Jingqin Fang

P2X7R suppression promotes glioma growth through epidermal growth factor receptor signal pathway

P2X7 receptor (P2X7R) has been shown to mediate an anticancer effect via apoptosis in different types of cancer. However, whether P2X7R exerts a promoting or suppressive effect on brain glioma is still a controversial issue and its underlying mechanism remains unknown. We showed here that P2X7R suppression exerted a pro-growth effect on glioma through directly promoting cells proliferation and pro-angiogenesis, which was associated with epidermal growth factor receptor (EGFR) signaling. The P2X7R was markedly downregulated by cells exposure to the P2X7R antagonist, brilliant blue G (BBG), moreover, the cells proliferation was enhanced in a dose-dependent manner and the expression of EGFR or p-EGFR protein was significantly upregulated. By constructing C6 cells with reduced expression of P2X7R using shRNA, we also demonstrated strong upregulation in cells proliferation and EGFR/p-EGFR expression. However, this effect of BBG was reversed in the presence of gefitinib or suramin. Magnetic resonance imaging and computed tomography perfusion showed that the BBG or P2X7R shRNA promoted the tumor growth by about 40% and 50%, respectively, and significantly increased angiogenesis. Nissl and Ki-67 staining also confirmed that BBG or P2X7R shRNA notably increased the tumor growth. More importantly, either BBG or P2X7R shRNA could markedly upregulated the expression of EGFR, p-EGFR, HIF-1α and VEGF in glioma cells. In conclusion, P2X7R suppression exerts a promoting effect on glioma growth, which is likely to be related to upregulated EGFR, HIF-1α and VEGF expression. These findings provide important clues to the molecular basis of anticancer effect of targeting purinergic receptors.

Effects of magnetically labeled exogenous endothelial progenitor cells on cerebral blood perfusion and microvasculature alterations after traumatic brain injury in rat model

Background Increasing evidence suggests that endothelial progenitor cells (EPCs), a subgroup of bone marrow hematopoietic stromal cells, play a critical role in neovascularization and tissue repair. Purpose To explore the effect of exogenous EPCs on the cerebral blood perfusion and microvessels in the injured region in rat model with traumatic brain injury (TBI). Material and Methods: EPCs were collected from the spleens of healthy Sprague-Dawley rats. Fifty-four Sprague-Dawley rats were randomly divided into six groups. The controlled cortical impact TBI was performed. Spleen-derived exogenous EPCs labeled with superparamagnetic iron oxide (SPIO) (SPIO-EPCs) were transplanted into the blood by tail vein of rats at 6 and 12 h after TBI, respectively. Magnetic resonance imaging (MRI) and computed tomography perfusion imaging were performed at various time points. Microvascular density was determined by immunohistochemistry. Results In SPIO-EPCs group, patchlike hypointensities were detected in the injured region at 24 h after transplantation, and the range of hypointensities tended to expand gradually over time on MRI, which was confirmed by Prussian blue staining. Computed tomography perfusion imaging parameters were gradually developed from hyperperfusion to normal, while, microvascular density was gradually increased during 72 to 168 h after injury. The values of these indices in SPIO-EPCs group were significantly lower than those in SPIO-alone group at the same time point, but no significant differences were found in different time groups. Conclusion The intravenously transplanted EPCs diminish the brain injury through restoring cerebral blood perfusion and increasing the cerebral microvasculature in the injured region in rat model with TBI.

The expression of P2X7 receptors in EPCs and their potential role in the targeting of EPCs to brain gliomas

In order to use endothelial progenitor cells (EPCs) as a therapeutic and imaging probe to overcome antiangiogenic resistance for gliomas, how to enhance proliferation and targeting ability of transplanted EPCs is a high priority. Here, we confirmed, for the first time, the expression of P2X7 receptors in rat spleen-derived EPCs. Activation of P2X7 receptors in EPCs by BzATP promoted cells proliferation and migration, rather than apoptosis. In vivo, the homing of transplanted EPCs after long-term suppression of P2X7 receptors by persistent BBG stimulation was evaluated by MRI, immunohistochemistry and flow cytometry. Compared to the group without BBG treatment, less transplanted EPCs homed to gliomas in the group with BBG treatment, especially integrated into the vessels containing tumor-derived endothelial cells in gliomas. Moreover, western blot showed that CXCL1 expression was downregulated in gliomas with BBG treatment, which meant P2X7 receptors suppression inhibited the homing of EPCs to gliomas through down-regulation of CXCLl expression. Further, effects of P2X7 receptors on C6 glioma cells or gliomas were evaluated at the same dose of BzATP or BBG used in EPCs experiments in vitro and in vivo. MTT assay and MRI revealed that P2X7 receptors exerted no significant promoting effect on C6 glioma cells proliferation, gliomas growth and angiogenesis. Taken together, our findings imply the possibility of promoting proliferation and targeting ability of transplanted EPCs to brain gliomas in vivo through P2X7 receptors, which may provide new perspectives on application of EPCs as a therapeutic and imaging probe to overcome antiangiogenic resistance for gliomas.

The effects of magnetically labeled rat spleen-originated endothelial progenitor cells on growth of glioma in vivo an experimental study.

RATIONALE AND OBJECTIVES The aim of this study was to investigate the effects of exogenous endothelial progenitor cells (EPCs) on the growth and invasiveness of glioma in vivo to provide an experimental basis for the value and safety of using magnetically labeled EPCs as target vectors to detect early infiltration of glioma. MATERIALS AND METHODS EPCs were collected from the spleens of healthy Sprague-Dawley rats, made EPCs conditioned medium after identification. Four models of Sprague-Dawley rat glioma (60 rats in total) were established as a control and three experimental groups (group A, B, and C). In the control group, orthotopic transplantation of C6 glioma cells was performed. Compared to the control group, EPCs conditioned medium was added in group A and P7228-labeled EPCs were added in group B. In group C, P7228-labeled EPCs were transplanted via the tail vein. Magnetic resonance imaging and perfusion-weighted imaging were performed on several days. Tumor microvascular density and vascular endothelial growth factor expression were determined through immunohistochemistry. RESULTS In group C, hypointense areas were detected at the periphery of the tumor on the first day after transplantation of EPCs, and more hypointense areas were found inside the tumor over time. Tumor size in all four groups developed significantly with increasing time (P < .01), but there was no marked difference among these groups at the same time (P > .05). No remarkable differences in microvascular density and cells positive for vascular endothelial growth factor were found at the same time among the four groups (P > .05). CONCLUSIONS Both magnetic resonance imaging and immunohistochemical findings confirmed that exogenous EPCs could not affect the biologic behavior of C6 glioma cells in vivo through a paracrine effect or by direct cellular interaction. Therefore, exogenous EPCs could not exert significant promoting effects on glioma growth.

Textural features of dynamic contrast-enhanced MRI derived model-free and model-based parameter maps in glioma grading

Presurgical glioma grading by dynamic contrast‐enhanced MRI (DCE‐MRI) has unresolved issues.

Quantitative in vivo imaging of tissue factor expression in glioma using dynamic contrast-enhanced MRI derived parameters

OBJECTIVE Tissue Factor (TF) has been well established in angiogenesis, invasion, metastasis, and prognosis in glioma. A noninvasive assessment of TF expression status in glioma is therefore of obvious clinical relevance. Dynamic contrast-enhanced (DCE) MRI parameters have been used to evaluate microvascular characteristics and predict molecular expression status in tumors. Our aim is to investigate whether quantitative DCE-MRI parameters could assess TF expression in glioma. MATERIALS AND METHODS Thirty-two patients with histopathologically diagnosed supratentorial glioma who underwent DCE-MRI were retrospectively recruited. Extended Tofts linear model was used for DCE-MRI post-processing. Hot-spot, whole tumor cross-sectional approaches, and histogram were used for analysis of model based parameters. Four serial paraffin sections of each case were stained with TF, CD105, CD34 and α-Sooth Muscle Actin, respectively for evaluating the association of TF and microvascular properties. Pearson correlation was performed between percentage of TF expression area and DCE-MRI parameters, multiple microvascular indexes. RESULTS Volume transfer constant (Ktrans) hot-spot value best correlated with TF (r=0.886, p<0.001), followed by 90th percentile Ktrans value (r=0.801, p<0.001). Moreover, histogram analysis of Ktrans value demonstrated that weak TF expression was associated with less heterogeneous and positively skewed distribution. Finally, pathology analysis revealed TF was associated with glioma grade and significantly correlated with these two dynamic angiogenic indexes which could be used to explain the strong correlation between Ktrans and TF expression. CONCLUSION Our results indicate that Ktrans may serve as a potential clinical imaging biomarker to predict TF expression status preoperatively in gliomas.

Posttraumatic Basilar Artery Herniation Associated with Dissecting Aneurysm Formation: Follow-up Over 20 Months

The fracture in the clivus of occipital bone, which might be longitudinal, transverse, or oblique, accounts for 0.36–0.57% of traumatic brain injuries found by computed tomography (CT) [1, 2]. Among these, the longitudinal clivus fracture has the highest mortality rate as a result of the direct injury of brain stem and vertebrobasilar artery [1–3]. Longitudinal clival fracture with the incarceration of vertebrobasilar artery at the fracture site is unusual. Only 15 cases had been reported previously [3, 4–15], and 10 patients died [3, 4, 6–11]. Here we report reported a rare case of the basilar artery incarceration at the fracture site involving herniation into the sphenoid sinus due to a longitudinal clival multifracture. The patient had no corresponding clinical manifestations. To our knowledge, this is the first case to report the manifestations of arterial injury in longitudinal clival fractures and prognosis.

Dynamic MR imaging for functional vascularization depends on tissue factor signaling in glioblastoma

ABSTRACT Glomeruloid vascular proliferation (GVP) is a diagnostic hallmark and links to aggressive behavior, therapy resistance and poor prognosis in glioblastoma (GBM). It lacks clinical approaches to predict and monitor its formation and dynamic change. Yet the mechanism of GVPs also remains largely unknown. Using an in situ GBM xenograft mouse model, combined clinical MRI images of pre-surgery tumor and pathological investigation, we demonstrated that the inhibition of tissue factor (TF) decreased GVPs in Mouse GBM xenograft model. TF shRNA reduced microvascular area and diameter, other than bevacizumab. TF dominantly functions via PAR2/HB-EGF-dependent activation under hypoxia in endothelial cells (ECs), resulting in a reduction of GVPs and cancer cells invasion. TF expression strongly correlated to GVPs and microvascular area (MVA) in GBM specimens from 56 patients, which could be quantitatively evaluated in an advanced MRI images system in 33 GBM patients. This study presented an approach to assess GVPs that could be served as a MRI imaging biomarker in GBM and uncovered a molecular mechanism of GVPs.

Increasing of Blood-Brain Tumor Barrier Permeability through Transcellular and Paracellular Pathways by Microbubble-Enhanced Diagnostic Ultrasound in a C6 Glioma Model

Most of the anticancer agents cannot be efficiently delivered into the brain tumor because of the existence of blood-brain tumor barrier (BTB). The objective of this study was to explore the effect of microbubble-enhanced diagnostic ultrasound (MEUS) on the BTB permeability and the possible mechanism. Glioma-bearing rats were randomized into three groups as follows: the microbubble-enhanced continued diagnostic ultrasound (MECUS) group; the microbubble-enhanced intermittent diagnostic ultrasound (MEIUS) group and the control group. The gliomas were insonicated through the skull with a diagnostic ultrasound and injected with microbubbles through the tail veins. Evans Blue (EB) and dynamic contrast-enhanced-MRI were used to test changes in the BTB permeability. Confocal laser scanning microscopy was used to observe the deposition of the EB in the tumor tissues. The distribution and expression of junctional adhesion molecule-A (JAM-A) and calcium-activated potassium channels (KCa channels) were detected by a Western blot, qRT-PCR, and immunohistochemical staining. In the MEUS groups, the EB extravasation (11.0 ± 2.2 μg/g in MECUS group and 17.9 ± 2.3 μg/g in MEIUS group) exhibited a significant increase compared with the control group (5.3 ± 0.9 μg/g). The MEIUS group had more EB extravasation than the MECUS group. The Ktrans value of the dynamic contrast-enhanced-MRI in the MEUS groups was higher than that of the control group and correlated strongly with the EB extravasation in the tumor (R2 = 0.97). This showed that the Ktrans value might be a non-invasive method to evaluate the BTB permeability in rat glioma after microbubble-enhanced ultrasound treatment.Western blot, qRT-PCR and immunohistochemical staining revealed that MEUS increased the KCa channels expression and reduced JAM-A expression in glioma. This change was more obvious in the MEIUS group than in the MECUS group. The results demonstrated that MEUS effectively increased the BTB permeability in glioma. The mechanisms might involve the up-regulation of KCa channels expression and affecting the formation of tight junctions in the BTB by a reduction of JAM-A expression. These findings might provide some new guidance for glioma drug therapy.