To verify the affected gene expression caused by SAHA regarding inflammation and angiogenesis, we measured cytokine levels in the HConF supernatant. The results of multiplex immunoassay indicated that SAHA actually increased IL-8 and MCP-1 protein levels, suggesting potential augmentation of the inflammatory response. In contrast, other inflammatory cytokines, CXCL1, CXCL6, and BMP2, were unaffected by SAHA. Previous studies demonstrated that two HDAC inhibitors, SAHA and trichostatin A (TSA), up-regulated IL-8 gene expression in NIH 3T3 cells by activating NF-κB signaling.
36,37 In contrast, there were other contradictory reports that HDAC inhibitors (including SAHA) have anti-inflammatory effects. For example, Wang et al.
38 reported that SAHA inhibited cytokine secretion (TNF-α, IL-8, IL-13, and IL-10) from human peripheral blood mononuclear cells and lymphocytes. Similarly, Choo et al.
39 reported that SAHA suppressed IL-1, IL-6, IL-18, and TNF-α secretion in LPS-stimulated THP-1 monocytic cells. Interestingly, TGF-β2 differentially affected IL-8 and MCP-1 levels in the presence of SAHA, although treatment with TGF-β2 alone did not significantly alter the IL-8 or MCP-1 levels; TGF-β2 significantly increased IL-8 levels in the presence of SAHA, whereas TGF-β2 significantly decreased MCP-1 levels. This discrepancy may be due to differences in their transcriptional regulation. IL-8 is regulated by the transcription factors NF-κB, activating protein (AP-1), CAAT/enhancer-binding protein β (C/EBPβ, also known as NF-IL-6), C/EBP homologous protein (CHOP), and cAMP response element binding protein (CREB),
40 whereas MCP-1 is regulated by NF-κB, AP-1, and Sp1.
41 As HDAC regulate the expression of not only histones, but also nonhistone proteins including various transcription factors, it is reasonable to suggest that treatment with TGF-β2 may result in differential expression of cytokines in the presence of SAHA. Thus, it is suggested that SAHA (and other HDAC inhibitors) have complex effects on inflammatory responses, probably depending on the experimental conditions and cell types. Although our results clearly showed that the levels of IL-8 and MCP-1 secretion by cultured HConFs were increased by SAHA treatments, previous animal experiments for GFS showed no inflammation by subconjunctival injection of SAHA.
16 This discrepancy may be explained by the fact that, in wound healing of GFS, inflammatory cytokines are produced mainly by macrophages, lymphocytes, and platelets, or that postoperative steroid treatments diminished the effect in the animal model. That is, minor production of inflammatory cytokines by fibroblasts may be masked by major secretion from infiltrated inflammatory cells and/or postoperative anti-inflammatory medication in animal experiments. However, as previous reports suggested complicated effects of SAHA on inflammatory responses, further studies are required to fully understand the role of SAHA in inflammatory response after GFS.