Jingzhou Hu

Blood vessel formation in the tissue-engineered bone with the constitutively active form of HIF-1α mediated BMSCs

The successful clinical outcome of the implanted tissue-engineered bone is dependent on the establishment of a functional vascular network. A gene-enhanced tissue engineering represents a promising approach for vascularization. Our previous study indicated that hypoxia-inducible factor-1α (HIF-1α) can up-regulate the expression of vascular endothelial growth factor (VEGF) and stromal-derived factor 1 (SDF-1) in bone mesenchymal stem cells (BMSCs). The angiogenesis is a co-ordinated process that requires the participation of multiple angiogenic factors. To further explore the angiogenic effect of HIF-1α mediated stem cells, in this study, we systematically evaluated the function of HIF-1α in enhancing BMSCs angiogenesis in vitro and in vivo. A constitutively active form of HIF-1α (CA5) was inserted into a lentivirus vector and transduced into BMSCs, and its effect on vascularization and vascular remodeling was further evaluated in a rat critical-sized calvarial defects model with a gelatin sponge (GS) scaffold. The expression of the key angiogenic factors including VEGF, SDF-1, basic fibroblast growth factor (bFGF), placental growth factor (PLGF), angiopoietin 1 (ANGPT1), and stem cell factor (SCF) at both mRNAs and proteins levels in BMSCs were significantly enhanced by HIF-1α overexpression compared to the in vitro control group. In addition, HIF-1α-over expressing BMSCs showed dramatically improved blood vessel formation in the tissue-engineered bone as analyzed by photography of specimen, micro-CT, and histology. These data confirm the important role of HIF-1α in angiogenesis in tissue-engineered bone. Improved understanding of the mechanisms of angiogenesis may offer exciting therapeutic opportunities for vascularization, vascular remodeling, and bone defect repair using tissue engineering strategies in the future.

Combination of β‐TCP and BMP‐2 gene‐modified bMSCs to heal critical size mandibular defects in rats

OBJECTIVE To investigate the effects of mandibular defects repaired by a tissue engineered bone complex with beta-tricalcium phosphate (beta-TCP) and bone morphogenic protein-2 (BMP-2) gene-modified bone marrow stromal cells (bMSCs). MATERIALS AND METHODS bMSCs derived from Fisher 344 rats were cultured and transduced with adenovirus AdBMP-2, AdEGFP gene in vitro. Osteogenic differentiation of bMSCs was determined by alkaline phosphatase staining, von Kossa assay and reverse transcription-polymerase chain reaction. Gene transduced or untransduced bMSCs were seeded on beta-TCP scaffolds to repair mandibular full thickness defects with a diameter of 5 mm. Eight weeks post-operation, X-ray examination, micro-computerized tomography and histological and histomorphological analysis were used to evaluate the bone healing effects. RESULTS Alkaline phosphatase staining and mineralized nodules formation were more pronounced in AdBMP-2 group 14 days after gene transduction when compared with that of AdEGFP or untransduced group. The mRNA expression of osteopontin and osteocalcin also significantly increased 9 days after AdBMP-2 gene transduction. Mandibular defects were successfully repaired with AdBMP-2-transduced bMSCs/beta-TCP constructs. The percentage of new bone formation in AdBMP-2 group was significantly higher than that of other control groups. CONCLUSIONS Bone morphogenic protein-2 regional gene therapy together with beta-TCP scaffold could be used to promote mandibular repairing and bone regeneration.

A new image correction method for live cell atomic force microscopy

During live cell imaging via atomic force microscopy (AFM), the interactions between the AFM probe and the membrane yield distorted cell images. In this work, an image correction method was developed based on the force-distance curve and the modified Hertzian model. The normal loading and lateral forces exerted on the cell membrane by the AFM tip were both accounted for during the scanning. Two assumptions were made in modelling based on the experimental measurements: (1) the lateral force on the endothelial cells was linear to the height; (2) the cell membrane Youngs modulus could be derived from the displacement measurement of a normal force curve. Results have shown that the model could be used to recover up to 30% of the actual cell height depending on the loading force. The accuracy of the model was also investigated with respect to the loading force and mechanical property of the cell membrane.

The feasibility and significance of preservation of the lobular branch of the great auricular nerve in parotidectomy.

This study was designed to evaluate the feasibility and significance of preserving the lobular branch of the great auricular nerve (GAN) during parotidectomy. Ninety-three patients with benign tumour undergoing parotidectomy were separated randomly into three groups. Thirty-one patients underwent a parotidectomy with the main trunk of GAN sacrificed (group A), 29 patients had the posterior-auricular branch preserved (group B), and 33 patients had the lobular branch preserved (group C). The operating times were recorded. Tactile sensitivity and pain sensitivity were evaluated preoperatively and at 1 week, 1 month, 3 months, 6 months and 1 year after surgery. Eighty-three patients were followed-up. Preservation of the lobular branch required no extra operating time. In group C, sensitivity in the lobular region reached preoperative levels by 6 months after surgery. In the other groups, recovery of sensory function in the lobular region was partial. Patients with the lobular branch of GAN preserved had significantly better sensory recovery in the lobular region 1 year after surgery (P < 0.05). These results demonstrate that preservation of the lobular branch of GAN is technically feasible during parotidectomy. The preservation of the lobular branch of GAN guarantees improvement of postoperative sensitivity of the lobular region.

Dose Dependent Activation of Retinoic Acid-Inducible Gene-I Promotes Both Proliferation and Apoptosis Signals in Human Head and Neck Squamous Cell Carcinoma

The retinoic-acid-inducible gene (RIG)-like receptor (RLR) family proteins are major pathogen reorganization receptors (PRR) responsible for detection of viral RNA, which initiates antiviral response. Here, we evaluated the functional role of one RLR family member, RIG-I, in human head and neck squamous cell carcinoma (HNSCC). RIG-I is abundantly expressed both in poorly-differentiated primary cancer and lymph node metastasis, but not in normal adjacent tissues. Activation of RIG-I by transfection with low dose of 5′-triphosphate RNA (3p-RNA) induces low levels of interferon and proinflammatory cytokines and promotes NF-κB- and Akt-dependent cell proliferation, migration and invasion. In contrast, activation of RIG-I by a high dose of 3p-RNA induces robust mitochondria-derived apoptosis accompanied by decreased activation of Akt, which is independent of the interferon and TNFα receptor, but can be rescued by over-expression of constitutively active Akt. Furthermore, co-immunoprecipitation experiments indicate that the CARD domain of RIG-I is essential for inducing apoptosis by interacting with caspase-9. Together, our results reveal a dual role of RIG-I in HNSCC through regulating activation of Akt, in which RIG-I activation by low-dose viral dsRNA increases host cell surviral, whereas higher level of RIG-I activation leads to apopotosis. These findings highlight the therapeutic potential of dsRNA mediated RIG-I activation in the treatment of HNSCC.

Autocrine epiregulin activates EGFR pathway for lung metastasis via EMT in salivary adenoid cystic carcinoma

Salivary adenoid cystic carcinoma (SACC) is characterized by invasive local growth and a high incidence of lung metastasis. Patients with lung metastasis have a poor prognosis. Treatment of metastatic SACC has been unsuccessful, largely due to a lack of specific targets for the metastatic cells. In this study, we showed that epidermal growth factor receptors (EGFR) were constitutively activated in metastatic lung subtypes of SACC cells, and that this activation was induced by autocrine expression of epiregulin (EREG), a ligand of EGFR. Autocrine EREG expression was increased in metastatic SACC-LM cells compared to that in non-metastatic parental SACC cells. Importantly, EREG-neutralizing antibody, but not normal IgG, blocked the autocrine EREG-induced EGFR phosphorylation and the migration of SACC cells, suggesting that EREG-induced EGFR activation is essential for induction of cell migration and invasion by SACC cells. Moreover, EREG-activated EGFR stabilized Snail and Slug, which promoted EMT and metastatic features in SACC cells. Of note, targeting EGFR with inhibitors significantly suppressed both the motility of SACC cells in vitro and lung metastasis in vivo. Finally, elevated EREG expression showed a strong correlation with poor prognosis in head and neck cancer. Thus, targeting the EREG-EGFR-Snail/Slug axis represents a novel strategy for the treatment of metastatic SACC even no genetic EGFR mutation.

Surgical approaches for tongue base schwannoma.

Schwannomas (neurilemmomas) are benign nerve sheath tumor originating from Schwann cells. They are well circumscribed and rarely infiltrate and metastasize. Schwannomas of the head and neck commonly occur in the tongue followed by the palate, floor of mouth, buccal mucosa, and mandible. Tongue base schwannomas could extend to the pharyngeal cavity or the floor of the mouse, and it is difficult to differentiate between tumors of the lingual, hypoglossal, and glossopharyngeal nerves.Surgical treatment of tongue base schwannomas is difficult because of limited operative exposure. Although mandibulotomy with lip splitting could obtain good exposure, surgeons might strike a balance between exposure obtaining and morbidity following because there are intricate neurovascular anatomical relationships in this region, and mandibulotomy with lip splitting would cause significant morbidity. Surgical approach options are important for tongue base schwannoma removal. From March 2008 to March 2010, 8 patients were clinically and pathologically diagnosed with tongue base schwannomas in our department, and all underwent surgical treatment. In our experience, transoral approach was used for tongue base schwannomas extending to the floor of the mouse and suprahyroid pharyngotomy approach for those extending to the pharyngeal cavity. Follow-up was made until now. One patient who experienced transoral excision still experienced numbness in the region of the lateral tongue tip, and the other 7 patients had no postoperative long-term complications.

Nuclear survivin promoted by acetylation is associated with the aggressive phenotype of oral squamous cell carcinoma

ABSTRACT Defects in apoptotic pathway contribute to development and progression of oral cancer. Survivin, a member of the inhibitors of apoptosis protein (IAP) family, is increased in many types of cancers. However, it is unclear whether increased survivin is associated with oral squamous cell carcinomas (OSCC), and what mechanisms may involve in. In this study, we examined survivin expression in OSCC compared with normal oral tissues via immunohistochemical staining. The results showed that, not only total survivin is increased in OSCCs, but also the subcellular location of survivin is changed in OSCCs compared with normal oral tissues. In most of normal oral tissues, survivin staining was either negative, or cytoplasmic positive/nuclear negative; whereas in most of OSCC tissues, survivin staining was nuclear positive. Statistic analysis indicates that nuclear survivin, rather than total or cytoplasmic one, correlates with tumor TNM stage and differentiation grade. Consistently, in vitro analysis showed that survivin is in cytoplasm in normal human oral kinotinocyte (HOK) cells; whereas it is in nucleus in OSCC HN6 cells. Importantly, treatment of HOK cells with HDAC inhibitor Trichostatin A (TSA) induces survivin acetylation and promotes its nuclear localization. Moreover, nuclear survivin in OSCC cells was acetylated at K129 in its C-terminal, suggesting that the acetylation is important for nuclear location of survivin. Our study demonstrates that it is nuclear survivin, rather than total or cytoplasmic one, associates with TNM stage and tumor grade of OSCC. Thus, we propose nuclear survivin as a prognostic marker for the progression of OSCC.

Tongue reconstruction with tongue base island advancement flap.

AbstractReconstruction of a medium-sized defect of the tongue remains a challenge if aesthetic impairment is to be avoided. In this study, 19 tongue base island advancement flaps were developed to reconstruct medium-sized defects after the tongue squamous cell carcinoma ablations: 13 cases were T1N0M0, and 6 cases were T2N0∼1M0. The largest size amounts to 5.4 × 4.8 cm (length × width), with a mean of 4.6 × 4.4 cm. The tongue base island advancement flap reduces the volume of the tongue base without causing function impairment of the tongue. All patients recovered with good objective and subjective speech and swallowing and aesthetics. No patient developed local recurrence or lymphatic metastasis. The technique of tongue base advancement flap is ideal for functional and aesthetic repair of medium-sized tongue defects after cancer ablation.

Interferon-alpha enhances the antitumour activity of EGFR-targeted therapies by upregulating RIG-I in head and neck squamous cell carcinoma

Background:The epidermal growth factor receptor (EGFR)-targeted therapies have been tested in the clinic as treatments for head and neck squamous cell carcinoma (HNSCC). Owing to intrinsic or acquired resistance, EGFR-targeted therapies often lead to a low response rate and treatment failure. Interferon-alpha (IFNα) is a chemosensitising agent and multi-functional cytokine with a tumour inhibitory effect. However, the synergic effect of IFNα and EGFR-targeted therapies (erlotinib and nimotuzumab) and their mechanisms in HNSCC remain unclear.Methods:The interactions between IFNα, erlotinib, and nimotuzumab were evaluated in vitro in HNSCC cells. The synergistic effect of IFNα (20 000 IU per day, s.c.), erlotinib (50 mg kg−1 per day, i.g.) and nimotuzumab (10 mg kg−1 per day, i.p.) was further confirmed in vivo using HNSCC xenografts in nude mice. The upregulation of retinoic-acid inducible gene I (RIG-I) induced by IFNα and EGFR-targeted therapies and its mechanism were detected in vitro and in vivo.Results:IFNα enhances the antitumour effects of erlotinib and nimotuzumab on HNSCC cells both in vitro and in vivo. Importantly, both IFNα and EGFR-targeted therapies promote the expression of RIG-I by activating signal transducers and activators of transcription 1 (STAT1) in HNSCC cells. RIG-I knockdown reduced the sensitivity of HN4 and HN30 cells to IFNα, erlotinib, and nimotuzumab. Moreover, IFNα transcriptionally induced RIG-I expression in HNSCC cells through STAT1.Conclusions:IFNα enhances the effect of EGFR-targeted therapies by upregulating RIG-I, and its expression may represent a predictor of the effectiveness of a combination treatment including IFNα in HNSCC.