Telencephalic stab wound injury induces regenerative angiogenesis and neurogenesis in zebrafish:unveiling the role of vascular endothelial growth factor signaling and microglia

After brain damage,regenerative angiogenesis and neurogenesis have been shown to occur simultaneously in mammals,suggesting a close link between these processes.However,the mechanisms by which these processes interact are not well understood.In this work,we aimed to study the correlation between angiogenesis and neurogenesis after a telencephalic stab wound injury.To this end,we used zebrafish as a relevant model of neuroplasticity and brain repair mechanisms.First,using the Tg(fli1:EGFP×mpeg1.1:mCherry)zebrafish line,which enables visualization of blood vessels and microglia respectively,we analyzed regenerative angiogenesis from 1 to 21 days post-lesion.In parallel,we monitored brain cell proliferation in neurogenic niches localized in the ventricular zone by using immunohistochemistry.We found that after brain damage,the blood vessel area and width as well as expression of the fli1 transgene and vascular endothelial growth factor(vegfaa and vegfbb)were increased.At the same time,neural stem cell proliferation was also increased,peaking between 3 and 5 days post-lesion in a manner similar to angiogenesis,along with the recruitment of microglia.Then,through pharmacological manipulation by injecting an anti-angiogenic drug(Tivozanib)or Vegf at the lesion site,we demonstrated that blocking or activating Vegf signaling modulated both angiogenic and neurogenic processes,as well as microglial recruitment.Finally,we showed that inhibition of microglia by clodronate-containing liposome injection or dexamethasone treatment impairs regenerative neurogenesis,as previously described,as well as injury-induced angiogenesis.In conclusion,we have described regenerative angiogenesis in zebrafish for the first time and have highlighted the role of inflammation in this process.In addition,we have shown that both angiogenesis and neurogenesis are involved in brain repair and that microglia and inflammation-dependent mechanisms activated by Vegf signaling are important contributors to these processes.This study paves the way for a better understanding of the effect of Vegf on microglia and for studies aimed at promoting angiogenesis to improve brain plasticity after brain injury.

Effect of Danzhi decoction on expression of angiogenesis factors in patients with sequelae of pelvic inflammatory disease

Objective:To investigate the efteets of traditional Chinese medicine.Danzhi decoction,on the expression angiogenesis factors in human endometrial cells during the sequelae of pelvic inflammatoty disease(SPID) and explore the role of Danzhi decction in improving the blood stasis microenvironment of SPID.Methods:A three-dimensional(3D) co—culture system including human vascular endothelial cells(VECs),endometrial stromal cells and glandular epithelial cells was established in vitro and treated with Danzhi decoction,sterilized water and aspirin respectively.A Milliplex multifunctional liquid chip technique was used to measure the expression levels of vascular endothelial growth factor(VEGF)—A/C/D,fibroblast growth factor-1/2.angiopoietin-2.epidermal growth factor(EGF) HB-EGF,bone morphogenetic protein-9.endoglin.endothehn-l.granulocyte colony stimulating factor,hepatocyte growth factor,interleukin-8,follistatin.placenta growth factor and leptin.The location of angiogenesis factors was monitored by immunofluorescence labeling and confocal laser scanning microscope3 D reconstruction.Results:Endometrial stromal cells and glandular epithelial cells were isolated and primary cultured for establishing a 3D co-culture system.The levels of VEGF—A/C/D in Danzhi decoction group and aspirin group were significantly lower than those in mock group(P<0.05).while there was no significant difference between Danzhi decoction group and aspirin group(P>0.05).Furthermore,the alterative location of VEGF—A/C/D was observed in the cytoplasm of endometrial glandular epithelial cells.Conclusions:Danzhi decoction mav inhibit the expression of VEGF in the blood stasis microenvironment of SPID by targeting the cytoplasm of endometrial glandular epithelial cell.

Skeletal myoblast based delivery of angiogenic growth factors:a comparison between angiopoietin-1 and VEGF gene delivery for therapeutic angiogenesis in the heart

Objectives This study investigated the efficacy of human skeletal myoblasts (SkM) mediated either human vascular endothelial growth factor-165 (hVEGF165) or angiopoietin-1 (Ang-1) on vascular development and myocardial regional perfusion. Methods A porcine heart model of chronic infarction was created in 28 female swine by coronary artery ligation. The animals were randomized into: (1) group-1, DMEM injected (n=6), (2) group-2, Ad-null transduced SkM transplanted (n=6), (3) group-3, Ad-hVEGF165 transduced SkM transplanted (n=8), and (4) group-4, Ad-Ang-1 transduced SkM (n=8). Three weeks later, 5 ml DMEM containing 3×108 SkM carrying exogenous genes were intramyocardially injected into 20 sites in left ventricle in groups-2, -3 and -4. Animals in group-1 were injected 5 ml DMEM without cells. Animals were kept on 5 mg/kg cyclosporine per day for 6 weeks. Regional blood flow was measured using fluorescent microspheres. The heart was explanted at 2, 6 and 12 weeks after transplantation for histological studies. Results Histological examination showed survival of lac-z expressing myoblasts in host tissue. Capillary density based on Von Willebrand factor-VIII (vWF-VIII) at low power field (×100) was 57.13±11.85 in group-3 at 6 weeks and declined to 32.1±5.21 at 12 weeks, while it was 39.9±10.26 at 6 weeks and increased to 45.14±6.54 at 12 weeks in group-4. The mature blood vessel index was highest in group- 4 at 6 and 12 weeks after transplantation. The regional blood flow in the center and peri-infarct area was significantly increased in animals of groups-3 and -4. Conclusions SkM carrying either hVEGF165 or Ang-1 induced neovascularization with increased blood flow. Ang-1 overexpression resulted in mature and stable blood vessel formation and may be a more potent arteriogenic inducer for neovascularization.

Effect of medical nutrition combined with exercise intervention on the placental ischemic hypoxic injury and serum angiogenesis factors in patients with gestational hypertension

Objective: To discuss the effect of medical nutrition combined with exercise intervention on the placental ischemic hypoxic injury and serum angiogenesis factors in patients with gestational hypertension. Methods: A total of 90 patients with gestational hypertension who received antenatal care and gave birth in our hospital between July 2014 and July 2016 were collected and divided into control group and observation group according to random number table, 45 cases in each group. Control group of patients received routine therapy, observation group of patients received routine therapy + nutrition combined with exercise intervention, and the ischemic hypoxic injury index expression in placenta tissue and serum angiogenesis factor levels before delivery were compared between two groups of patients. Results: After intervention, ischemic hypoxic injury index NO level in placental grinding fluid of observation group was higher than that of control group while ET-1, HIF-1α, Bax, Caspase-3 and MDA levels were lower than those of control group;serum angiogenesis factors TGFβ1, HGF, bFGF, VEGF and Ang-2 levels were significantly higher than those of control group while sFlt-1 level was lower than that of control group. Conclusion: The combination of medical nutrition and exercise can effectively reduce the placental ischemic hypoxic injury and reduce the angiogenesis in patients with gestational hypertension.

Angiogenesis Factors Associated with New Breast Cancer Cell Line AMJ13 Cultured <i>in Vitro</i>

Background: AMJ13 is a new breast cancer cell line that has been established from a 70-year-old Iraqi woman with a histological diagnosis of infiltrating ductal carcinoma. It is the first for an Iraqi population. In breast cancer, angiogenesis provides the tumor tissue, which is rapidly proliferated with oxygen and nutrients, removes wastes and increases the opportunity of cancer cells to invade other organs. Methods: The AMJ13 breast cancer cell line was represented at three different passages and incubated for interval times. Microarray panel of 43 different angiogenesis markers was used to scan the supernatant for the factors. ELISA was used to quantify some of the important angiogenesis factors released in the culture medium and to confirm absence of those who was not detected by the antibody array. RT-PCR was used to confirm the gene expression (mRNA) of studied factors. Results: Microarray analysis showed that TIMP1 and two secreted at highest levels compared to the rest of the factors with low presence of endostatin. Other non-detectable factors by microarray examined by ELISA assay that showed highest expression level of VEGF-A were obtained at earliest passage, while the highest levels of FGF-b were obtained at late passage. The VEGF-D secretion was shown low concentrations at all studied passages. There is no detectable level of EGF protein in different passages and times interval tested. There are no significant differences in secretion of sICAM between different passages and incubation periods. Conclusion is that AMJ13 cell line depends on VEGF-A as main angiogenesis factor to induce micro-vessels supported by low levels of VEGF-D for lymphatic vessels formation. AMJ13 cell line depends on FGF as growth factors as in late passages it was shifted to depend mainly on FGF completely. All of this process may be regulated by TGF-β. TIMP-1 has proangiogentic effect and has feedback talk with TIMP-2. Understanding the angiogenesis process for breast cancer can give us better targets for therapy and more effective treatments.

Effect of kanglaite injection combined with chemotherapy on tumor factors, angiogenesis and immunoglobulin in patients with lung adenocarcinoma

Objective:To explore the effect of Kanglaite injection combined with chemotherapy on the levels of tumor factors, angiogenesis and immunoglobulin in patients with lung adenocarcinoma.Method: A total of 88 patients with lung adenocarcinoma in our hospital and the Fifth Peoples Hospital of Wuxi from February 2014 to December 2017 were divided randomly into observation group and control group according to the visiting time, 44 cases for each group. The observation group was treated with chemotherapy combined with Kanglaite injection, and the control group was treated with chemotherapy only.Both groups were treated for 6 weeks and then the levels of CEA, CA125, CA199, VEGFA, VEGFB, VEGFC, and IgA, IgM, IgG after treatment in both groups were compared.Result: Before treatment, there were no significant differences in the levels of CEA, CA125 and CA199 between two groups. The level of CEA in the observation group (29.88±7.07) μg/L was lower than that in the control group,and the difference was statistically significant. The levels of CA125 and CA199 after treatment were lower than those before treatment, but with no significant difference. Between the two groups, there was no significant difference of the levels of VEGFA,VEGFB and VEGFC before treatment, the level of VEGFA after treatment in the observation group (80.49±12.29) ng/mL was lower than that in the control group and the difference was statistically significant, and the levels of VEGFB and VEGFC after treatment were also lower than those before treatment, but with no significant difference. The levels of IgA, IgM and IgG before treatment in two groups had no significant difference, The level of IgM after treatment in the observation group (1.52±0.30) g/L was lower than that in the control group, with a statistical difference. The levels of IgA and IgG after treatment were lower than those before treatment, but with no significant difference;No serious adverse reaction was observed after treatment in both groups.Conclusion: Kanglaite injection combined with chemotherapy has a good clinical effect in lung adenocarcinoma, which can effectively inhibit the high expression of tumor markers, reduce angiogenesis, enhance body immunity, and can be recommended for clinical application.

Effects of Xiaocangping tablets combined with 5-fluorouracil and cisplatin on immunity, tumor markers, angiogenesis and related factors in patients with advanced esophageal cancer

Objective:To explore the effects of Xiaocangping tablets combined with 5-fluorouracil and cisplatin on immunity, tumor markers, angiogenesis and related factors in patients with advanced esophageal cancer.Methods:86 patients with advanced esophageal cancer admitted to our hospital from March 2014 to February 2017 were randomly divided into control group (43 cases) and observation group (43 cases). All the subjects were treated with 5-fluorouracil plus cisplatin, and the observation group was treated with Xiaocangping tablets on this basis. The changes of immune function, tumor markers, angiogenesis and related factors in the two groups were compared and analyzed.Results:After treatment, the levels of CD4+, CD4+/CD8 + in both groups were significantly higher than those before treatment (P<0.05), while the levels of CD8+ were significantly lower than those before treatment (P<0.05), the levels of CD4+ and CD4+/CD8+ in the observation group were significantly higher than those in the control group (P<0.05), while the levels of CD8+ in the observation group was significantly lower than that in the control group (P<0.05);the levels of CA125, CEA and CA19-9 in the two groups were significantly lower than those before treatment (P<0.05), and the levels of CA125, CEA and CA19-9 in the observation group were significantly lower than those in the control group (P<0.05);the levels of VEGF, TGF-β1, MMP-9, NGAL in the two groups were significantly lower than those before treatment (P<0.05), and the levels of VEGF, TGF-β1, MMP-9, NGAL in the observation group were significantly lower than the control group (P<0.05).Conclusions:Chemotherapy combined with fluorouracil + cisplatin chemotherapy for advanced esophageal cancer can effectively improve the immune function of patients, reduce the level of tumor markers, inhibit the proliferation of tumor blood vessels, and have significant anti-cancer effects, which is of positive significance for controlling the development of patients' conditions.

Buyang Huanwu decoction increases vascular endothelial growth factor expression and promotes angiogenesis in a rat model of local cerebral ischemia

Angiogenesis in the infarct periphery can improve blood flow. Vascular endothelial growth factor （VEGF） has been considered a potential therapeutic target for stroke. Buyang Huanwu decoction （BYHWD） is a classic traditional formula in traditional Chinese medicine and is used to treat stroke; in addition, the promotion effects on VEGF protein expression have been confirmed. However, little is known about how BYHWD regulates angiogenesis, or about the effects of BYHWD on VEGF mRNA expression. For this reason, the present study measured microvessel density in rats with cerebral ischemia using immunohistochemistry. In addition, VEGF expression was measured by re-verse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay to determine the effects of BYHWD on angiogenesis and VEGF expression in rats with cerebral ischemia. Results demonstrated that microvessel density, as well as VEGF mRNA and protein expression, increased after 7 and 14 days of BYHWD treatment, which suggests that BYHWD promoted angiogenesis following cerebral ischemia and upregulated VEGF mRNA and protein expression in ischemic cerebral regions.

Inhibitory effect of arsenic trioxide on angiogenesis and expression of vascular endothelial growth factor in gastric cancer

AIM: To investigate the inhibitory effect of As2O3 on angiogenesis of tumor and expression of vascular endothelial growth factor (VEGF) in tumor cells in vivo and in vitro. METHODS: The solid tumor model was formed in nude mice with the gastric cancer cell line SGC-7901. The animals were randomly divided into three groups. As2O3 was injected into the arsenic-treated groups (2.5 mg/kg and 5 mg/kg) and the same volume of saline solution was injected into the control group. Microvessel density (MVD) and expression of VEGF were detected with immunofluorescence laser confocal technology. Further expression of VEGF protein and VEGF mRNA was measured with Western bloting and fluorescence quantitative RT- PCR in SGC-7901 cells treated with As2O3. RESULTS: In nude mice, after treatment with 5 mg/kg and 2.5 mg/kg As2O3 respectively, about 50% and 30% tumor growth inhibition were observed correspondingly (P < 0.05, P < 0.05). Decrease in MVD appeared in As2O3-treated tumors compared with control group (P < 0.001, P < 0.001). MVD in tumors was significantly lower in 5 mg/kg group than in 2.5 mg/kg group (P < 0.01). The fluorescence intensity levels of VEGF in tumor cells were significantly lowered in the arsenic-treated groups (P < 0.01, P < 0.01). The fluorescence intensity level of VEGF in 5 mg/kg group was lower than that in 2.5 mg/ kg group (P < 0.01). In vitro, the expression of VEGF protein decreased in dose- and time-dependent manner after the treatment with As2O3, but in VEGF mRNA no significant difference was found between the control group and the treated groups. CONCLUSION: As2O3 can inhibit solid tumor growth by inhibiting the formation of new blood vessels. One of the mechanisms is that As2O3 can inhibit VEGF protein expression.

Expression of angiopoietins, Tie2 and vascular endothelial growth factor in angiogenesis and progression of hepatocellular carcinoma

AIM： TO investigate the significance of angiopoietins, Tie2 and vascular endothelial growth factor （VEGF） expression in the angiogenesis and progress of hepatocellular carcinoma （HCC）. METHODS： Fresh surgically resected specimens of HCC and noncancerous liver （NCL） tissue from 38 patients with HCC were obtained, and expression of angiopoietin-1 （Ang-1）, angiopoietin-2 （Ang-2）, Tie2, and VEGF messenger RNA （mRNA） was examined by real-time quantitative reverse transcription-polymerase chain reaction （RT-PCR）. Expression pattern of each gene in HCC and NCL tissue specimens was compared and the potential role and interaction in angiogenesis of HCC were analyzed. Genes＇ expression level and its relationship with tumor＇s clinicopathological parameters were also investigated. Immunohistochemical staining of CD34 was performed to determine the microvessel density （MVD） and Ang-2/Ang-1 ratio was calculated. Relationships between Ang-2/Ang-1 ratio, VEGF and MVD and clinicopathological features were also tested so as to evaluate their significance in the progression of HCC. RESULTS： Ang-2 and VEGF mRNAs in HCC were significantly higher than those in NCL tissue （P 〈 0.05）, whereas the Ang-1 and Tie2 mRNAs showed no statistical significance （P 〉 0.05）, though slightly lower level of Ang-1 mRNA in HCC was observed. Ang-2/ Ang-1 ratio and VEGF were both positively correlated to MVD. The Ang-2/Ang-1 ratio, Ang-2 and VEGF were all associated with tumor＇s clinicopathological parameters （P 〈 0.05） except for histological grades （P 〉 0.05）. Ang-1 and Tie2 levels in different clinicopathological groups were not significantly different （P 〉 0.05）. CONCLUSION： Dominant Ang-2 expression against Ang-1 through Tie2 receptor in the presence of VEGF plays a critical role in initiating early neovascularization and transformation of noncancerous liver to hepatocellular carcinoma. Its consequently constant operation in formed HCC induces further angiogenesis and progression of HCC.

Krüppel-like factor 8 overexpression is correlated with angiogenesis and poor prognosis in gastric cancer

AIM:To investigate Krüppel-like factor 8 (KLF8) expression in gastric cancer and its relationship with angiogenesis and prognosis of gastric cancer. METHODS:One hundred and fifty-four patients with gastric cancer who underwent successful curative resection were retrospectively enrolled in the study. Fifty tumor-adjacent healthy gastric tissues (≥ 5 cm from the tumor margin) obtained during the original resection were randomly selected for comparative analysis. In situ expression of KLF8 and CD34 proteins were examined by immunohistochemistry. The intratumoral microvessel density (MVD) was determined by manually counting the immunostained CD34-positive endothelial cells in three consecutive high-magnification fields (× 200). The relationship between differential KLF8 expression and MVD was assessed using Spearman's correlation coefficient test. χ2 test was performed to evaluate the effects of differential KLF8 expression on clinicopathologic factors. Kaplan-Meier and multivariate Cox survival analyses were used to assess the prognostic value of differential KLF8 expression in gastric cancer. RESULTS:Significantly higher levels of KLF8 protein were detected in gastric cancer tissues than in the adjacent non-cancerous tissues (54.5% vs 34.0%, P < 0.05). KLF8 expression was associated with tumor size (P < 0.001), local invasion (P = 0.005), regional lymph node metastasis (P = 0.029), distant metastasis (P = 0.023), and tumor node metastasis (TNM) stage (P = 0.002), as well as the MVD (r = 0.392, P < 0.001). Patients with KLF8 positive expression had poorer overall survival (P < 0.001) and cancer-specific survival (P < 0.001) than those with negative expression. Multivariate analysis demonstrated that KLF8 expression independently affected both overall and cancer-specific survival of gastric cancer patients (P = 0.035 and 0.042, respectively). CONCLUSION:KLF8 is closely associated with gastric tumor progression, angiogenesis and poor prognosis, suggesting it may represent a novel prognostic biomarker and therapeutic target for gastric cancer.

Hepatocyte Growth Factor Gene-modified Bone Marrow-derived Mesenchymal Stem Cells Transplantation Promotes Angiogenesis in a Rat Model of Hindlimb Ischemia

Summary： Angiogenic gene therapy and cell-based therapy for peripheral arterial disease （PAD） have been studied intensively currently. This study aimed to investigate whether combining mesenchymal stem cells （MSCs）transplantation with ex vivo human hepatocyte growth factor （HGF） gene transfer was more therapeutically efficient than the MSCs therapy alone in a rat model of hindlimb ischemia. One week after establishing hindlimb ischemia models, Sprague-Dawley （SD） rats were randomized to receive HGF gene-modified MSCs transplantation （HGF-MSC group）, untreated MSCs transplantation （MSC group）, or PBS injection （PBS group）, respectively. Three weeks after injection, angiogenesis was significantly induced by both MSCs and HGF-MSCs transplantation, and capillary density was the highest in the HGF-MSC group. The number of transplanted cell-derived endothelial cells was greater in HGF-MSC group than in MSC group after one week treatment. The expression of angiogenic cytokines such as HGF and VEGF in local ischemic muscles was more abundant in HGF-MSC group than in the other two groups. In vitro, the conditioned media obtained from HGF-MSCs cultures exerted proproliferative and promigratory effects on endothelial cells. It is concluded that HGF gene-modified MSCs transplantation therapy may induce more potent angiogenesis than the MSCs therapy alone. Engraftment of MSCs combined with angiogenic gene delivery may be a promising therapeutic strategy for the treatment of severe PAD.

Effect of Antisense Oligodeoxynucleotide of Vascular Endothelial Growth Factor C on Lymphangiogenesis and Angiogenesis of Pancreatic Cancer

In order to investigate the effect of antisense oligonucleotide （ASODN） of vascular endothelial growth factor C （VEGF-C） on lymphangiogenesis and angiogenesis of pancreatic cancer, antisense and scamble-sense oligonucleotide of VEGF-C were constructed, and the model of nude mice with orthotopically xenografied human pancreatic cancer cells （Panc-1） was established. Thirty nude mice were randomly divided into 3 groups： PBS control group （group A）, scramble-sense control group （group B） and antisense group （group C）. All nude mice were treated once every 2 days as 3 times per week, for 3 weeks （oligonucleotide 10 mg/kg every time）. After treatments were completed, ELISA method was used to examine the concentration of VEGF-C in plasma and immunohistochemical method to examine microvessel density （MVD）, lymphtic vessel density （LVD） of pancreatic cancer. The results showed that the expression of VEGF-C was inhibited significantly in group C. The concentrations were 237.5±41.5, 221.5±52.3 and 108.6±14.9 pg/mL in groups A, B and C respectively （P〈0.01）. LVD in groups A, B and C was 13.8±2.1, 12.4±1.9 and 4.2±1.6 respectively （P〈0.01）. MVD in groups A, B and C was 27.5±8.7, 25.9±4.2 and 19.4±5.6 respectively with no significant difference among the groups （P〉0.05）. It was suggested that VEGF-C ASODN decreased the expression levels of VEGF-C in nude mice with orthotopically xenografted human pancreatic cancer, and it could inhibit lymphangiogenesis, but had no significant effect on angiogenesis.

Effect of non-anticoagulant N-desulfated heparin on expression of vascular endothelial growth factor, angiogenesis and metastasis of orthotopic implantation of human gastric carcinoma

AIM： To investigate the effect of N-desulfated heparin on tumor metastasis and angiogenesis, and expression of vascular endothelial growth factor （VEGF） of orthotopic implantation of human gastric carcinoma in male severe combined immune deficiency （SCID） mice. METHODS： Human gastric cancer SGC-7901 cells were orthotopically implanted into the stomach of SC/D mice. The mice were randomly divided into normal saline group and N-desulfated heparin group. One week after operation, the mice in N-desulfated heparin group reo ceived i.v. injections of N-desulfated heparin （Shanghai Institute of Cell Biology, Chinese Academy of Sciences, 10 mg/kg.d） twice weekly for 3 wk. The mice in normal saline group received i.v. injections of normal saline （100 μL） twice weekly for 3 wk. The mice were sacrificed six weeks after implantation. Tumor metastasis was evaluo ated histologically for metastasis under microscope. Intratumoral microvessel density （MVD） and VEGF expression were evaluated immuohistochemically. VEGF mRNA expression in gastric tissue of SC/D mice was detected by real time PCR. RESULTS： The tumor metastasis rate was 80% in normal saline group and 20% in N-desulfated heparin group （P 〈 0.05）. MVD was 8.0 ± 3.1 in normal saline group and 4.3 ± 1.8 in N-desulfated heparin group （P 〈 0.05）. VEGF positive immunostaining was found in cytoplasm of cancer cells. The rate of VEGF positive expression was higher in normal saline group than in N-desulfated hepa- rin treated group （90% vs 20%, P 〈 0.05）. VEGF mRNA expression was significantly inhibited by N-desulfated heparin and was higher in normal saline group than in N-desulfated heparin group （Ct value 19.51 ± 1.01 vs 22.55± 1.36, P 〈 0.05）. N-desulfated heparin significantly inhibited the expression of VEGF mRNA in cancer cells. No bleeding occurred in N-desulfated heparin group. CONCLUSION： N-desulfated heparin can inhibit metastasis of gastric cancer by suppressing tumor VEGF expression and tumor angiogenesis, but has no obvious anticoagulant activity.

Bone marrow cells produce nerve growth factor and promote angiogenesis around transplanted islets

AIM:To clarify the mechanism by which bone marrow cells promote angiogenesis around transplanted islets.METHODS: Streptozotocin induced diabetic BALB/ c mice were transplanted syngeneically under the kidney capsule with the following: (1) 200 islets (islet group: n=12), (2) 1-5×106 bone marrow cells (bone marrow group: n=11), (3) 200 islets and 1-5×106 bone marrow cells (islet + bone marrow group: n= 13), or (4) no cells (sham group:n=5). All mice were evaluated for blood glucose, serum insulin, serum nervegrowth factor (NGF) and glucose tolerance (GTT) up to postoperative day (POD) 14. Histological assessment for insulin, von Willebrand factor (vWF) and NGF was performed at POD 3, 7 and 14.RESULTS: Blood glucose level was lowest and serum insulin was highest in the islet + bone marrow group. Serum NGF increased in islet, bone marrow, and islet + bone marrow groups after transplantation, and there was a significant difference (P=0.0496, ANOVA) between the bone marrow and sham groups. The number of vessels within the graft area was signif icantly increased in both the bone marrow and islet + bone marrow groups at POD 14 as compared to the islet alone group (21.2 ± 3.6 in bone marrow, P=0.01, vs islet group, 22.6 ± 1.9 in islet + bone marrow, P = 0.0003, vs islet group, 5.3 ± 1.6 in islet-alone transplants). NGF was more strongly expressed in bone marrow cells compared with islets. CONCLUSION: Bone marrow cells produce NGF and promote angiogenesis. Islet co-transplantation with bone marrow is associated with improvement of islet graft function.

Beta-nerve growth factor promotes neurogenesis and angiogenesis during the repair of bone defects

We previously showed that the repair of bone defects is regulated by neural and vascular signals. In the present study, we examined the effect of topically applied β-nerve growth factor（β-NGF） on neurogenesis and angiogenesis in critical-sized bone defects filled with collagen bone substitute. We created two symmetrical defects, 2.5 mm in diameter, on either side of the parietal bone of the skull, and filled them with bone substitute. Subcutaneously implanted osmotic pumps were used to infuse 10 μgβ-NGF in PBS（β-NGF ＋ PBS） into the right-hand side defect, and PBS into the left（control） defect, over the 7 days following surgery. Immunohistochemical staining and hematoxylin-eosin staining were carried out at 3, 7, 14, 21 and 28 days postoperatively. On day 7, expression of β III-tubulin was lower on the β-NGF ＋ PBS side than on the control side, and that of neurofilament 160 was greater. On day 14, β III-tubulin and protein gene product 9.5 were greater on the β-NGF ＋ PBS side than on the control side. Vascular endothelial growth factor expression was greater on the experimental side than the control side at 7 days, and vascular endothelial growth factor receptor 2 expression was elevated on days 14 and 21, but lower than control levels on day 28. However, no difference in the number of blood vessels was observed between sides. Our results indicate that topical application of β-NGF promoted neurogenesis, and may modulate angiogenesis by promoting nerve regeneration in collagen bone substitute-filled defects.

The Effect of Brain-derived Neurotrophic Factor on Angiogenesis

To investigate the in vitro and in vivo proangiogenic effects of brain-derived neurotrophic factor （BDNF）, human umbilical vein endothelial cells （HUVECs） were isolated and cultured in primary culture. The effect of BDNF on the proliferation of HUVECs was examined by MTT assay. The effects of BDNF on HUVEC migration and tube formation were studied by modified Boyden cham- ber assay and tube formation assay, respectively. Matrigel plug assay and chorioallantoic membrane assay were used to evaluate the effects of BDNF on angiogenesis in vivo. Our results showed that BDNF substantially stimulated the migration and tube formation of HUVECs in vitro, although it did not induce HUVEC proliferation. BDNF also induced angiogenesis both in matrigel plug of mouse model and in chick chorioallantoic membrane. In conclusion, BDNF can promote angiogenesis both in vitro and in vivo, and may be a proangiogenic factor.

THE EFFECT OF BASIC FIBROBLAST GROWTH FACTOR SLOW-RELEASE MICROCAPSULES ON ANGIOGENESIS IN INFARCTED RABBIT MYOCARDIUM

Objectives To observe the effect of basic fibroblast growth factor (bFGF) slow-release microcapsules on angiogenesis in infarcted myocardial regions. Methods.Myocardial infarction was induced in 24 New Zealand rabbits by ligating the root of left anterior descending coronary artery.Group Ⅰ(n=8) served as control, group Ⅱ(n=8) as a blank microcapsule group, group Ⅲ(n=8, each microcapsule contains 1μg bFGF) as micrpcapsule group.In group Ⅱ and Ⅲ, 5 blank microcapsules or bFGF slow-release microcapsules were implanted into myocardium underneath the epicardium between the left anterior descending coronary artery and left circumflex artery.Infarct size was evaluated by infarcted weight/left ventricle weight ratio and angiogenesis was evaluated by immunohistochemical examinations 5 weeks later. [WT5”BX] Results.As compared with group Ⅰ and Ⅱ, rabbits treated with bFGF slow-release microcapsules showed higher microvessel counts (group Ⅰ3775±450, group Ⅱ3837±498,vs.group Ⅲ 13550±481,P<0001) and less infarcted weight /left ventricle weight (group Ⅰ168%±04%,group Ⅱ167%±05%,vs.group Ⅲ 70%±02%,P<0001). Conclusions.Subepicardial administration of bFGF slow-release microcapsule in the infarcted rabbit model results in effective angiogenesis and reduction in infarct size.

Role of Brain-derived Neurotrophic Factor in Bone Marrow Angiogenesis in Multiple Myeloma

Summary： This study examined the expression of brain-derived neurotrophic factor （BDNF） in multiple myeloma （MM） and its role in bone marrow angiogenesis. The peripheral blood plasma was harvested from 71 MM patients and 63 patients without hematological malignancy. The BDNF level in the blood plasma was determined by ELISA. Human bone marrow endothelial cells （HBMECs） were cul- tured. The mRNA and protein expression levels of the BDNF receptor TrkB in HBMECs were detected by using RT-PCR and flow cytometry, respectively. The viability of HBMECs treated with recombinant human （rh） BDNF or not was measured by using MTT assay. The migration of HBMECs in the presence of rhBDNF or not was determined by modified Boyden chamber assay. In vitro tube formation assay was used to assess the effect of rhBDNF on HBMECs differentiation. The results of ELISA revealed that the BDNF level was significantly higher in peripheral blood plasma of MM patients than in that of control patients （4.39±0.67 vs. 1.96±0.39 ng/mL, P〈0.05）. The BDNF receptor TrkB was ex- pressed in HBMECs at mRNA and protein level. MTT assay manifested that rhBDNF could signifi- cantly concentration-dependently promote the HBMECs proliferation. The number of HBMECs treated with 160 ng/mL rhBDNF for 48 h was 1.57±0.10 folds higher than that in control group （P〈0.05）. Moreover, rhBDNF could enhance HBMECs migration in a concentration-dependent manner and the maximal migration was reached in the presence of 100 ng/mL rhBDNF. The migration indexes were 1.40±10.11, 1.64±0.16, 2.06±0.25 and 2.18±0.21 in 25, 50, 100 ng/mL rhBDNF groups and 25 ng/mL rhVEGF group, respectively. In vitro tube formation assay demonstrated that the area of the formed tu- bular structure was increased with the rhBDNF concentration. In control group, there was no formation of intact tubular structure and the HBMECs on the matrigel were irregularly dispersed. HBMECs treated with 100 ng/mL rhBDNF could form intact tubular structure and the area and the diameter of tubes were significantly greater than those in control group （P〈0.05）. There was no significant differ- ence in the formed tubular area between 25 ng/mL VEGF group and 100 ng/mL rhBDNF group. It was concluded that BDNF plays an important role in myeloma cell-induced angiogenesis, and it may be- come a new target of anti-angiogenesis treatment for MM.

Study on Angiogenesis Factor of Human Osteosarcoma

Angiogenesis factor of human osteosarcoma was partially purified and its biological features were studied. The active peptide with 8000 to 10 000 u molecular weight in the conditioned medium obtained from the cultivation of human osteosarcoma cells were partially purified by ultrafiltration, chromatography and dialysis. The angiogenic effects of the fractions were assessed by proliferation assay of human umbilical vein and pig aorta thoracic endothelial cells. The results showed that the chromatography fractions of 4 to 6 could significantly promote the proliferation of the endothelial cells. It was suggested that the human osteosarcoma cells could synthesize and secrete angiogenesis factor with a molecular weight of 8000 to 10 000 u.