Jian-Gang Ren

Hypoxia-induced autophagy in endothelial cells: a double-edged sword in the progression of infantile haemangioma?

AIMS The aim of this study was to investigate the precise role of hypoxia-induced autophagy in endothelial cells, and whether it contributes to the distinctive progression of infantile haemangioma (IH). METHODS AND RESULTS The endothelial cells (EOMA and HUVECs) were cultured under hypoxic conditions for indicated times (0-72 h). The results showed that short exposure of the endothelial cells to hypoxia resulted in increased cell survival and proliferation, accompanied by occurrence of autophagy. Prolonged hypoxia-induced autophagy, correlating with increased cell death, was also detected afterwards. Correspondingly, autophagy inhibition prevented the enhanced cell survival and proliferation capacity, advanced the occurrence of cell-death in early hypoxic stage, and meanwhile attenuated the ability of prolonged hypoxia in cell-death induction. Moreover, our data demonstrated that the functional transformation of hypoxia-induced autophagy, pro-survival to pro-death, was rigorously regulated by the switch between hypoxia-inducible factor-1α (HIF-1α) and mammalian target of rapamycin (mTOR) pathways. Importantly, we also revealed the activation levels of HIF-1α and mTOR, as well as the autophagy status during the progression of IH. CONCLUSION This study unmasks the functional switch between HIF-1α and mTOR in regulating hypoxia-induced autophagy in endothelial cells and, more importantly, indicates its potential role in the progression of IH.

Transformation of Cell‐Derived Microparticles into Quantum‐Dot‐Labeled Nanovectors for Antitumor siRNA Delivery

Cell-derived microparticles (MPs) have been recently recognized as critical intercellular information conveyors. However, further understanding of their biological behavior and potential application has been hampered by the limitations of current labeling techniques. Herein, a universal donor-cell-assisted membrane biotinylation strategy was proposed for labeling MPs by skillfully utilizing the natural membrane phospholipid exchange of their donor cells. This innovative strategy conveniently led to specific, efficient, reproducible, and biocompatible quantum dot (QD) labeling of MPs, thereby reliably conferring valuable traceability on MPs. By further loading with small interference RNA, QD-labeled MPs that had inherent cell-targeting and biomolecule-conveying ability were successfully employed for combined bioimaging and tumor-targeted therapy. This study provides the first reliable and biofriendly strategy for transforming biogenic MPs into functionalized nanovectors.

Bleomycin induces endothelial mesenchymal transition through activation of mTOR pathway: a possible mechanism contributing to the sclerotherapy of venous malformations.

Bleomycin (BLM), one of the most common sclerosants, is often used to treat venous malformations (VMs). The present study was designed to investigate whether endothelial mesenchymal transition (EndoMT) contributes to the therapeutic effects of BLM.

M2-polarised macrophages in infantile haemangiomas: correlation with promoted angiogenesis

Aims The pathogenesis of infantile haemangiomas (IHs) is still far from clear despite the fact that they are common vascular tumours distinctive for their perinatal presentation, rapid growth during the first year of life and subsequent slow involution. Aims To determine the role of M2-polarised macrophages in IHs. Methods M2-polarised macrophages were initially identified in 20 specimens of IHs by both immunochemistry and immunofluorescence for CD68 and CD163. The immunopositive M2-polarised macrophages in different phases of IHs were quantified, and further analysed for their correlations with the expression levels of Ki67, vascular endothelial growth factor (VEGF) and macrophage colony-stimulating factor (M-CSF). Results The infiltrating macrophages in proliferative IHs were predominantly CD68/CD163, thus of the M2-polarised phenotype, whereas the density of these cells was significantly decreased in the involuting IHs. The high density of M2-polarised macrophages in proliferative IHs was closely correlated with overexpression of M-CSF, one of the cytokines considered to induce macrophages to polarise towards an M2 phenotype. The infiltrating M2-polarised macrophages probably contributed to the proliferation and angiogenesis of haemangioma endothelial cells, as evidenced by their close correlations with the immunoreactivities of Ki67 and VEGF. Conclusions Results indicate that the infiltrating M2-polarised macrophages may contribute to the progression of IHs by promoting the angiogenic process.

The Adaptor Protein p62 Is Involved in RANKL-induced Autophagy and Osteoclastogenesis

Previous studies have implicated autophagy in osteoclast differentiation. The aim of this study was to investigate the potential role of p62, a characterized adaptor protein for autophagy, in RANKL-induced osteoclastogenesis. Real-time quantitative PCR and western blot analyses were used to evaluate the expression levels of autophagy-related markers during RANKL-induced osteoclastogenesis in mouse macrophage-like RAW264.7 cells. Meanwhile, the potential relationship between p62/LC3 localization and F-actin ring formation was tested using double-labeling immunofluorescence. Then, the expression of p62 in RAW264.7 cells was knocked down using small-interfering RNA (siRNA), followed by detecting its influence on RANKL-induced autophagy activation, osteoclast differentiation, and F-actin ring formation. The data showed that several key autophagy-related markers including p62 were significantly altered during RANKL-induced osteoclast differentiation. In addition, the expression and localization of p62 showed negative correlation with LC3 accumulation and F-actin ring formation, as demonstrated by western blot and immunofluorescence analyses, respectively. Importantly, the knockdown of p62 obviously attenuated RANKL-induced expression of autophagy- and osteoclastogenesis-related genes, formation of TRAP-positive multinuclear cells, accumulation of LC3, as well as formation of F-actin ring. Our study indicates that p62 may play essential roles in RANKL-induced autophagy and osteoclastogenesis, which may help to develop a novel therapeutic strategy against osteoclastogenesis-related diseases.

Downregulation of the transforming growth factor‐β/connective tissue growth factor 2 signalling pathway in venous malformations: its target potential for sclerotherapy

Previous studies have implicated vascular destabilization and changes in extracellular matrix (ECM) composition in venous malformations (VMs).

Magnetic and Folate Functionalization Enables Rapid Isolation and Enhanced Tumor-Targeting of Cell-Derived Microvesicles

Cell-derived microvesicles (MVs), which are biogenic nanosized membrane-bound vesicles that convey bioactive molecules between cells, have recently received attention for use as natural therapeutic platforms. However, the medical applications of MV-based delivery platforms are limited by the lack of effective methods for the efficient isolation of MVs and the convenient tuning of their targeting properties. Herein, we report the development of magnetic and folate (FA)-modified MVs based on a donor cell-assisted membrane modification strategy. MVs inherit the membrane properties of their donor cells, which allows them to be modified with the biotin and FA on their own membrane. By conjugating with streptavidin-modified iron oxide nanoparticles (SA-IONPs), the MVs can be conveniently, efficiently, and rapidly isolated from the supernatant of their donor cells using magnetic activated sorting. Moreover, the conjugated magnetic nanoparticles and FA confer magnetic and ligand targeting activities on the MVs. Then, the MVs were transformed into antitumor delivery platforms by directly loading doxorubicin via electroporation. The modified MVs exhibited significantly enhanced antitumor efficacy both in vitro and in vivo. Taken together, this study provides an efficient and convenient strategy for the simultaneous isolation of cell-derived MVs and transformation into targeted drug delivery nanovectors, thus facilitating the development of natural therapeutic nanoplatforms.

Disorganized vascular structures in sporadic venous malformations: a possible correlation with balancing effect between Tie2 and TGF-β

Venous malformations (VMs) are among the most common slow-flow vascular malformations characterized by irregular venous channels, luminal thrombi, and phleboliths. To systematically manifest the disorganized vascular structures in sporadic VMs, we initially evaluated histopathological characteristics, perivascular cell coverage, adhesion molecules expression and vascular ultrastructures. Then, the expression of Tie2 and TGF-β in VMs was detected. Meanwhile, the in vitro studies were performed for mechanism investigation. Our data showed that the perivascular α-SMA+ cell coverage and expression of adhesion molecules in VMs were significantly decreased compared with those in the normal skin tissues. We also found that the expression and phosphorylation levels of Tie2 were upregulated, whereas TGF-β was downregulated in VMs, and they were negatively correlated. Moreover, the in vitro results also revealed a possible balancing effect between Tie2 and TGF-β, as demonstrated by the findings that Ang-1 (agonist of Tie2) treatment significantly downregulated TGF-β expression, and treatment with recombinant TGF-β could also suppress Tie2 expression and phosphorylation. This study provided strong evidence supporting the disorganized vascular structures and dysregulation of related molecules in sporadic VMs, and demonstrated a possible balancing effect between Tie2 and TGF-β, which might help to develop novel therapeutics for vascular disorganization-related disorders.

Elevated Level of Circulating Platelet-derived Microparticles in Oral Cancer

Numerous studies have demonstrated that circulating microparticles (MPs) play important roles in a variety of diseases (e.g., atherosclerosis, hypertension, and diabetes), but the association between circulating MPs and oral squamous cell carcinoma (OSCC) remains largely unknown. In the present study, the circulating platelet-derived MPs (PMPs) in 63 patients with OSCC, 22 patients with infected keratocystic odontogenic tumor, and 31 healthy volunteers were characterized and quantified by flow cytometric analysis. The coagulation function of patients with OSCC was correspondingly evaluated. Meanwhile, the inflammation-related cytokines were detected in plasma by enzyme-linked immunosorbent assay and in tumor tissues by immunohistochemistry. Our results showed that the plasma level of circulating PMPs was significantly higher in OSCC patients compared with healthy volunteers and patients with infected keratocystic odontogenic tumor, and they showed positive correlation with the increased level of fibrinogen. Moreover, the coagulation time was significantly shorter after the MPs were added to the MP-free plasma. Most important, the levels of interleukin 6 and tumor necrosis factor α in plasma and tumor tissues were significantly increased in OSCC patients, which were closely correlated with the elevated level of circulating PMPs. In summary, this study suggests that the elevated level of circulating PMPs, showing close correlation with the secretion of inflammation-related factors, may contribute to the increased procoagulant activity in patients with OSCC.

Blockage of glycolysis by targeting PFKFB3 suppresses tumor growth and metastasis in head and neck squamous cell carcinoma

BackgroundMany cancers including head and neck squamous cell carcinoma (HNSCC) are characterized by a metabolic rewiring with increased glucose uptake and lactate production, termed as aerobic glycolysis. Targeting aerobic glycolysis presents a promising strategy for cancer therapy. This study investigates the therapeutic potential of glycolysis blockage by targeting phosphofructokinase-2/fructose-2, 6-bisphosphatase 3 (PFKFB3) in HNSCC.Methods1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15) was used as a selective antagonist of PFKFB3. Glycolytic flux was determined by measuring glucose uptake, lactate production and ATP yield. PFKFB3 expression was examined using HNSCC tissue arrays. Cell proliferation, apoptosis and motility were analysed. HNSCC xenograft mouse model and metastasis mouse model were established to examine the therapeutic efficacy of PFK15 in vivo.ResultsHNSCC showed an increased PFKFB3 expression compared with adjacent mucosal tissues (P < 0.01). Targeting PFKFB3 via PFK15 significantly reduced the glucose uptake, lactate production and ATP generation in HNSCC cell lines. PFK15 suppressed cell proliferation, halted cell cycle progression and induced cell apoptosis. The invadopodia of HNSCC cells was markedly reduced after PFK15 treatment, thereby impairing cell motility and extracellular matrix degradation ability. The in vivo data from the xenograft mice models proved that PFK15 administration suppressed the tumor growth. And the results from the metastatic mice models showed administration of PFK15 alleviated the lung metastasis of HNSCC and extended the life expectancy of mice.ConclusionsThe pharmacological inhibition of PFKFB3 via PFK15 suppressed tumor growth and alleviated metastasis in HNSCC, offering a promising strategy for cancer therapy.