Supplementary Materials Supporting Information supp_293_19_7195__index. in GMVECs than in BMVECs, coupled with an increase in C3aR production in TNF-stimulated GMVECs, provides a possible explanation for the predominance of renal damage, and the absence of cerebral injury, in individuals with episodes of aHUS and TA-TMA. indicate the deletions of complement factor H-related genes 1 and 3. host disease????(deletion; plus autoantibody to FH) Open in a separate window Infectious/inflammatory events have been demonstrated to trigger initial or recurrent episodes of aHUS and TA-TMA (6, 47, 48, 50,C52). Furthermore, TNF has been demonstrated to be elevated in aHUS and TA-TMA patients clinically (53, 54). We have previously shown that TNF contributes to AP activation in human glomerular microvascular endothelial cells (GMVECs), as demonstrated by higher levels of activation products C3a, Ba, and C5a in these cells compared with levels in HUVECs after TNF stimulation (55). We also found that TNF caused substantial down-regulation of GMVEC expression, resulting in diminished PC activation by CD141-bound thrombin (55). The vulnerability of the kidney to AP-mediated injury in aHUS and TA-TMA led us to hypothesize that there is a difference in AP activation and regulation in GMVECs (the cell type that is predominantly involved in these two types of TMA) compared with brain microvascular endothelial cells (BMVECs), a microvascular endothelial cell type that is not affected. To achieve our objectives, we compared AP activation and regulation in GMVECs and in BMVECs that were either unstimulated or, as a model for inflammation/infection, stimulated by TNF. These MVECs serve as ideal models for our studies as they produce and secrete all AP components and regulators, as well as VWF (18, 55). Results Gene expression of AP components in unstimulated and TNF-stimulated BMVECs relative to GMVECs We compared in BMVECs and GMVECs the expression of genes that encode essential proteins in the activation of the AP: as a control. Unstimulated GW788388 distributor BMVECs had 14-fold higher GW788388 distributor mRNA levels for and compared with levels in unstimulated GMVECs. Unstimulated BMVECs had 4-fold lower mRNA levels for both and compared with unstimulated GMVECs (Fig. 1and were 7-, 4-, 5-, and 8-fold higher, respectively, and expression levels were 3-fold lower in TNF-stimulated BMVECs compared with TNF-stimulated GMVECs (Fig. 1and the classical complement component in unstimulated BMVECs and GMVECs (was used for normalization. *, 0.05; **, 0.001. Quantitative gene expression of AP components by TNF-stimulated GMVECs and BMVECs We additionally quantified changes in gene expression of each AP component in BMVECs and GMVECs after exposure to TNF from unstimulated gene levels in each MVEC type (Fig. 2). AP component expression of both cell types changed with TNF stimulation in a similar pattern (but not in magnitude). Both GMVECs and BMVECs had increased gene expression of (150- and 50-fold, respectively) and of (60- and 80-fold, respectively) and reduced expression of (10- and 2-fold respectively). expression changed minimally in GMVECs and BMVECs with TNF stimulation ( 2-fold increases), and and mRNA levels did not change substantially in either cell type with TNF. Open in a separate window Figure 2. Quantitative gene expression of AP components by TNF-stimulated GMVECs and BMVECs. The mRNA levels of the genes for the AP components and the classical complement component in TNF-stimulated BMVECs and GMVECs were quantified relative to the same genes in unstimulated BMVECs and GMVECs. RNA was extracted from unstimulated BMVECs and GMVECs that were maintained in serum-free medium for 24 h and in these cells after incubation with 10 ng/ml TNF for 48 h (24 h in complete medium and 24 h in serum-free medium). Fold changes in TNF-stimulated BMVEC and GMVEC mRNA levels were calculated relative to levels in unstimulated MVECs. RNA was extracted in 4C6 separate experiments from each cell type. Data are means plus standard deviations (S.D.) from RT-qPCR runs with triplicate measurements. was used for normalization. Gene expression of AP surface and soluble regulatory protein genes in unstimulated and TNF-stimulated BMVECs relative to GMVECs Gene expression levels of surface (and and and Table 2). TNF-stimulated BMVECs also had higher expression levels of all five AP regulatory protein genes studied relative to TNF-stimulated GMVECs, and TNF magnified the relative differences for and and Table 2). Open in a separate window Figure 3. Gene expression of AP surface and soluble regulatory protein genes in unstimulated and TNF-stimulated BMVECs relative to GMVECs. The mRNA levels of the genes for three membrane-bound regulatory receptors (and was used for normalization. *, GW788388 distributor 0.05; **, 0.001. Table 2 BMVEC complement regulatory gene expression levels relative to GMVECs Summary of regulatory gene expression levels in MMP9 unstimulated and TNF-stimulated BMVECs relative to levels in unstimulated and TNF-stimulated GMVECs. gene expression was higher in BMVECs compared with GMVECs (Fig. 3). A possible explanation for this enigma is that the mAb used to detect surface CD46 does.