and IFNγ in hCMEC/D3 cells. These include the positive regulation of chemokines, CAMs, apoptosis genes, complement and coagulation cascades [11, 12, 14]. Furthermore, among the CAMs upregulated by TNFα and IFNγ we identified CEACAM-1, a molecule involved in angiogenesis and vascular permeability , although its role in brain endothelial barrier function have not been explored yet. In addition, we recently reported that the ability of CEC to respond to inflammatory stimuli by changing the pattern of gene expression is in part controlled by transcriptional activity [8, 9] and in part at the post-transcriptional level via small non coding RNAs termed microRNAs [16, 17]. For instance, miR-155 overexpression in hCMEC/D3 cells reflects the activated state of CEC induced by TNFα and IFNγ, these include upregulation of genes associated with antigen presentation, CAMs, complement pathways, cytokine activity and barrier breakdown .
One molecular mechanism associated with cytokine-induced endothelial barrier dysfunction is reorganization of both cytoplasmic and transmembrane JCM from the cell-cell contacts [8, 18]. Here, we observed by gene expression profiling in hCMEC/D3 cells that stimulation with TNFα and IFNγ for 24 h altered mRNA levels of several JCM including claudin-5 and MARVELD-2 (also known as tricellulin), both previously reported to be enriched in CECs . Indeed, we recently reported that changes in paracellular permeability and transendothelial electrical resistance correlated with changes in the expression of claudin-5 at the transcript and protein level [8, 9]. Another molecular mechanism associated with loss of endothelial barrier integrity is rearrangement of integrin-focal adhesion complexes [20, 21]. Our data supports the hypothesis that FA constitute a novel pathway that is critical for BBB maintenance  as many FA components were modulated by cytokines in CECs. For instance, integrin ß1 serves as a platform to establish focal contacts and is downregulated by cytokines in hCMEC/D3 cells. In addition, loss of integrin ß1 might also affect claudin-5 protein levels  and thus the brain endothelial barrier .
We have considered some potential criticisms of the interpretations given above. First, high concentrations of TNFα and IFNγ induce an increase in CEC paracellular permeability associated with caspase-3/-7 activation and apoptotic cell death . We cannot discard the possibility that some of the effects observed in this analysis might be secondary to a small loss of cell viability. However, our gene expression profile and the altered expression of BBB-associated genes in hCMEC/D3 cells after cytokine treatment correlate well with a recent report . A second possible drawback is that most of the changes in the gene expression pattern reported here need to be further validated. However, the altered levels observed in 12 genes after cytokine-treatment within 4 functional pathways were validated by qRT-PCR. We observed that three genes analyzed, ELMO1, ITGB1 and ABCB1, were always downregulated after cytokine-treatment, but their fold changes were variable. In addition, some of the functional consequences of TNFα and IFNγ on CEC have been reported. For instance, in agreement with our findings, Poller et al.  showed that TNFα reduced expression of BCRP (ABCG2) mRNA levels, protein levels and functional activity . Additionally, we previously demonstrated that CCL2, CXCL10 and CCL5 protein expression is upregulated by stimulation with TNFα and IFNγ in hCMEC/D3 cells  and that these chemokines have also been detected in MS plaques . Similar to our results Pan et al.  reported that TNFα induced an increased in IL-15 and its receptor, IL15RA, in rat brain endothelial cells suggesting an important role of this interleukin in the brain endothelial response to cytokines  that is conserved between species.
In summary, pro-inflammatory cytokines might alter the highly selective barrier permeability of brain endothelial cells by establishing a new pattern of gene expression. Changes in expression of CEC genes involve biological processes associated with regulation of leukocyte infiltration, inter-brain endothelial junctions, integrin-focal adhesions and transport systems. This analysis provides insight into key molecular and cellular processes altered during neuroinflammation.