T cell-mediated immunity plays a significant role in the development of atherosclerosis (AS). There is increasing evidence that CD8+ T cells are also involved in AS but their exact roles remain unclear. The inhibitory receptors programmed cell death-1 (PD-1) and T cell immunoglobulin and mucin domain 3 (Tim-3) are well known inhibitory molecules that play a crucial role in regulating CD8+ T cell activation or tolerance. Here, we demonstrate that the co-expression of PD-1 and Tim-3 on CD8+ T cells is up-regulated in AS patients. PD-1+ Tim-3+ CD8+ T cells are enriched for within the central T (TCM) cell subset, with high proliferative activity and CD127 expression. Co-expression of PD-1 and Tim-3 on CD8+ T cells is associated with increased anti-atherogenic cytokine production as well as decreased pro-atherogenic cytokine production. Blockade of PD-1 and Tim-3 results in a decrease of anti-atherogenic cytokine production by PD-1+ Tim-3+ CD8+ T cells and in an augmentation of TNF-α and IFN-γ production. These findings highlight the important role of the PD-1 and Tim-3 pathways in regulating CD8+ T cells function in human AS.

Angiogenesis is a process of development and growth of new capillary blood vessels from pre-existing vessels. Angiogenic growth factors play important roles in the development and maintenance of some malignancies, of which vascular endothelial growth factor (VEGF)/VEGFR2 interactions are involved in proliferation, migration, and survival of many cancer cells. The aim of this study was to investigate the function of VEGFR2 in human hemangiomas (HAs). Using immunohistochemistry assay, we examined the expression levels of VEGF, VEGFR2, Ki-67, glucose transporter-1 (Glut-1), phosphorylated protein kinase B (p-AKT) and p-ERK in different phases of human HAs. Positive expression of VEGF, VEGFR2, Ki-67, Glut-1, p-AKT and p-ERK was significantly increased in proliferating phase HAs, while decreased in involuting phase HAs (P=0.001; P=0.003). In contrast, cell apoptotic indexes were decreased in proliferating phase HAs, but increased in involuting phase HAs (P<0.01). Furthermore, we used small hairpin RNA (shRNA)-mediated VEGFR2 knockdown in primary HA-derived endothelial cells (HemECs) to understand the role of VEGF/VEGFR2 signaling. Knockdown of VEGFR2 by Lv-shVEGFR2 inhibited cell viability and induced apoptosis in primary HemECs companied with decreased expression of p-AKT, p-ERK, p-p38MAPK and Ki-67 and increased expression of caspase-3 (CAS-3); Overexpression of VEGFR2 promoted cell viability and blocked apoptosis in Lv-VEGFR2-transfected HemECs. Taken together, our findings demonstrate that, increased expression of VEGFR2 is involved in the development of primary HemECs possibly through regulation of the AKT and ERK pathways, suggesting that VEGFR2 may be a potential therapeutic target for HAs.

MicroRNAs (miRNAs) exhibit a crucial role in the regulation of angiogenesis and tumor progression, of which miR-199a-5p (miR-199a) has been reported to function as a tumor suppressor in multiple malignancies. However, the precise mechanisms underlying miR-199a in hemangiomas (HAs) remain elusive. In this study, we found that miR-199a had low expression level, while proliferating cell nuclear antigen (PCNA) had high expression level in proliferating-phase HAs compared with the involuting-phase HAs and normal tissues. Spearman correlation analysis revealed the negative correlation of miR-199a with PCNA expression in proliferating-phase HAs. In vitro experiments showed that restoration of miR-199a suppressed cell proliferation capability and induced cell apoptosis in HA-derived endothelial cells (HDEC) and CRL-2586 EOMA cells, followed with decreased PCNA expression and increased cleaved caspase-3 expression, but miR-199a inhibitor reversed these effects. Furthermore, HIF1A was identified as a target of miR-199a and had negative correlation with miR-199a expression in proliferating-phase HAs. Overexpression of HIF1A attenuated the anti-proliferation effect of miR-199a mimic in HAs cells. Taken together, our findings demonstrate that miR-199a may inhibit proliferation and induce apoptosis in HAs cells via targeting HIF1A and provide a potential therapeutic target for HAs.

Background/Aims: Dysregulation of long noncoding RNAs (lncRNAs) is associated with the proliferation and metastasis in a variety of cancers, of which lncRNA maternally expressed gene 3 (MEG3) has been indicated as a tumor suppressor in multiple malignancies. However, the underlying mechanisms by which MEG3 contributes to human hemangiomas (HAs) remain undetermined. Methods: qRT-PCR analysis was performed to examine the expression levels of MEG3 and VEGF in proliferating or involuting phase HAs. MTT, colony formation assay, flow cytometry analysis and a subcutaneous xenograft tumor model were conducted to assess the effects of MEG3 on the HAs tumorigenesis. The interaction between MEG3 and miRNAs or their downstream pathways was evidenced by bioinformatic analysis, luciferase report assays, RNA immunoprecipitation (RIP) assay. and Western blot analysis. Results: The expression of MEG3 was substantially decreased and had a negative correlation with VEGF expression in proliferating phase HAs, as compared with the involuting phase HAs and normal skin tissues. Ectopic expression of MEG3 suppressed cell proliferation, colony formation and induced cycle arrest in vitro and in vivo, followed by the downregulation of VEGF and cyclinD1, but knockdown of MEG3 reversed these effects. Furthermore, MEG3 was verified to act as a sponge of miR-494 in HAs cells, and miR-494 counteracted MEG3-caused anti-proliferative effects by regulating PTEN/PI3K/AKT pathway, and exhibited the negative correlation with MEG3 and PTEN expression in proliferating phase HAs. Conclusion: Our findings suggested that lncRNA MEG3 inhibited HAs tumorigenesis by sponging miR-494 and regulating PTEN/PI3K/AKT pathway.

Graphene is a two-dimensional structured material with a hexagonal honeycomb lattice composed of carbon atoms. The biological effects of graphene oxide (GO) have been extensively investigated, as it has been widely used in biological research due to its increased hydrophilicity/biocompatibility. However, the exact mechanisms underlying GO-associated lung toxicity have not yet been fully elucidated. The aim of the present study was to determine the role of GO in lung injury induction, as well as its involvement in oxidative stress, inflammation and autophagy. The results revealed that lower concentrations of GO (5 and 10 mg/kg) did not cause significant lung injury, but the administration of GO at higher concentrations (50 and 100 mg/kg) induced lung edema, and increased lung permeability and histopathological lung changes. High GO concentrations also induced oxidative injury and inflammatory reactions in the lung, demonstrated by increased levels of oxidative products [malondialdehyde(MDA) and 8-hydroxydeoxyguanosine (8-OHdG)] and inflammatory factors (TNF-α, IL-6, IL-1β and IL-8). The autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CLQ) inhibited autophagy in the lung and attenuated GO-induced lung injury, as demonstrated by a reduced lung wet-to-dry weight ratio, lower levels of protein in the bronchoalveolar lavage fluid, and a reduced lung injury score. Furthermore, 3-MA and CLQ significantly reduced the levels of MDA, 8-OHdG and inflammatory factors in lung tissue, suggesting that autophagy also mediates the development of oxidative injury and inflammation in the lung. Finally, autophagy was directly inhibited in BEAS-2B cells by short hairpin RNA-mediated autophagy protein 5 (ATG5) knockdown, which were then treated with GO. Cell viability, as well as the extent of injury (indicated by lactate dehydrogenase level) and oxidative stress were determined. The results revealed that ATG5 knockdown-induced autophagic inhibition significantly decreased cellular injury and oxidative stress, suggesting that autophagy induction is a key event that leads to lung injury during exposure to GO. In conclusion, the findings of the present study indicated that GO causes lung injury in a dose-dependent manner by inducing autophagy.

Gene expression was examined in hemangiomas (HA), benign, birthmark-like tumors occurring in infancy, and confirmed in HA-derived endothelial cells (HDEC), for which cell proliferation and apoptosis were also assessed. Protein and mRNA accumulation of Rho-associated protein kinase (ROCK), vascular endothelial growth factor (VEGF), Ki-67 and proliferating cell nuclear antigen was significantly higher in proliferating phase HAs than in involuting phase HAs. In contrast, p53 and caspase-3 exhibited higher levels of accumulation in involuting than proliferating HAs. Cell apoptotic indexes were low in proliferating phase HAs and increased in involuting phase HAs. HDECs were treated with the ROCK inhibitor Y-27632. Y-27632 induced p53 expression and downregulated VEGF expression, significantly inhibited cell proliferation, and induced cell apoptosis in HA cells. The inhibitor effects were confirmed in HAs from HDEC-injected nude mice. These results indicated that ROCK is involved in p53-mediated apoptosis and VEGF expression in HA cells and suggested that such inhibition may be exploited for future HA therapies.

Endothelial injury and dysfunction followed by endothelial activation and inflammatory cell recruitment are factors contributing to the initiation and progression of atherosclerosis. Oxidized low-density lipoprotein (ox-LDL) promotes inflammation during atherogenesis and lipid deposition in the arterial wall. We observed that stimulation of human umbilical vein endothelial cells (HUVECs) with ox-LDL activated pro-inflammatory cytokine production and apoptosis, inhibited cell migration, and upregulated T-cell immunoglobulin and mucin domain 3 (Tim-3) expression. Tim-3, in turn, protected HUVECs from ox-LDL-induced apoptosis via the JNK pathway and reversed the inhibition of migration. Tim-3 also inhibited ox-LDL-induced inflammatory cytokine production by suppressing NF-κB activation. In addition, Tim-3 increased production of type 2 T helper cells (Th2) and regulatory T cell (Treg)-associated cytokines. Blocking Tim-3 reversed its effects on the inflammatory response to ox-LDL. Thus, Tim-3 signaling may be a “self-control” mechanism in ox-LDL-triggered inflammation in HUVECs. These results identify Tim-3 as a factor in HUVEC activity and suggest its potential in the treatment of atherosclerosis.

Hemangioma (HA) is a type of benign tumor common in infancy. The main feature of HA is the abnormal proliferation of vascular endothelial cells. To date, the pathogenesis of HA remains unclear. Fully understanding the process of HA tumorigenesis is essential for developing novel treatment for HAs. Dysregulation of microRNAs (miRNAs/miR) has been reported to be involved in the development of various diseases, including HA. In the present study, the expression of miR‑424 decreased in HA‑derived endothelial cells (HemECs). To elucidate the role of miR‑424 in HAs development, the present study overexpressed or inhibited miR‑424 in HemECs, revealing that miR‑424 overexpression significantly inhibited HemEC growth and promoted apoptosis, while the downregulation of miR‑424 promoted cell growth and inhibited cell apoptosis. To elucidate the underlying mechanism, bioinformatic analyses were performed, the result of which demonstrated that the 3'‑untranslated region of vascular endothelial growth factor receptor 2 (VEGFR‑2) may be a target of miR‑424. The result of a dual luciferase reporter assay confirmed that the expression of VEGFR‑2 was inhibited by miR‑424. In addition, it was revealed that the hyper‑phosphorylation of protein kinase B (AKT) and extracellular signal‑regulated kinase (ERK) in HemECs, and the restoration of miR‑424 markedly inhibited the activation of AKT and ERK. In conclusion, these results indicated that miR‑424 may target VEGFR‑2 and inhibit HemECs growth, and that low expression of miR‑424 in HemECs may lead to an increase in cell growth and a decrease in cell apoptosis. Thus, it was proposed that miR‑424 may serve as a tumor suppressor in HemECs, and that VEGFR‑2 may be a potential tumor suppressive target in HemECs and for the treatment of HA.