Jun-Yi Zhu

Autophagy regulates hypoxia‐induced osteoclastogenesis through the HIF‐1α/BNIP3 signaling pathway

Previous studies have implicated that hypoxic stress could enhance osteoclast differentiation; however, the underlying mechanism remains poorly understood. Autophagy is a dynamic lysosomal degradation process that has emerged as an important regulator under hypoxic environment. In the present study, we demonstrate for the first time that autophagy regulates hypoxia‐induced osteoclastogenesis in vitro. We found that exposure of RAW264.7 cells to hypoxia (0.2% oxygen) resulted in enhanced osteoclast differentiation, accompanied by the observation of several specific features of autophagy, including appearance of membranous vacuoles, formation of acidic vesicular organelles, cleavage and recruitment of microtubule‐associated protein 1 light chain 3 (LC3) to autophagosomes, increase in autophagic flux, as well as up‐regulation of autophagy‐related gene (Atg) expression. Moreover, suppression of autophagy with DN‐Atg5K130R or 3‐methyladenine (3‐MA) significantly attenuated the osteoclast differentiation under hypoxic conditions, indicating the functional significance of autophagy in hypoxia‐induced osteoclastogenesis. The data also showed that the activation of autophagy under hypoxic conditions was caused by up‐regulated expression of hypoxia‐inducible factor‐1α (HIF‐1α)‐dependent Bcl‐2 adenovirus E1a 19 kDa interacting protein 3 (BNIP3). Importantly, knockdown of HIF‐1α or BNIP3 obviously abrogated hypoxia‐induced autophagy activation and osteoclastogenesis enhancement. Collectively, our results highlight the fact that autophagy is a pivotal regulator for hypoxia‐induced osteoclast differentiation, which may provide new insight into the pathological processes of osteoclastogenesis under hypoxic stress and help develop new therapeutic strategies for abnormal osteoclastogenesis. J. Cell. Physiol. 227: 639–648, 2012.

Hyperbranched-hyperbranched polymeric nanoassembly to mediate controllable co-delivery of siRNA and drug for synergistic tumor therapy.

This study reported a flexible nanoplatform constructed on the pH-dependent self-assembly of two kinds of hyperbranched polymers, and then validated its potency as the controllable siRNA/drug co-delivery vehicle for the combination of chemotherapy with RNA interfering (RNAi) therapy. By virtue of pH-reversible phenylboronate linking, phenylboronic acid-tethered hyperbranched oligoethylenimine (OEI600-PBA) and 1,3-diol-rich hyperbranched polyglycerol (HBPO) can be spontaneously interlinked together into a core-corona nanoconstruction. The special buildup of compactly clustering OEI600-PBA units around hydrophobic HBPO aggregate offered significant advantages over parent OEI600-PBA, including strengthened affinity to siRNA, ability of further loading anticancer drug, easier cellular transport, and acidity-responsive release of payloads. To evaluate the co-delivery capability, Beclin1 siRNA and antitumor DOX were used as the therapeutic models in order to suppress the post-chemotherapy survival of tumor cells caused by drug-induced autophagy. The nanoassembly-mediated single delivery of DOX displayed even better anticancer effects than free DOX, demonstrating the superiority of our pH-responsive nano-design. The nanoassembly-mediated co-delivery of siRNA together with DOX can effectively silence Beclin1 gene, suppress DOX-induced autophagy, and consequently provide strong synergism with a significant enhancement of cell-killing effects in cultured cancerous cells. The in vivo combinational treatment was shown to make the tumor more sensitive to DOX chemotherapy while displaying substantially improved safety as compared with the monochemotherapy.

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.

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.

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).

Overexpression of macrophage migration inhibitory factor in adenoid cystic carcinoma: correlation with enhanced metastatic potential

ObjectivesAdenoid cystic carcinoma (ACC) is a malignant tumor frequently arising in salivary glands with poor long-term prognosis due to high rates of local recurrences and distant metastases. Macrophage migration inhibitory factor (MIF) is a multi-functional cytokine and has recently emerged as a pro-tumorigenic factor in various cancers. This study is designed to investigate the expression status and functional significance of MIF in ACC.MethodsImmunohistochemical staining was performed to evaluate the expression levels of MIF, HIF-1α, MMP-9, p53, and p-JNK in ACC tissues. In vitro, ACC-2 cells were exposed to recombinant human MIF (rMIF) or ISO-1 (an inhibitor of MIF) at different concentrations and times, followed by the detection of cell growth, viability, migration, and invasion, as well as the expression levels of several cellular signals.ResultsThe immunohistochemical results demonstrated the overexpression of MIF in ACC tissues as well as its association with the distant metastasis. Further analyses showed a significant correlation between the staining of MIF and p-JNK. Moreover, the in vitro studies revealed that the treatment for ACC cells with ISO-1 significantly attenuated cell migratory and invasive capacity, as opposed to the limited promotive effects of rMIF. More importantly, MIF inhibition could cause the activation of JNK, correlating with the immunohistochemical findings on ACC tissues.ConclusionsThe results suggest that MIF is likely to be an important player in the pathogenesis of ACC and may promote cancer metastasis, which possibly involves JNK inactivation. Further investigation of MIF-mediated molecular events may provide novel insights into the treatment for ACC.

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.

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.

Macrophages Contribute to the Progression of Infantile Hemangioma by Regulating the Proliferation and Differentiation of Hemangioma Stem Cells

Macrophage infiltration has been implicated in infantile hemangioma (IH), the most common tumor of infancy. However, the exact role of macrophages in IH remains unknown. This study aims to clarify the functional significance of macrophages in the progression of IH. The distribution of macrophages in human IH was analyzed, and our results revealed that polarized macrophages were more prevalent in proliferating IHs than in involuting IHs, which was consistent with the increased macrophage-related cytokines in proliferating IHs. In vitro results further demonstrated that polarized macrophages effectively promoted the proliferation of hemangioma stem cells (HemSCs) and suppressed their adipogenesis in an Akt- and extracellular signal-regulated kinase 1/2 (Erk1/2)-dependent manner. Moreover, M2- but not M1-polarized macrophages promoted the endothelial differentiation of HemSCs. Furthermore, mixing macrophages in a murine hemangioma model elevated microvessel density and postponed fat tissue formation, which was concomitant with the activation of Akt and Erk1/2 signals. Cluster analysis revealed a close correlation among the macrophage markers, Ki67, vascular endothelial growth factor (VEGF), p-Akt, and p-Erk1/2 in human IH tissues. Collectively, our results suggest that macrophages in IH contribute to tumor progression by promoting the proliferation and endothelial differentiation while suppressing the adipogenesis of HemSCs. These findings indicate that targeting the infiltrating macrophages in IH is a promising therapeutic approach to accelerate IH regression.

Clinical Significance and Roles in Angiogenesis of Circulating Microparticles in Oral Cancer

Our recent study established the increased circulating microparticles (MPs) and their procoagulant activity in oral squamous cell carcinoma (OSCC). In the present study, we further evaluated different phenotypes of circulating MPs in OSCC patients and explored their clinical significance and effects on angiogenesis (a critical event in tumor progression). To conduct the study, circulating MPs in 45 OSCC patients and 18 healthy volunteers were characterized and quantified by transmission electron microscopy and flow cytometry. Correlations between circulating MPs and clinicopathologic data, microvessel density, and proangiogenic factor levels in patients with OSCC were analyzed by immunohistochemistry and Spearman rank correlation test. Additionally, the in vitro studies were performed with use of human umbilical vein endothelial cells. Our results showed that the levels of circulating MPs as well as the subsets of platelet-derived, endothelium-derived, and pan-leukocyte MPs in stages III to IV OSCC were significantly higher than stages I to II and healthy subjects. Moreover, these increased circulating MPs were significantly correlated with tumor size, TNM stages, microvessel density, and expression levels of vascular endothelial growth factor (VEGF) and matrix metallopeptidase 9 (MMP9) in OSCC patients. The in vitro studies revealed that circulating MPs isolated from OSCC patients could be effectively taken up by human umbilical vein endothelial cells and could promote the proliferation, migration, invasion, and tube formation of recipient endothelial cells, accompanied by increased expression of proangiogenic factors. In summary, circulating MPs play important roles in angiogenesis and local tumor progression of OSCC. Our results shed new light on the progression of OSCC and might be helpful to explore novel treatment strategies targeting tumor angiogenesis.