PD-L1 Expression Correlated with Clinicopathological Factors and Akt/Stat3 Pathway in Oral SCC.

Programmed cell death ligand 1 (PD-L1) is an immune checkpoint molecule that inhibits immune responses. The physiological and prognostic role of the PD-L1 signaling pathway in the oral maxillofacial region is unclear. This study aimed to investigate the role of PD-L1 in the progression of oral squamous cell carcinoma (OSCC). Furthermore, clinicopathological factors related to PD-L1 expression were examined in patients with OSCC through immunohistochemistry (IHC) of tissue sections and through an in vitro study in OSCC cells. The medical records, radiographic findings, and mortality referrals of 81 patients obtained from the National Statistical Office were reviewed. IHC was performed on tissue specimens of these patients to determine the expression levels of PD-L1, which showed significant statistical differences based on age, tumor size, TNM stage, cervical lymph node metastasis, and locoregional recurrence. Patients with a high PD-L1 expression had significantly poorer survival rates. Multivariate analysis using the Cox proportional model confirmed the high relative risk ratio for high PD-L1 expression, TNM stage, and neck node metastasis, all of which were significantly associated with a poor prognosis in patients with OSCC. The in vitro study showed that SAS and YD38 cells transfected with PD-L1 siRNA had significantly increased apoptosis, reduced proliferative capacity, and tumorigenicity.

SIRT1 inhibits monocyte adhesion to the vascular endothelium by suppressing Mac-1 expression on monocytes.

SIRT1 signaling pathways modulate vascular inflammation; however, the precise role of SIRT1 in monocyte adhesion to the vascular endothelium, a key event initiating vascular inflammation, is unclear. Thus, this study investigated the roles and molecular interaction of SIRT1 and TLR2 in regulating monocyte adhesion to the vascular endothelium. In vitro, both Mac-1 expression and the endothelial adhesion of THP-1 cells stimulated with Pam3CSK4, a TLR2 ligand, were markedly increased in association with a decreased expression of SIRT1. In THP-1 cells stimulated with Pam3CSK4, the promoter activity and expression of SIRT1 were decreased. The TLR2-dependent suppression of SIRT1 expression in THP-1 cells was mediated by the transcription factors NF-κB and CREB, suggesting that the TLR2-mediated NF-κB and CREB signaling downregulated SIRT1 expression in monocytes. In peripheral blood monocytes (PBMCs) isolated from SIRT1 transgenic (TG) mice and THP-1 cells treated with recombinant SIRT1, both the increased Mac-1 expression and endothelial adhesion induced by Pam3CSK4 were significantly attenuated. In addition, the en face immunohistochemical study showed a marked increase in monocyte adhesion to the aortic endothelium of WT mice treated with Pam3CSK4, which was significantly attenuated in Pam3CSK4-treated SIRT1 TG mice. Moreover, a greater number of atherosclerotic plaques formed in WT mice fed a high-fat diet than in SIRT1 TG mice, indicating a pivotal role for SIRT1 in preventing vascular inflammation. Based on these results, SIRT1 might be a potential target for researchers aiming to develop therapeutic interventions for vascular inflammation, including atherosclerosis.

HMGB1 Increases IL-1ß Production in Vascular Smooth Muscle Cells via NLRP3 Inflammasome.

Vascular smooth muscle cells (VSMCs) are the major cell type in the blood vessel walls, and their phenotypic modulation is a key cellular event driving vascular remodeling. Although high mobility group box-1 (HMGB1) plays a pivotal role in inflammatory processes after vascular injuries, the importance of the links between VSMCs, HMGB1 and vascular inflammation has not been clarified. To prove the hypothesis that VSMCs might be active players in vascular inflammation by secreting inflammatory cytokines, we investigated the proinflammatory effects of HMGB1 and its intermediary signaling pathways in VSMCs. When cultured human VSMCs were stimulated with HMGB1 (10-500 ng/ml), IL-1ß production was markedly increased. HMGB1 also increased the expression of NLRP3 inflammasome components including NLRP3, ASC and caspase-1. Among these components, HMGB1-induced expressions of NLRP3 and caspase-1 were markedly attenuated in TLR2 siRNA-transfected cells, whereas ASC and caspase-1 expressions were reduced in RAGE-deficient cells. In TLR4-deficient cells, HMGB1-induced caspase-1 expression was significantly attenuated. Moreover, IL-1ß production in HMGB1-stimulated cells was significantly reduced in cells transfected with caspase-1 siRNA as well as in cells treated with monoclonal antibodies or siRNAs for TLR2, TLR4 and RAGE. Overall, this study identified a pivotal role for NLRP3 inflammasome and its receptor signaling involved in the production of IL-1ß in VSMCs stimulated with HMGB1. Thus, targeting HMGB1 signaling in VSMCs offers a promising therapeutic strategy for treating vascular remodeling diseases.

5-Lipoxygenase in monocytes emerges as a therapeutic target for intimal hyperplasia in a murine wire-injured femoral artery.

Given the importance of leukotrienes in vascular inflammation induced by local tissue injury, this study investigated the role for 5-lipoxygenase (5-LO) in monocytes in the development of intimal hyperplasia. As a mechanistic study, the importance of monocyte 5-LO in monocyte-macrophage differentiation with subsequent infiltration in neointima was evaluated. In a mouse model of wire-injured femoral artery, intimal hyperplasia started as early as 2wks after injury, and luminal area and blood flow were reduced due to increased neointima formation. Time-dependent increases in macrophage infiltration were observed in neointima and showed a positive relationship with neointima volume. In 5-LO-deficient (KO) mice or wild-type (WT) mice treated with an inhibitor of 5-LO activating protein (MK886, 1 and 10mg/kg), intimal hyperplasia and macrophage infiltration into neointima were reduced, but monocyte adhesion to injured luminal surface was not inhibited, which suggested 5-LO participates in monocyte-macrophage differentiation. In an in vitro study, monocyte-macrophage differentiation was found to be increased by high mobility group box 1 protein (HMGB1), but this effect was attenuated in cells isolated from 5-LO-KO mice. Furthermore, macrophage infiltration and intimal hyperplasia were more prominent in 5-LO-KO mice transplanted with monocytes from WT mice than in 5-LO-KO mice transplanted with monocytes from 5-LO-KO mice. Taken together, it was suggested that 5-LO in monocytes played a pivotal role in monocyte-macrophage differentiation and subsequent infiltration of macrophage in neointima, leading to vascular remodeling after vascular injury.

α-Iso-Cubebene Inhibits PDGF-Induced Vascular Smooth Muscle Cell Proliferation by Suppressing Osteopontin Expression.

α-Iso-cubebene (ICB) is a dibenzocyclooctadiene lignin contained in Schisandra chinensis (SC), a well-known medicinal herb that ameliorates cardiovascular symptoms. Thus, we examined the effect of ICB on vascular smooth muscle cell (VSMC) proliferation, a key feature of diverse vascular diseases. When VSMCs primary cultured from rat thoracic aorta were stimulated with PDGF (1-10 ng/ml), cell proliferation and osteopontin (OPN) expression were concomitantly up-regulated, but these effects were attenuated when cells were treated with MPIIIB10, a neutralizing monoclonal antibody for OPN. In aortic tissues exposed to PDGF, sprouting VSMC numbers increased, which was attenuated in tissues from OPN-deficient mice. Furthermore, VSMC proliferation and OPN expression induced by PDGF were attenuated dose-dependently by ICB (10 or 30 µg/ml). Reporter assays conducted using OPN promoter-luciferase constructs showed that the promoter region 538-234 bp of the transcription start site was responsible for transcriptional activity enhancement by PDGF, which was significantly inhibited by ICB. Putative binding sites for AP-1 and C/EBPß in the indicated promoter region were suggested by TF Search, and increased binding of AP-1 and C/EBPß in PDGF-treated VSMCs was demonstrated using a ChIP assay. The increased bindings of AP-1 and C/EBPß into OPN promoter were attenuated by ICB. Moreover, the PDGF-induced expression of OPN was markedly attenuated in VSMCs transfected with siRNA for AP-1 and C/EBPß. These results indicate that ICB inhibit VSMC proliferation by inhibiting the AP-1 and C/EBPß signaling pathways and thus downregulating OPN expression.

Osteopontin plays a key role in vascular smooth muscle cell proliferation via EGFR-mediated activation of AP-1 and C/EBPß pathways.

Osteopontin (OPN) is known as an active player in the progression of vascular remodeling diseases, however, the precise role in the proliferation of vascular smooth muscle cells (VSMC) is unclear. Thus, this study investigated the role of OPN in VSMC proliferation induced by 4-hydroxynonenal (HNE), and identified the intracellular signaling pathways involved in 4-HNE-induced OPN production. In VSMC primary cultured from rat thoracic aorta as well as in VSMC in the media of aorta, HNE enhanced OPN expression in concentration-dependent manners. Both the proliferation of cultured VSMC and PCNA positive cells in aortic tissues were also increased by HNE, which were attenuated in OPN-deficient cells and aortic tissues isolated from OPN-deficient mice, indicating a pivotal role of OPN in HNE-induced VSMC proliferation. In the promoter assay, HNE increased OPN promoter activity, which was attenuated when the regions harboring AP-1 and C/EBPß binding sites were mutated. The increased bindings of AP-1 and C/EBPß to the OPN promoter were also demonstrated by ChIP analysis. In addition, the increases in both OPN expression and the activities of AP-1 and C/EBPß by HNE were attenuated by AG1478, an EGFR antagonist. Based on these results, it was suggested that HNE induced OPN expression in VSMC via signaling pathways involving AP-1 and C/EBPß, leading to increases in VSMC proliferation and subsequent vascular remodeling.