The mechanism(s) by which 7KCh induces inflammation have been studied in human fibroblasts,
54 human aortic and embryonic vascular endothelial cells (HUVECs),
8,16,54,55 cultured neuroretinal cells,
56 human monocytic U937 cells,
57–60 THP-1 cells,
44 aortic smooth muscle cells,
18,37,54,60,61 human macrophages,
20,38,62 and human RPE cells.
8,27 In aortic smooth muscle cells, 7KCh has been reported to induce the expression and activity of NOX-4, which in turn increases the formation of ROS.
37 In the mouse J774A.1 macrophage cell line, 7KCh was demonstrated to induce apoptosis by increasing ROS formation and caspase-3 activity.
62 ROS formation is known to activate a series of proapoptotic pathways (Bax, p53, p21, phosphorylated JNK, and others) and to downregulate antiapoptotic genes (BcL-2, BcL-xL, AKT).
38 In the human macrophage THP-1 cell line, 7KCh treatment increased the phosphorylation of p38 MAPK and ERK and decreased total AKT protein.
38 In cultured human aortic endothelial cells, ROS-dependent translocation of NFκB into the nucleus was observed 2 hours after 7KCh exposure.
39 In these cell types, the pharmacologic effects elicited by 7KCh seem to be preceded by the formation of ROS. The prevention of ROS formation by antioxidants such as β-carotene,
38,63 treatment with NOX-4 inhibitors (diphenyleneiodonium chloride, DPI), or siRNA targeting are sufficient to block all the downstream effects of 7KCh.
37 In the RPE-derived cells, ROS formation does not seem to be involved in the 7KCh-induced inflammatory response or cell death (
Fig. 3). ARPE-19 cells treated with 15 μM 7KCh failed to form ROS (
Fig. 3A) or to induce NOX-4 (
Fig. 3B). Moreover, treatment with N-acetyl-cysteine (a known ROS scavenger) failed to attenuate the 7KCh-induced VEGF response (
Fig. 3C). Other investigators have also previously demonstrated that 7KCh did not increase ROS formation in ARPE-19 cells, even at concentrations in excess of 100 μM.
31 To further verify this result, we measured ROS formation in RPE-J cells, HMVECs, and HAoSMCs using CoCl
2 as a positive control (
Fig. 4). 7KCh failed to induce ROS formation or NOX-4 induction (data not shown) in any of the four cell types tested. Other investigators using primary porcine RPE cells observed an increase in ROS formation in response to 7KCh
21 but concluded that the cytokine induction was mediated by LXR, not by ROS.