Streptozotocin induced diabetic retinopathy in rat and the expression of vascular endothelial growth factor and its receptor

·AIM: To establishtherat model of streptozotocin (STZ)- induced diabetic retinopathy (DR), which is the most common cause of visual loss and blindness in patients with diabetes, and observe the gene expression of vascular endothelial growth factor (VEGF) and its receptors during the development of DR. ·METHODS: A rat model of diabetes was established by intraperitoneal injection of STZ. The diabetic rats were housed for 2, 3 and 4 months after the development of diabetes. Retinal histopathological observation was performed. The retinal vessels were observed by immunofluorescence staining by CD31. The mRNA expression of VEGF, VEGF receptor 1 and 2 (VEGFR1/2) in rat retina was detected by reverse transcription - polymerase chain reaction (RT-PCR) analysis. · RESULTS: Retinal histopathological observation showed the morphological changes of inner nuclear layer (INL) and outer nuclear layer (ONL) at any time -point, and also demonstrated the increased new vessels at both 3, 4 months after the development of diabetes. The CD31 staining results showed that the number of vessels was increased in the retinas of diabetic rats at both 3 and 4 months after the development of diabetes. As compared to the normal rats, the mRNA expression of VEGF was increased in retinas of diabetic rats at 3 months after the development of diabetes, while VEGFR1 and VEGFR2 mRNA expression was increased at 2, 3 and 4 months after the development of diabetes.·CONCLUSION:Takentogether,ourresultsdemonstrated that DR was occurred at 3 months after the development of diabetes, and the mRNA expression of VEGF, VEGFR1 and VEGFR2 were increased in the process of DR. The present study further evidenced the involvement of VEGF and its receptors in the process of DR. ·

Molecular Modeling Studies of Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibitors Combining Molecular Docking and 3D-QSAR Methods

The vascular endothelial growth factor （VEGF） and its receptor tyrosine kinases VEGFR-2 or kinase insertdomain receptor （KDR） have emerged as attractive targets for the design of novel anticancer agents. In the present work, molecular docking method combined with three dimensional quantitative structure-activity relationships （comparative molecular field analysis （CoMFA） and comparative molecular similarity indice analysis （CoMSIA）） to analyze the possible interactions between KDR and those derivatives which acted as selective inhibitors. The CoMFA and CoMSIA models gave a cross-validated coefficient Q2 of 0.713 and 0.549, non-cross-validated R2 values of 0.974 and 0.878, and predicted R2 values of 0.966 and 0.823, respectively. The 3D contour maps generated by the CoMFA and CoMSIA models were used to identify the key structural requirements responsible for the biological activity. The information obtained from 3D-QSAR and docking studies were very helpful to design novel selective inhibitors of KDR with desired activity and good chemical property.

Induction of vascular endothelial growth factor receptor expression in human umbilical vein endothelial cells after repeated bevacizumab treatment in vitro

AIM： To investigate the mechanism underlying the loss of responsiveness to anti-vascular endothelial growth factor（VEGF） treatment after repeated injections for choroidal neovascularization, VEGF and VEGF receptor（VEGFR） expressions were evaluated following repeated bevacizumab treatments in hypoxic human umbilical vein endothelial cells（HUVECs） in vitro.METHODS： HUVECs were incubated under hypoxic conditions in two media of different bevacizumab concentrations（1.0 or 2.5 mg/m L） for 17 h, and then in a new medium without bevacizumab for 7h. This procedure was repeated twice more. A culture with an identical volume of excipients served as the control. Cytotoxicity and cell proliferation were assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and Ki-67 assays, respectively. Levels of VEGF and VEGFR were assessed using enzyme-linked immunosorbent assay and Western blot respectively.RESULTS： Cytotoxic effects were not reported for either bevacizumab concentration. Cell proliferation was not reduced after anti-VEGF treatments. VEGF level after single treatment was significantly higher than that of the control and after repeated treatments. Phosphorylated VEGFR-2 expression increased significantly after singleand repeated bevacizumab treatments compared with the control. The 1.0 mg/m L bevacizumab induced significantly higher expressions of VEGFR-2 than the 2.5 mg/m L in single and repeated treatment groups.CONCLUSION： Bevacizumab treatment of HUVECs elevated VEGFR expression in both single and repeated treatments, indicating a mechanism for the reduced efficacy of anti-VEGF therapy in ocular neovascular disorders.

Intravitreal injection of resveratrol inhibits laser-induced murine choroidal neovascularization

AIM:To determine the effects of intravitreal resveratrol(RSV)on murine laser-induced choroidal neovascularization(CNV).METHODS:The toxicity of RSV to choroidal endothelial cell(CEC)was measured using thiazolyl blue tetrazolium bromide(M一)assay.Effects of RSV on choroidal endothelial cell(CEC)migration were evaluated with a modified Boyden chamber assay,while tube formation was evaluated in a 2-D gel assay.CNV was induced by laser photocoagulation in mice.The effects of intravitreal injection of RSV on CNV development were evaluated by fluorescein angiography(FA),confocal analysis of isolectin B4 labeled choroidal flat mounts,and histologic examination of CNV membranes.Immunostaining was used to analyze the expression and phosphorylation of vascular endothelial growth factor receptor 2(VEGFR2).RESULTS:No significant cell toxicity was observed in CEC if the concentration of RSV was less than 200 pmol/L(P>0.05).RSV inhibited vascular endothelial growth factor(VEGF)-induced CEC migration(P<0.05)and tube formation(P<0.05)invitro.Furthermore,intravitrealinjectionof RSV significantly inhibited laser induced CNV formation in mice.The FA leakage,CNV volume and CNV area analysis revealed that there were 41%,45%,and 58%reduction in RSV-treated eyes(1.691±0.1032,178163±78623μm^3 and 6508±619.0μm^2,respectively)compared with those in control(2.724±0.08447,379676±98382μm3and16576±2646μm^2,respectively;P<0.05).Phospho-VEGFR2expression was much weaker in the sections of CNV lesions in RSV injected mice compared with that in control(P<0.05).CONCLUSION:Intravitreal injection of RSV exerts an inhibitory effect on CNV,which may through suppressing endothelial cell migration,tube formation and VEGFR2 phosphorylation.

Clinicopathological and Prognostic Significance of Hypoxia-inducible Factor-1 alpha in Lung Cancer: a Systematic Review with Meta-analysis

Hypoxia-inducible factor-1 alpha（HIF-1α） plays a vital role in the initiation, evaluation and prognosis in lung cancer. The prognostic value of HIF-1α reported in diverse study remains disputable. Accordingly, a meta-analysis was implemented to further understand the prognostic role of HIF-1α in lung cancer. The relationship between HIF-1α and the clinicopathological characteristics and prognosis of lung cancer were investigated by a meta-analysis. Pub Med and Embase were searched from their inception to January 2015 for observational studies. Fixed-effects or random-effects meta-analyses were used to calculate odds ratios and 95% confidence intervals of different comparisons. A total of 20 studies met the criteria. The results showed that HIF-1α expression in lung cancer tissues was significantly higher than that in normal lung tissues. Expression of HIF-1α in patients with squamous cell carcinoma was significantly higher than that of patients with adenocarcinomas. Similarly, non-small cell lung cancer（NSCLC） patients had higher HIF-1α expression than small cell lung cancer（SCLC） patients. Moreover, lymph node metastasized tissues had higher HIF-1α expression than non-lymph node metastasized tissues. A high level HIF-1α expression was well correlated with the expression of vascular endothelial growth factor and epidermal growth factor receptor in the NSCLC. Notably, NSCLC or SCLC patients with positive HIF-1α expression in tumor tissues had lower overall survival rate than patients with negative HIF-1α expression. It was suggested that HIF-1α expression may be a prognostic biomarker and a potential therapeutic target for lung cancer.

Ischemic preconditioning protects against ischemic brain injury

In this study, we hypothesized that an increase in integrin αβand its co-activator vascular endothelial growth factor play important neuroprotective roles in ischemic injury. We performed ischemic preconditioning with bilateral common carotid artery occlusion for 5 minutes in C57BL/6J mice. This was followed by ischemic injury with bilateral common carotid artery occlusion for 30 minutes. The time interval between ischemic preconditioning and lethal ischemia was 48 hours. Histopathological analysis showed that ischemic preconditioning substantially diminished damage to neurons in the hippocampus 7 days after ischemia. Evans Blue dye assay showed that ischemic preconditioning reduced damage to the blood-brain barrier 24 hours after ischemia. This demonstrates the neuroprotective effect of ischemic preconditioning. Western blot assay revealed a significant reduction in protein levels of integrin αβ, vascular endothelial growth factor and its receptor in mice given ischemic preconditioning compared with mice not given ischemic preconditioning 24 hours after ischemia. These findings suggest that the neuroprotective effect of ischemic preconditioning is associated with lower integrin αβand vascular endothelial growth factor levels in the brain following ischemia.

Clinical Significance of VEGF-C and VEGFR-3 Expression in Non-small Cell Lung Cancer

The relationship between vascular endothelial growth factor-C （VEGF-C）/vascular endothelial growth factor receptor 3 （VEGFR-3） expression and clinicopathologic features of the patients with non-small cell lung cancer （NSCLC） was investigated. The expression of VEGF-C and VEGFR-3 was assessed in 65 patients with NSCLC by immunohistochemistry. The significance of VEGF-C and VEGFR-3 expression was analyzed statistically. The results showed that VEGF-C and VEGFR-3 were highly expressed in cytoplasm and membrane in lung cancer tissues with the positive rate being 55.4 % and 52.3 % respectively, while there was no expression in the normal lung tissues. The expression of VEGF-C was significantly increased in adenocacinoma as compared to other types of NSCLC （P〈0.05）. The VEGFR-3 expression was closely related with lymph node metastasis （P〈0.01） and TNM stage （P〈0.05）. There was a positive correlation between the VEGF-C and VEGFR-3 expression in NSCLC patients （r=-0.658, P〈0.01）. It is suggested that VEGFR-3 plays an important role in the lymphatic metastasis of NSCLC. The interaction between VEGF-C and VEGFR-3 may be deeply involved in the mechanism of lung cancer metastasis.

Apatinib as an alternative therapy for advanced hepatocellular carcinoma

Angiogenesis plays an important role in the occurrence and development of tumors.Registered tyrosine kinase inhibitors targeting vascular endothelial growth factor reduce angiogenesis.Apatinib,a tyrosine kinase inhibitor,can specifically inhibit vascular endothelial growth factor receptor 2,showing encouraging anti-tumor effects in a variety of tumors including advanced hepatocellular carcinoma(HCC).This article intends to review the clinical research and application prospects of apatinib in the field of HCC.

Effect of VEGF on Neural Differentiation of Human Embryonic Stem Cells in vitro

The effects of vascular endothelial growth factor （VEGF） on neural differentiation of human embryonic stem cells （hESCs） in vitro and the possible mechanism were observed. The hESCs lines, TJMU1 and TJMU2, were established and stored by our laboratory, hESCs differentiated into neuronal cells through embryonic body formation. In this induction process, hESCs were divided into three groups： group A, routine induction; group B, routine induction＋10 ng/mL VEGF; group C, routine in- duction＋10 ng/mL VEGF＋10 ng/mL VEGFR2/Fc. OCT4, Nestin and GFAP in each group were de- tected by RT-PCR, and the cells expressing Nestin and GFAP were counted by immunofluorescence. The percentage of Nestin positive cells in group B was significantly higher than in groups A and C, while the percentage of GFAP positive cells in group B was significantly lower than in groups A and C （P〈0.01）. There was no significant difference between groups A and C （P〉0.05）. It was concluded that VEGF, via VEGFR2, stimulated the neural differentiation of hESCs in vitro.

Expression of VEGFR2 and NRP-1 in non-small cell lung cancer and their clinical significance

Objective： Vascular-targeted therapy is gradually becoming more appealing for patients with lung cancer. It is unclear whether vascular endothelial growth factor receptor 2（VEGFR2） and neuropilin-1（NRP-1） can be biomarkers for clinical treatment. We aimed to investigate the expression levels of VEGFR2 and NRP-1 in human non-small cell lung cancer（NSCLC） and their clinical significance by observing patient prognosis. Methods： VEGFR2 and NRP-1 were assessed by immunohistochemistry（IHC） in 40 patients with NSCLC and in 10 patients with benign lesions of lung; kinase insert domain receptor（KDR） and NRP-1 copy number gain（CNG） was assessed by fluorescence in situ hybridization（FISH）. The distributions of overall survival（OS） and progression-free survival（PFS） were estimated using the Kaplan-Meier method and compared between groups by log-rank test.Results： Rates of positive immunostaining for VEGFR2 and NRP-1 were 58% and 55%, respectively. KDR and NRP-1 CNG（＋） were detected in 32.5% and 30% of tumors, respectively. Levels of both VEGFR2 and NRP-1 in lung tumors were significantly different than in the control tissue（χ2=11.22, P=0.001; χ2=9.82, P=0.001, respectively）; similar results were obtained using CNGs（χ2=4.39, P=0.036; χ2=3.95, P=0.046, respectively）. Statistically significant correlations were observed with histological grade, clinical TNM stage and the lymph node status（P〈0.05）, but not age, gender or pathology type（P〉0.05）. VEGFR2 showed a strong correlation with NRP-1（Rs=0.68, P=0.00）; similar results were observed with KDR and NRP-1 CNG（Rs=0.32, P=0.04）. Significant differences in OS and PFS were observed between the groups with higher VEGFR2 and NRP-1 and those with lower expression（P〈0.05）. Conclusions： According to these data, VEGFR2 and NRP-1 are highly expressed in NSCLC. We can conclude that they play a key role in NSCLC occurrence, development and metastasis and are associated with patient prognosis（P〈0.05 for OS and PFS）. This information will be beneficial for clinical antiangiogenic treatment in NSCLC.

Age-related changes in cerebral angiogenesis and fetal liver kinase-1 expression after cerebral ischemia/reperfusion

Cerebral angiogenesis in the early stages after cerebral ischemia injury is essential for the recovery of nerve function,in which fetal liver kinase-1（Flk-1）,as a major regulator of vasculogenesis and angiogenesis,plays a very important role.Microvessel density（MVD）was greater in an aged model group compared with the young sham operated group（P 〈 0.01）.MVD and the sum of the lumen area were decreased in the aged group at 1,3,6 and 12 days following ischemia/reperfusion（I/R）injury compared with the young model group（P 〈 0.05 and P 〈 0.01,respectively）.Flk-1 protein and mRNA expression was greater in the aged model group when compared with the young sham operated group（P 〈 0.01）.Flk-1 protein and mRNA expression was lower in the aged group at 1,3,6 and 12 days after I/R compared with the young model group（P 〈 0.01）.Flk-1 expression in aged rats attenuated rapidly,but was still maintained at relatively higher levels at 12 days following I/R in younger rats.The results suggest that angiogenesis was weakened after cerebral I/R in aged rats,and the mechanism of which might be correlated with attenuated expression of Flk-1 protein and mRNA.

Case Report: Pazopanib Treatment Response in a Patient with Metastatic Pleomorphic Dermal Sarcoma (Atypical Fibroxanthoma) with Circulating Tumor Cell-Derived Colonies as a Predictive Marker

Atypical fibroxanthomas (AFX) are rare skin tumors. These generally are superficial tumors, usually <3 cm red, fleshy, ulcerated skin lesions, that characteristically occur on sun-damaged skin, sometimes in immunocompromised or previously irradiated patients. These are part of a spectrum of more aggressive fibro-histiocytic neoplasms. In the older literature, these have been termed aggressive or metastatic AFX, but currently these have been reclassified as pleomorphic dermal sarcomas (PDS) and systemic undifferentiated pleomorphic sarcoma (UPS, formerly malignant fibrohistiocytic sarcoma, MFH). We present the case of a 64-year old woman who developed a deeply invasive PDS on the vertex of her scalp invading to the galea, with in-transit scalp metastases. Very little information is available about optimal treatment of metastatic PDS lesions. The patient was initially treated with 2 cycles of epirubicin/ifosfamide chemotherapy, resulting in life-threatening complications. A pretreatment peripheral blood sample was sent for CTC-derived colony assay. This sample grew 8 colonies from 10 ml blood. The tumor failed to respond to epirubicin and ifosfamide, and after several months of hospitalization, a second peripheral blood CTC-derived colony assay grew >376 colonies. The patient could not tolerate additional chemotherapy. She was therefore treated with the oral targeted agent pazopanib. The patient developed a dramatic biopsy-confirmed complete response. After 11 months of pazopanib treatment, a repeat CTC-derived culture sample grew only 8 colonies/10 ml blood. The complete response to pazopanib is still ongoing at over 41 months. To our knowledge, this is the first demonstration of clinical complete response of a PDS tumor following targeted therapy. An additional novel feature was the demonstration that CTC-derived colonies could be grown from the blood of a PDS patient. The number of colonies appeared to correlate with the clinical treatment response and seemed to function as a potential prognostic marker.

CD4＋VEGFR1HIGH T cell as a novel Treg subset regulates inflammatory bowel disease in lymphopenic mice

Regulatory T cells （Tregs） are a specialized subpopulation of T cells that control the immune response and thereby maintain immune system homeostasis and tolerance to self-antigens. Many subsets of CD4＋ Tregs have been identified, including Foxp3~, Trl, Th3, and Foxp3neg iT（R）35 cells. In this study, we identified a new subset of CD4＋VEGFR1high Tregs that have immunosuppressive capacity. CD4＋VEGFRlhigh T cells, which constitute approximately 1.0% of CD4＋ T cells, are hyporesponsive to T-cell antigen receptor stimulation. Surface marker and FoxP3 expression analysis revealed that CD4＋VEGFR1high T cells are distinct from known Tregs. CD4＋VEGFR1high T cells suppressed the proliferation of CD4＋CD25- T cell as efficiently as CD4＋CD25high natural Tregs in a contact-independent manner. Furthermore, adoptive transfer of CD4＋VEGFR1＋ T cells from wild type to RAG-2-deficient C57BL/6 mice inhibited effector T-cell-mediated inflammatory bowel disease. Thus, we report CD4＋ VEGFR1high T cells as a novel subset of Tregs that regulate the inflammatory response in the intestinal tract.