Effect of VEGF,P53 and telomerase on angiogenesis of gastric carcinoma tissue

Objective:To investigate the effect of vascular endothelial growth factor(VEGF),P53 and telomerase on angiogenesis in gastric carcinoma tissue.Methods:A total of 95 surgical resection samples of gastric cancer tissue after pathological diagnosis are collected to observe the VEGF,P53 and telomerase expression using immunohistochemical methods.Relationship between their expression and its influence on angiogenesis in gastric carcinoma tissue were analyzed.Results:Microvascular density(MVD)and the expression of VEGF,P53 and telomerase were positively correlated.Expression of VEGF and P53 protein were related to tumor type and lymph metastasis,and also a correlation was observed between P53 and VEGF.The telomerase expression had no correlation with VEGF,and P53.Conclusions:VEGF angiogenesis has a angiogenesis promoting effect on gastric cancer tissue development and plays an important role in tumor generation and metastasis.Mutant P53 promotes the tumor angiogenesis generation by adjusting VEGF.Telomerase has a certain role in promoting activity of angiogenesis through different way rather than P53.

Over-expression of VEGF in Marrow Stromal Cells Promotes Angiogenesis in Rats with Cerebral Infarction via the Synergistic Effects of VEGF and Ang-2

This study explored whether the transplantation of modified marrow stromal cells (MSCs) has angiogenic effects in a left middle cerebral artery occlusion infarction/reperfusion (MCAO I/R) rat model and preliminarily examined the mechanism of angiogenesis following cerebral infarction.MSCs were isolated by using a direct adherent method and cultured.Vascular endothelial growth factor (VEGF) was transfected into MSCs by employing the liposome transfection.The transfection efficiency was measured by the optical density method.The protein expression of VEGF gene before and after transfection was measured by Western blotting.SD rat model of transient occlusion of the left middle cerebral artery was established by using an approach of intra-luminal occlusion.Tetrazolium (TTC) and HE staining were performed to observe the cerebral infarction.ELISAs were used to measure the levels of VEGF in the rat cerebral tissues.The expression patterns of angiopoietin-2 (Ang-2) and CD34 in cells surrounding the area of infarction were immunohistochemistrically oserved.Ang-2 protein expression in the tissue surrounding the area of infarction was measured by Western blotting.VEGF expression in the MSCs increased after transfection at a rate of approximately 28%±3.4%.ELISA showed that the expression of VEGF in the cerebral tissue was significantly increased after induction of infarction,peaking on the 4th day and decreasing to the levels of the sham surgery group (normal) within 7 to 10 days.The VEGF level was significantly higher at each time point in the VEGF-MSC and MSC groups compared to the model group.Moreover,the VEGF level was higher in the VEGF-MSC group than in the MSC group and stayed relatively high until the 10th day.The immunohistochemical results showed that 10 days after the infarction,the number of Ang-2 and CD34-expressing cells in the area surrounding the infarction was significantly higher in the VEGF-MSC group and the MSC group compared to the model group.Moreover,the VEGF level was higher in the VEGF-MSC group than the MSC group.A similar trend in Ang-2 protein expression was revealed by Western blotting.In the MCAO rat model transfected with modified MSCs over-expressing VEGF,compared to the MSC transplantation group,the concentration of VEGF was significantly increased in the brain tissue after cerebral infarction.In addition,the level of Ang-2 was up-regulated,with angiogenesis promoted,the blood supply to the areas surrounding the cerebral infarction increased,and neurological function improved.We are led to speculate that the synergistic effects of VEGF and Ang-2 may be responsible for the angiogenesis following cerebral infarction.

Overexpression of ACE2 ameliorates Aβ-induced blood–brain barrier damage and angiogenesis by inhibiting NF-κB/VEGF/VEGFR2 pathway

Background:Pathological angiogenesis and blood–brain barrier damage may play an important role in Alzheimer's disease(AD).ACE2 is mainly expressed on the surface of endothelial cells in brain.Recent studies have shown that the expression of ACE2 in AD is reduced,but its role in AD is still unclear.Method:We induced AD damage in endothelial cells using Aβ25-35 and overexpressed ACE2 in bEend.3 cells through lentiviral transfection.We detected the effect of Aβ25-35 on cell viability using the CCK-8 assay and examined the effect of overexpressing ACE2 on angiogenesis using an angiogenesis assay.We used western blot and cell immunofluorescence to detect changes in the expression of the VEGF/VEGFR2 pathway,tight junction protein,and NF-κB pathway.Results:Aβ25-35 treatment significantly decreased the expression of ACE2 and reduced cell viability.ACE2 overexpression(1)reduced the number of branches and junctions in tube formation,(2)inhibited the activation of the VEGF/VEGFR2 pathway induced by Aβ25-35,(3)increased the expression of TJPs,including ZO-1 and claudin-5,and(4)restored Aβ25-35-induced activation of the NF-κB pathway.Conclusion:Overexpression of ACE2 can improve pathological angiogenesis and blood–brain barrier damage in AD models in vitro by inhibiting NF-κB/VEGF/VEGFR2 pathway activity.ACE2 may therefore represent a therapeutic target for endothelial cell dysfunction in AD.

Hypoxia-induced factor-1 alpha upregulates vascular endothelial growth factor C to promote lymphangiogenesis and angiogenesis in breast cancer patients

Hypoxia-induced factor-1 alpha （HIF-1α） affects many effector molecules and regulates tumor lymphangio- genesis and angiogenesis during hypoxia. The aim of this study was to investigate the role of HIF-1α in the regu- lation of vascular endothelial growth factor C （VEGF-C） expression and its effect on lymphangiogenesis and an- giogenesis in breast cancer. Lymphatic vessel density （LVD）, microvessel density （MVD） and the expressions of HIF-1α and VEGF-C proteins were evaluated by immunohistochemistry in 75 breast cancer samples. There was a significant correlation between HIF-1α and VEGF-C （P = 0.014, r = 0.273, Spearman＇s coefficient of correlation）. HIF-1α and VEGF-C overexpression was significantly correlated with higher LVD （P = 0.003 and P = 0.017, re- spectively）, regional lymph nodal involvement （P = 0.002 and P = 0.004, respectively） and advanced tumor, node, metastasis （TNM） classification （P = 0.001 and P = 0.01, respectively）. Higher MVD was observed in the group expressing higher levels of HIF-1α and VEGF-C （P = 0.033 and P = 0.037, respectively）. Univariate analysis showed shorter survival time in patients expressing higher levels of HIF-1α and VEGF-C. HIF-1α was also found to be an independent prognostic factor of overall survival in multivariate analysis. The results suggest that HIF-1α may affect VEGF-C expression, thus acting as a crucial regulator of lymphangiogenesis and angiogenesis in breast cancer. This study highlights promising potential of HIF- 1α as a therapeutic target against tumor lymph node me- tastasis.

Clinical implication of VEGF serum levels in cirrhotic patients with or without portal hypertension

AIM To determine whether serum vascular endothelial growth factor (VEGF) levels correlates with the severity of liver cirrhosis and whether portal hypertension impacts on the expression of serum VEGF protein. METHODS Fifty three patients (mean age 56±2 years) with HCV ( n =26), HBV ( n =13), and cryptogenic liver cirrhosis ( n =14) (Child Pughs class A: 24, B: 19 and C: 12) and normal renal function constitute the patient population, who were all diagnosed by clinical, histological and radiological findings. Six healthy people and six patients with acute hepatitis served as controls. Severity of liver disease was evaluated by the CP score. Serum levels of IGF 1 and VEGF were measured by radioimmunoassay and ELISA, respectively. Portal hypertension was assessed using pulsed Doppler ultrasound. RESULTS The mean serum VEGF levels in all cirrhotic patients (73±58) were significantly lower than those of healthy controls (360±217, P <0 01) and acute hepatitis (1123±1261, P <0 01) respectively. No significant difference in median serum VEGF levels were noted among the different Child Pughs classes (class A: median, 49 4ng/L , range, 21ng/L - 260ng/L , Class B: median 59 9ng/L ; range 21-92, and Class C: median 69; range 20ng/L - 247ng/L ). A significant correlation was noted between serum VEGF and two accurate parameters of portal hypertension: portal blood flow velocity ( r =0 6) and spleen size ( r =0 55). No correlation was found between VEGF serum levels and serum albumin, IGF 1, platelets count and aminotrasnferases ( r =0 2, r =0 1, r =0 2 and r =0 2, respectively). CONCLUSION Circulating VEGF level in patients with liver cirrhosis could not serve as an indicator of the progression of chronic liver disease but rather, they may reflect increased portal hypertension or decreased hepatic regenerative activity or the combination of both.

The molecular mechanism underlying angiogenesis in hepatocellular carcinoma: the imbalance activation of signaling pathways

OBJECTIVE: To explore the effect of two dominating signaling pathways, VEGF/KDR and angiopoietins/Tie2, on the formation of new blood vessel in hepatocellular carcinoma (HCC) growth and metastasis. METHODS: RT-PCR and Western blot were employed to evaluate the VEGF/KDR and angiopoietins/Tie2 expression in samples from 23 patients with HCC. Meanwhile, microvessel density (MVD) was determined as a marker of angiogenesis by counting CD34 positive cells with the method of immunohistochemistry. RESULTS: The two pathways were activated in all HCC samples. The expressions of vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang2) were significantly higher (P<0.05) in hepatocellular carcinoma tissues and the margin of the tumor than those in control groups, and so did CD34 positive cells. Although significant difference in the expression of kinase insert domain containing receptor (KDR) and Ang1/Tie2 was not observed in all groups, their distinct high levels were seen in hepatoma and its margin compared with normal and cirrhotic liver. VEGF and Ang2 expressions were seen up-regulated in HCC with vascular invasion and satellite lesion. CONCLUSIONS: The two signaling pathways, VEGF/KDR and angiopoietins/Tie2 are activated in the process of angiogenesis in HCC and modulate the formation of new blood vessels. The imparity of the two signaling pathways' activation is to benefit HCC metastasis. In the two pathways, VEGF and Ang2 may play an important role in the process of angiogenesis, and are necessary indicators for the prognosis and metastasis of HCC. This study provides another clue for the exploration of anti-angiogenic agents.

Impaired tumor angiogenesis and VEGF- induced pathway in endothelial CD146 knockout mice

CD146 is a newly identified endothelial biomarker that has been implicated in angiogenesis. Though in vitro angio- genic function of CD146 has been extensively reported, in vivo evidence is still lacking. To address this issue, we generated endothelial-specific CD146 knockout （CD146 EC-Ko） mice using the Tg（Tek-cre） system. Surprisingly, these mice did not exhibit any apparent morphological defects in the development of normal retinal vasculature. To evaluate the role of CD146 in pathological angiogenesis, a xenograft tumor model was used. We found that both tumor volume and vascular density were significantly lower in CD146Ec-KO mice when compared to WT littermates. Additionally, the ability for sprouting, migration and tube formation in response to VEGF treatment was impaired in endothelial cells （ECs）of CD146Ec-Ko mice. Mechanistic studies further confirmed that VEGF- induced VEGFR-2 phosphorylation and AKT/p38 MAPKs/ NF-KB activation were inhibited in these CD146-null ECs, which might present the underlying cause for the observed inhibition of tumor angiogenesis in CD146Ec-Ko mice. These results suggest that CD146 plays a redundant role in physiological angiogenic processes, but becomes essential during pathological angiogenesis as observed in tumorigenesis.

Indomethacin suppresses growth of colon cancer via inhibition of angiogenesis in vivo

AIM: It has been reported that regular consumption of nonsteroidal anti-inflammatory drugs like indomethacin decreases the incidence and mortality rate of a number of gastrointestinal cancers. We aimed to explore the efficacy and possible mechanisms of indomethacin on tumor growth and tumor angiogenesis of human colon cancer xenografts in nude mice. METHODS: MTT (thiazolyl blue) assay was used to assess the effect of indomethacin on cultured human colorectal cancer cell line HCT116. HCT116 cells were inoculated subcutaneously into BALB/c-nu/nu mice. After oral administration of indomethacin, 3 mg/kg·d for 4 wk, animals were sacrificed by cervical dislocation. Immunohistochemical staining was employed to determine the microvessel density (MVD) and vascular endothelial growth factor (VEGF) expression in tumor tissues. RESULTS: Indomethacin, a non-selective COX inhibitor, significantly decreased the viability of HCT116 cells in a dose-dependent manner (P<0.05) with 50% inhibition at approximately 318.2±12.7 μmol/L Growth of HCT116 cell tumor was significantly suppressed by indomethacin. The tumor volume was significantly decreased in the treated group (458.89±32.07 mm3) compared to the control group (828.21±31.59 mm3) (P<0.05). The MVD of the treated group (19.50±5.32) was markedly decreased compared to the control group (37.40±4.93) (P<0.001). The VEGF expression of the treated group (1.19±0.17) was obviously reduced as compared to the control group (1.90±0.48) (P<0.01). The decrease in MVD was positively correlated with the decrease of VEGF expression (rs = 0.714, P<0.05). We did not see gastrointestinal complications in the treated group and no differences were noted in the body weight of the mice between the two groups throughout the study CONCLUSION: Indomethacin can significantly decrease the viability of cultured HCT116 cells and retard human colorectal HCT116 cell tumor growth via inhibiting tumor angiogenesis, which might be through reduction of VEGF expression.

瘦素 瘦素受体 VEGF和CD34在结直肠癌中的表达

目的:探讨瘦素(LePtin)、瘦素受体(Ob-R)、血管内皮生长因子(VEGF)和CD34蛋白(标记组织微血管密度以反映血管形成活跃程度的特异性抗体)在结直肠癌中的表达及其生物学意义方法:应用免疫组化SP法检测68例结直肠癌患者的结直肠癌组织、癌旁组织和正常结直肠组织中瘦素、瘦素受体、VEGE和CD34的表达情况,结合临床病理资料进行分析、结果:瘦素、瘦素受体和VEGE在结直肠癌组织中的阳性表达率明显高于癌旁组织和正常结直肠组织,其表达与肿瘤的病理学分级肠壁浸润深度、淋巴结转移、Dukes分期、远处转移及有脉管瘤检明显相关微血管密度(MVD值)在结直肠癌组织中的表达明显高于癌旁组织和正常结直肠组织,癌旁组织高于正常组织在结直肠癌组织中瘦素、瘦素受体、VEGF的表达与CD34表达具有一致性。结论:微血管密度是衡量结直肠癌发展、浸润及转移的重要指标瘦素与瘦素受体结合促进结直肠癌细胞增殖瘦素与 VEGF协同作用可促进结直肠癌新生血管形成。

All-trans retinoic acid upregulates VEGF expression in glioma cells in vitro

All-trans retinoid acid （ATRA） is one of the most potent and most thoroughly studied differentiation inducers that induce the differentiation and apoptosis of glioma cells. However, the effect of ATRA on angiogenesis of glioma re- mains poorly understood. We examined the effect of ATRA on the expression of vascular endothelial growth fac- tor （VEGF） in different glioma cell lines and investigated the underlying mechanism, intending to partially reveal the effects of ATRA on angiogenesis of glioma. Glioma cells were treated by ATRA at 5 and 10 μmol/L. The VEGF mRNA transcript levels were determined by real-time RT-PCR and the protein levels of VEGF in glioma cells were evaluated by Western blotting assays. Moreover, hypoxia-inducible factor-1α （HIF-la） mRNA expression was analyzed by using real-time RT-PCR. After treatment with 5 and 10 μmol/L ATRA, the VEGF mRNA tran- script levels in glioma cells increased remarkably, compared with that in the control group, and the relative protein expression of VEGF was also up-regulated. Meanwhile, the HIF-la mRNA expression also increased. ATRA in- creases the expression of VEGF in glioma cells at both transcriptional and translational levels.

Inhibitory Effects of Parthenolide on the Angiogenesis Induced by Human Multiple Myeloma Cells and the Mechanism

The inhibitory effects of parthenolide （PTL） on angiogenesis induced by multiple myeloma （MM） cells in vitro and the mechanism were investigated. Human MM line RPMI8226 cells were cultured in vitro. The effects of MM culture supernatant on the migration and tubule formation ability of human umbilical vein endothelial cells （HUVECs） treated with PTL were observed. By using Western blot, the expression of p65 and IкB-α in MM cells was detected. RT-PCR was used to assay the expression of VEGF, IL-6, MMP2 and MMP9 mRNA in MM cells. ELISA was used to measure the levels of VEGF and IL-6 in MM cell culture supernatant. The expression of MMP2 and MMP9 in MM cells was examined by immunohistochemistry. （1） In 3.5, 5.0, 7.5 and 10 μmol/L PTL groups the number of migrated cells was 310±56, 207±28, 127±21 and 49±10 respectively, which was significantly different from that in positive control group （598±47） （P〈0.01）. In 3.5 and 5.0 μmol/L PTL groups the areas of capillary-like structures were 0.092±0.003 and 0.063±0.002 mm2, significantly less than in positive control group （0.262±0.012 mm2） （P〈0.01）, but in 7.5 and 10 μmol/L PTL groups no capillary-like structures were found; （2） After treatment with different concentrations of PTL for 48 h, the expression of p65 protein was gradually decreased, while that of IкB-α was gradually enhanced with the increased concentration of PTL; （3） After treatment with 3.5, 5.0, 7.5 and 10 μmol/L PTL for 48 h, the VEGF levels in the supernatant were 2373.4±392.2, 1982.3±293.3, 1247.0±338.4 and 936.5±168.5 pg/mL respectively, significantly different from those in positive control group （2729±440.0 pg/mL） （P〈0.05）. After treatment with 7.5 and 10 μmol/L PTL, the IL-6 levels in the culture supernatant were 59.6±2.8 and 41.4±9.8 pg/mL respectively, signifi- cantly lower than in positive control group （1287.3±43.5 pg/mL） （P〈0.05）; （4） RT-PCR revealed that PTL could significantly inhibit the expression of VEGF and IL-6 mRNA in MM cells, but not influence the expression of MMP2 and MMP9 mRNA.; （5） Immunohistochemistry indicated that PTL had no significant effects on the expression of MMP2 and MMP9 protein in MM cells. It was concluded that the abilities of the culture supernatant of MM cells treated with PTL to induce endothelial cells migration and tubule formation were significantly reduced, suggesting PTL could obviously inhibit the angiogenesis induced by MM cells. PTL could decrease NF-kappaB activity and significantly suppress the expression of VEGF and IL-6 mRNA and protein, which might contribute to the mechanism by which PTL inhibited the angiogenesis induced by MM cells.

Homoharringtonine induces apoptosis of endothelium and down－regulates VEGF expression of K562 cells

Homoharringtonine (HHT) has currently been used successfully in the treatment of acute and chronic myeloid leukemias and has been shown to induce apoptosis of different types of leukemic cells in vitro. Emerging evidence suggests that angiogenesis may play an important role in hematological malignancies, such as leukemia. However, whether HHT can relieve leukemia by anti-angiogenesis is still unknown. We investigated the anti-angiogenesis potential of HHT with the human umbilical vein endothelial cell line (ECV304) and leukemic cell line (K562) in vitro. Cellular proliferation was determined by MTT assay and apoptosis was analyzed by flow cytometry, The mRNA expression of vascular endothelial growth factor (VEGF) was assessed by RT-PCR and VEGF protein production was detected by Western blot. Inhibition of cell proliferation and induction of apoptosis by HHT were discovered in ECV304 cells, and appeared in a dose- and time-dependent manner, Also, treatment with HHT caused down-regulation of VEGF mRNA expression in K562 cells in similar dose- and time-dependent manner and inhibition of VEGF protein production in K562 cells in response to the enhancing concentration of HHT. The results demonstrated that HHT could also induce apoptosis in endothelium and down-regulate VEGF expression in K562 cells. In conclusion, we believe HHT has anti-angiogenesis potential and speculate that HHT might exert its anti-leukemia effects via reduction of angiogenesis.

Anticancer effect of Psidium guajava(Guava) leaf extracts against colorectal cancer through inhibition of angiogenesis

Objective:To evaluate the anti-angiogenic and anticancer activities of Psidium guajava leaf extracts against angiogenesis-dependent colorectal cancer.Methods:Three extracts were produced using distilled water,ethanol,and n-hexane as solvents.The extracts were physically characterised through gas chromatography–mass spectrometry,ultraviolet–visible spectroscopy,and Fourier transform infrared spectroscopy.Their antioxidant activity was evaluated using the 2,2-diphenyl-1-picrylhydrazyl,total phenolic content,and total flavonoid content assays.To assess their anti-angiogenic activity,cell viability and rat aortic ring assays were conducted,while cell migration,tube formation,colony formation,and VEGF ELISA assays were conducted to elucidate their effects on different aspects of angiogenesis.Molecular docking was used to assess the antiangiogenic potential of some possible compounds in the extracts.Tumour spheroid assay was used to assess the extracts’potential as a treatment for colorectal cancer.Results:The ethanol extract showed the best antioxidant activity.The distilled water and ethanol extracts exhibited more inhibitory activity against EA.hy926 cell viability and aortic ring microvessel growth.In addition,the ethanol extract performed significantly better than the distilled water extract against cell migration and colony formation,and VEGF expression of the cells was suppressed by the ethanol extract.Both the distilled water and ethanol extracts showed significant inhibitory effect on EA.hy926 tube formation and tumour spheroids consisting of EA.hy926 and HCT116 cells.The ethanol extract containedβ-caryophyllene andβ-elemene by phytochemical analysis and subsequent docking studies,which may contribute to its anti-angiogenic activity.Conclusions:The ethanol extract of Psidium guajava has potential in the treatment of colorectal cancer through the inhibition of angiogenesis.

Enhanced angiogenesis by the hyaluronic acid hydrogels immobilized with a VEGF mimetic peptide in a traumatic brain injury model in rats

Angiogenesis plays an important role in brain injury repair,which contributes to the reconstruction of regenerative neurovascular niche for promoting axonal regeneration in the lesion area.As a major component of developing brain extracellular matrix,hyaluronic acid(HA)has attracted more attention as a supporting matrix for brain repair.In the present study,HA-KLT hydrogel was developed via modifying HA with a VEGF mimetic peptide of KLT(KLTWQELYQLKYKGI).The characterization of the hydrogel shows that it could provide a porous,three-dimensional scaffold structure,which has a large specific surface area available for cell adhesion and interaction.Compared with the unmodified HA hydrogel,the HA-KLT hydrogel could effectively promote the attachment,spreading and proliferation of endothelial cells in vitro.Furthermore,the pro-angiogenic ability of hydrogels in vivo was evaluated by implanting them into the lesion cavities in the injured rat brain.Our results showed that the hydrogels could form a permissive interface with the host tissues at 4 weeks after implantation.Moreover,they could efficiently inhibit the formation of glial scars at the injured sites.The HA-KLT hydrogel could significantly increase the expression of endoglin/CD105 and promote the formation of blood vessels,suggesting that HA-KLT hydrogel promoted angiogenesis in vivo.Collectively,the HA-KLT hydrogel has the potential to repair brain defects by promoting angiogenesis and inhibiting the formation of glial-derived scar tissue.

Design and semisynthesis of oleanolic acid derivatives as VEGF inhibitors:Inhibition of VEGF-induced proliferation,angiogenesis,and VEGFR2 activation in HUVECs

Angiogenesis inhibitors targeting the VEGF signaling pathway are developed into drugs for the treatment of vaious diseases,such as cancer,rheumatoid arthritis,and age-related macular degeneration.Recent studies have revealed that oleanolic acid(OA),a natural pentacyclic triterpenoid,inhibited the VEGF/VEGFR2 signaling pathway and angiogenesis in HUVECs,which may represent an attractive VEGF inhibitor.In this paper,rational structural modification towards OA was performed in order to improve its inhibitory effects aganist VEGF and anti-angiogenesis potential.As a result,a series of novel OA derivatives,possessingα,β-unsat-urated ketone system in ring A and amide functional group at C-28,were prepared and evaluated for cytotoxicity and their ability to inhibit VEGF-induced abnormal proliferation of HUVECs.The results showed that two promising derivatives,OA-1 and OA-16,exhibited no in vitro cytotoxicity against HUVECs but showed more potent inhibitory activity against VEGF-induced proliferation and angiogenesis in HUVECs,compared with OA.The results of Western blot indicated that OA-1 and OA-16 inhibited VEGF-induced VE-GFR2 activation.Furthermore,small interfering RNA experiments were performed to confirm that both compounds inhibited VEGF-induced angiogenesis via VEGFR2.Thus,the present study resulted in the discovery of new promising OA-inspired VEGF inhibitors,which can serve as potential lead compounds for the treatment of angiogenesis-related diseases.

The Circadian System Is Essential for the Crosstalk of VEGF-Notch-mediated Endothelial Angiogenesis in Ischemic Stroke

Ischemic stroke is a major public health problem worldwide.Although the circadian clock is involved in the process of ischemic stroke,the exact mechanism of the circadian clock in regulating angiogenesis after cerebral infarction remains unclear.In the present study,we determined that environmental circadian disruption(ECD)increased the stroke severity and impaired angiogenesis in the rat middle cerebral artery occlusion model,by measuring the infarct volume,neurological tests,and angiogenesis-related protein.We further report that Bmal1 plays an irreplaceable role in angiogenesis.Overexpression of Bmal1 promoted tube-forming,migration,and wound healing,and upregulated the vascular endothelial growth factor(VEGF)and Notch pathway protein levels.This promoting effect was reversed by the Notch pathway inhibitor DAPT,according to the results of angiogenesis capacity and VEGF pathway protein level.In conclusion,our study reveals the intervention of ECD in angiogenesis in ischemic stroke and further identifies the exact mechanism by which Bmal1 regulates angiogenesis through the VEGF-Notch1 pathway.

A self-assembling peptide targeting VEGF receptors to inhibit angiogenesis

Vascular endothelial growth factor(VEGF)-vascular endothelial growth factor receptor(VEGFR)pathways are essential in tumor angiogenesis,growth and metastasis.Studies on anti-angiogenic therapy have been mostly focused on the blockage of VEGF-VEGFR pathways.We report an extracellularly transformable peptide-based nanomaterial to develop artificial extracellular matrix(ECM)-like networks for high-efficient blockage of natural VEGF-VEGFR interactions.The transformable peptide-based nanomaterial transforms from nanoparticles into nanofibers upon binding to VEGFR in solution.In addition,the transformable peptide-based nanomate rial forms ECM-like fibrous netwo rks on VEGFR overexpressed cells,inhibiting the VEGF-VEGFR interactions and the subsequent angiogenesis.The tube formation is reduced by nearly 85.1% after treatment.This strategy shows excellent potential for anti-angiogenesis,and inhibition of tumor invasion and metastasis.

Vascular endothelial growth factor induced angiogenesis following focal cerebral ischemia/reperfusion injury in rabbits

BACKGROUND： Therapeutic angiogenesis has opened up new pathway for the treatment of ischemic cerebrovascular disease in recent years. The exploration of the effect of vascular endothelial growth factor （VEGF） on inducing angiogenesis following ischemia/reperfusion injury can provide better help for the long-term treatment of cerebrovascular disease in clinic. OBJECTIVE： To observe the effect of VEGF on inducing angiogenesis following focal cerebral ischemia /reperfusion injury in rabbits through the angiogenesis of microvessels reflected by the expression of the factors of vascular pseudohemophilia. DESIGN： A randomized controlled animal tria SETTNG： Department of Medical Imaging, Second Hospital of Hebei Medical University MATERIALS： Sixty-five healthy male New Zealand rabbits of clean degree, weighing （2.6±0.2） kg, aged 4.5-5 months, were used. The polyclonal antibody against vascular pseudohemophilia （Beijing Zhongshan Company）, recombinant VEGF165 （Peprotech Company, USA）, biotinylated second antibody and ABC compound （Wuhan Boster Company） were applied. METHODS： The experiments were carried out in the Laboratory of Neuromolecular Imaging and Neuropathy, Second Hospital of Hebei Medical University from May to August in 2005. （1） The rabbits were randomly divided into three groups： sham-operated group （n=15）, control group （n=25） and VEGF-treated group （n=-25）. In the control group and VEGF-treated group, models were established by middle cerebral artery occlusion （MCAO） induced focal cerebral ischemia/reperfusion. In the VEGF-treated group, VEGF165 （2.5 mg/L） was stereotactically injected into the surrounding regions of the infarcted sites immediately after the 2-hour ischemia/reperfusion; Saline of the same dosage was injected in the control group. But the rabbits in the sham-operated group were only drilled but not administrated. （2） The experimental indexes were observed on the 3^rd 7^th, 14^th, 28^th and 70^th days of the experiment respectively, 3 rabbits in the sham-operated group and 5 in the control group and VEGF-treated group were observed at each time point. The brain tissues in the surrounding regions of the infarcted sites were collected. The positive expressions of the factors of vascular pseudohemophilia in vascular endothelial cells were analyzed with immunohistochemical method. The microvessels in unit statistical field were counted with the imaging analytical software. MAIN OUTCOME MEASURES： The changes of microvascular density in the brain tissue and the positive expressions of the factors of vascular pseudohemophilia in the surrounding regions of the infarcted sites were observed on the 3^rd 7^th, 14^th, 28^th and 70^th days of the experiment. RESULTS： All the 65 New Zealand rabbits were involved in the analysis of results without deletion. Changes of the number of microvessels at different time points in each group： There were no obvious changes at different time points in the sham-operated group. The numbers of microvessels at 7 and 14 days were obviously more in the control group than in the sham-operated group [（6.0±1.1）, （9.0±0.9） microvessels; （3.0±1.1）, （3.0±1.1） microvessels; P〈 0.05-0.01], and those at 3, 7, 14 and 28 days were obviously more in the VEGF-treated group than in the control group [（8.3±2.0）, （13.4±1.4）, （15.5±2.3）, （6.8± 1.0） microvessels; （3.4±0.6）, （6.0±1.1）, （9.0±0.9）, （3.2±0.8） microvessels; P 〈 0.01]. （2） Positive expressions of the factors of vascular pseudohemophilia in the surrounding regions of infarcted sites： There were no obvious changes at different time points in the sham-operated group. In the control group, the changing law of the expressions was the same as that for the number of microvessels that the expression began to mildly increase at 7 days, reached the peak value at 14 days, and began to reduce at 28 days. In the VEGF-treated group, the expression was obviously increased at 3 days, also reached the peak value at 14 days, and reduced to the normal level at 70 days, but the expressions were obviously stronger than those in the control group at the same time points. CONCLUSION： Angiogenesis can be obviously induced in rabbits after the focal cerebral ischemia/reperfusion injury is treated with VEGF for 18 days.

中药复方血清对HUVEC血管形成及Gli1 VEGF表达的影响

目的:观察中药复方肠复康(CFK)动物血清对HUVEC血管形成及Gli 1、VEGF表达的影响。方法:采用双室联合培养人脐静脉内皮细胞(HUVEC)和人结肠癌(Lovo)细胞,观察CFK含药血清对HUVEC血管形成的影响,并观察Gli 1及VEGF表达的变化。结果:CFK含药血清作用后,HUVEC增殖活性明显降低,其成管指数明显降低,Gli 1、VEGF表达明显减少,与对照组比较均有显著性差异(P<0.05或P<0.01)。结论:CFK具有抑制大肠癌血管生成作用。其机制与其抑制Hedgehog-Gli1信号通路激活,下调VEGF表达而发挥抗肿瘤血管形成作用有关。

Scopolin isolated from Erycibe obtusifolia Benth stems suppresses adjuvant-induced rat arthritis by inhibiting inflammation and angiogenesis

Objective To study the effects and mechanisms of scopolin isolated from the stems of Erycibe obtusifolia Benth in arthritis-associated inflammation and angiogenesis.Methods Adjuvant-induced arthritic rat,an animal model for human RA was used in this study for examining the potential remedial effect of scopolin.The swelling in both inoculated and non-inoculated paws,body weights and articular index(AI)scores were detected to evaluate the severity of the arthritis.Histologic assessment of tissue sections from rat ankles was also performed.Furthermore,the blood vessel density in the synovial tissues was quantitatively evaluated.In addition,expressions of VEGF,FGF-2,TNF-α,IL-1β and IL-6 in rat synovial tissues were determined by immunohistochemistry assay in an attempt to explain the mechanisms of scopolin for suppressing arthritis.Results Scopolin dose-dependently inhibited both inoculated and non-inoculated paw swelling in rat AIA.The mean AI scores of scopolin treated groups were also dose-dependently lower than that of model group.In addition,compared with the weights of model group,the mean body weights of rats treated with scopolin(50,100 mg·kg-1)were higher from day 13 to 22,perhaps indicative of healthier animals.The histologic architecture of the joint was highly abnormal in the model group rats,while high dose of scopolin treated rats preserved a nearly normal histologic architecture of the joint.Moreover,the new blood vessels were reduced dose-dependently in the synovial tissue of rat AIA treated with scopolin.Further,scopolin reduced the overexpression of IL-6,VEGF and FGF-2 in rat synovial tissues.Conclusions Scopolin is capable of reducing clinical symptoms of rat AIA by inhibiting inflammation and angiogenesis,and this compound may be a potent therapeutic agent for angiogenesis related diseases and can serve as structural base for screening for more potent synthetic analogs.