The formation of new lymphatic vessels provides an additional route for tumour cells to metastasize. Therefore, inhibiting lymphangiogenesis represents an interesting target in cancer therapy. First evidence suggests that histone deacetylase inhibitors (HDACi) may mediate part of their antitumor effects by interfering with lymphangiogenesis. However, the underlying mechanisms of HDACi induced anti-lymphangiogenic properties are not fully investigated so far and in part remain unknown. Human lymphatic endothelial cells (LEC) were cultured in vitro and treated with or without HDACi. Effects of HDACi on proliferation and cell cycle progress were analysed by BrdU assay and flow cytometry. Apoptosis was measured by quantifying mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates. In vitro lymphangiogenesis was investigated using the Matrigel short term lymphangiogenesis assay. The effects of TSA on cell cycle regulatory proteins and apoptosis-related proteins were examined by western blotting, immunofluorescence staining and semi-quantitative RT-PCR. Protein- and mRNA half-life of p21 were analysed by western blotting and quantitative RT-PCR. The activity of the p21 promoter was determined using a dual luciferase assay and DNA-binding activity of Sp1/3 was investigated using EMSA. Furthermore, siRNA assays were performed to analyse the role of p21 and p53 on TSA-mediated anti-lymphangiogenic effects. We found that HDACi inhibited cell proliferation and that the pan-HDACi TSA induced G0/G1 arrest in LEC. Cell cycle arrest was accompanied by up-regulation of p21, p27 and p53. Additionally, we observed that p21 protein accumulated in cellular nuclei after treatment with TSA. Moreover, we found that p21 mRNA was significantly up-regulated by TSA, while the protein and mRNA half-life remained largely unaffected. The promoter activity of p21 was enhanced by TSA indicating a transcriptional mechanism. Subsequent EMSA analyses showed increased constitutive Sp1/3-dependent DNA binding in response to HDACi. We demonstrated that p53 was not required for TSA induced p21 expression and growth inhibition of LECs. Interestingly, siRNA-mediated p21 depletion almost completely reversed the anti-proliferative effects of TSA in LEC. In addition, TSA induced apoptosis by cytochrome c release contributed to activating caspases-9, −7 and −3 and downregulating the anti-apoptotic proteins cIAP-1 and −2. In conclusion, we demonstrate that TSA - a pan-HDACi - has distinct anti-lymphangiogenic effects in primary human lymphatic endothelial cells by activating intrinsic apoptotic pathway and cell cycle arrest via p21-dependent pathways.

Background: Patients with chronic inflammatory diseases (e.g. psoriasis and rheumatoid arthritis) are at increased risk for the development of atherosclerosis and cardiovascular diseases (CVD). Previous studies have suggested that phosphodiesterase 4 (PDE4) inhibitors possess anti‐inflammatory properties.Objectives: Here we examined the effect of the PDE4 inhibitor apremilast, a well‐established anti‐psoriatic drug, on pro‐inflammatory responses in TNFα‐activated endothelial cells.Methods: Human umbilical vein endothelial cells (HUVEC) were treated with tumour necrosis factor‐α (TNFα) in the presence or absence of apremilast. Expression levels of pro‐inflammatory cytokines, chemokines and adhesion molecules were assessed by ELISA, western blot and RT‐PCR. Effects of apremilast on adhesion and transendothelial migration (TEM) of THP‐1 monocytic cells were analysed in transwell assays.Results: Apremilast suppressed TNFα‐induced expression and secretion of important endothelial and monocytic pro‐inflammatory factors, including granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), C‐X‐C motif chemokine ligand 10 (CXCL10), chemokine (C‐C motif) ligand 2 (CCL2), vascular cell adhesion molecule 1 (VCAM‐1), E‐selectin and matrix metalloproteinase‐9 (MMP9). Functionally, apremilast reduced adhesion of THP‐1 cells to activated HUVECs and TEM in response to TNFα. Mechanistically, apremilast suppressed activation of nuclear factor κB (NFκB) and mitogen‐activated protein kinases (MAPK) signalling in activated HUVECs. Furthermore, inhibition of p38, C‐Jun‐N‐terminale Kinase (JNK) and NFκB in activated HUVECs decreased expression of GM‐CSF, VCAM‐1 and E‐selectin. Additionally, apremilast decreased IL‐17A‐induced secretion of IL‐6 and CCL2.Conclusions: We demonstrate that apremilast has distinct anti‐inflammatory effects in activated HUVECs, indicating that apremilast could have the therapeutic potential to prevent higher risk for CVD in patients with chronic inflammatory diseases.