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1.  Fibroblast Growth Factor-2 and Vascular Endothelial Growth Factor Mediated Augmentation of Angiogenesis and Bone Formation in Vascularized Bone Allotransplants 
Microsurgery  2014;34(4):301-307.
We previously demonstrated recipient-derived neoangiogenesis to maintain viability of living bone allogeneic transplants without long-term immunosuppression. The effect of cytokine delivery to enhance this process is studied.
Vascularized femur transplantation was performed from DA to PVG rats. Poly(D,L-lactide-co-glycolide) microspheres loaded with buffer (N=11), basic fibroblast growth factor (FGF2) (N=10), vascular endothelial growth factor (VEGF) (N=11), or both (N=11) were inserted intramedullarly alongside a recipient-derived a/v bundle. FK-506 was administered for 2 weeks. At 18 weeks, bone blood flow, microangiography, histologic, histomorphometric and alkaline phosphatase measurements were performed.
Bone blood flow was greater in the combined group than control and VEGF groups (p=0.04). Capillary density was greater in the FGF2 group than in the VEGF and combined groups (p<0.05). Bone viability, growth and alkaline phosphatase activity did not vary significantly between groups.
Neoangiogenesis in vascularized bone allotransplants is enhanced by angiogenic cytokine delivery, with results using FGF2 that are comparable to isotransplant from previous studies. Further studies are needed to achieve bone formation similar to isotransplants.
doi:10.1002/micr.22221
PMCID: PMC3976711  PMID: 24395434
2.  Vascularized Bone Graft Chimerism Mediated by Vascular Endothelial Growth Factor 
Microsurgery  2014;35(1):45-51.
Background
Vascular Endothelial Growth Factor (VEGF) induces angiogenesis and osteogenesis in bone allotrasnplants. We aim to determine whether bone remodeling in VEGF-treated bone allotransplants results from repopulation with circulation-derived autogenous cells, or survival of allogenic transplant-derived cells.
Methods
Vascularized femoral bone grafts were transplanted from female Dark Agouti rats (DA;RT1a) to male Piebald Viral Glaxo (PVG;RT1c). Arteriovenous bundle implantation and short term immunosuppression were used to maintain cellular viability. VEGF was encapsulated in biodegradable microspheres and delivered intramedullary in the experimental group (n=22). In the control group (n=22), no VEGF was delivered. Rats were sacrificed at 4 or 18 weeks. Laser capture microdissection of bone remodeling areas was performed at the inner and outer cortex. Sex-mismatched genes were quantified with reverse transcription-polymerase chain reaction to determine the amount of male cells to total cells, defined as the relative Expression Ratio (rER).
Results
At 4 weeks rER was significantly higher at the inner cortex in VEGF treated transplants as compared to untreated transplants (0.622±0.225 vs 0.362±0.081, p=0.043). At 4 weeks, the outer cortex in the control group had a significantly higher rER (p=0.038), while in the VEGF group, the inner cortex had a higher rER (p=0.015). Over time, in the outer cortex the rER significantly increased to 0.634±0.106 at 18 weeks in VEGF treated rats (p=0.049). At 18 weeks, the rER was > 0.5 at all cortical areas in both groups.
Conclusion
These in vivo findings suggest a chemotactic effect of intramedullary applied VEGF on recipient derived bone and could imply that more rapid angiogenesis of vascularized allotransplants can be established with microencapsulated VEGF.
doi:10.1002/micr.22300
PMCID: PMC4308546  PMID: 25073635
3.  Effect of rhBMP-2 and VEGF in a Vascularized Bone Allotransplant Experimental Model Based on Surgical Neoangiogenesis 
We have demonstrated survival of living allogeneic bone without long-term immunosuppression using short-term immunosuppression and simultaneous creation of an autogenous neoagiogenic circulation. In this study bone morphogenic protein-2 (rhBMP-2), and/or vascular endothelial growth factor (VEGF), were used to augment this process. Femoral diaphyseal bone was transplanted heterotopically from 46 Dark Agouti to 46 Lewis rats. Microvascular repair of the allotransplant nutrient pedicle was combined with intra-medullary implantation of an autogenous saphenous arteriovenous (AV) bundle and biodegradable microspheres containing buffer (control), rhBMP-2 or rhBMP-2 + VEGF. FK-506 given daily for 14 days maintained nutrient pedicle flow during angiogenesis. After an 18 weeks survival period, we measured angiogenesis (capillary density) from the AV bundle and cortical bone blood flow. Both measures were greater in the combined (rhBMP-2 + VEGF) group than rhBMP-2 and control groups (p<0.05). Osteoblast counts were also higher in the rhBMP-2 + VEGF group (p<0.05). A trend towards greater bone formation was seen in both rhBMP2 + VGF and rhBMP2 groups as compared to controls (p=0.059). Local administration of VEGF and rhBMP-2 augments angiogenesis, osteoblastic activity and bone blood flow from implanted blood vessels of donor origin in vascularized bone allografts.
doi:10.1002/jor.22277
PMCID: PMC3972920  PMID: 23192572
bone; allotransplantation; microspheres; BMP; VEGF
4.  Basic fibroblast growth factor promotes VEGF-C-dependent lymphangiogenesis via inhibition of miR-381 in human chondrosarcoma cells 
Oncotarget  2016;7(25):38566-38578.
A chondrosarcoma is a common, primary malignant bone tumor that can grow to destroy the bone, produce fractures and develop soft tissue masses. Left untreated, chondrosarcomas metastasize through the vascular system to the lungs and ultimately lead to large metastatic deposits of the malignant cartilage taking over lung volume and function. Vascular endothelial growth factor (VEGF)-C has been implicated in tumor-induced lymphangiogenesis and elevated expression of VEGF-C has been found to correlate with cancer metastasis. bFGF (basic fibroblast growth factor), a secreted cytokine, regulates biological activity, including angiogenesis and metastasis. We have previously reported on the important role of bFGF in angiogenesis in chondrosarcomas. However, the effect of bFGF in VEGF-C regulation and lymphangiogenesis in chondrosarcomas is poorly understood. In this investigation, we demonstrate a correlation exists between bFGF and VEGF-C in tissue specimens from patients with chondrosarcomas. To examine the lymphangiogenic effect of bFGF, we used human lymphatic endothelial cells (LECs) to mimic lymphatic vessel formation. We found that bFGF-treated chondrosarcomas promoted LEC tube formation and cell migration. In addition, bFGF knockdown inhibited lymphangiogenesis in vitro and in vivo. We also found that bFGF-induced VEGF-C is mediated by the platelet-derived growth factor receptor (PDGFR) and c-Src signaling pathway. Furthermore, bFGF inhibited microRNA-381 expression via the PDGFR and c-Src cascade. Our study is the first to describe the mechanism of bFGF-promoted lymphangiogenesis by upregulating VEGF-C expression in chondrosarcomas. Thus, bFGF could serve as a therapeutic target in chondrosarcoma metastasis and lymphangiogenesis.
doi:10.18632/oncotarget.9570
PMCID: PMC5122411  PMID: 27229532
bFGF; chondrosarcoma; lymphangiogenesis; VEGF-C; miR-381
5.  Human adipose tissue-resident monocytes exhibit an endothelial-like phenotype and display angiogenic properties 
Introduction
Adipose tissue has the unique property of expanding throughout adult life, and angiogenesis is required for its growth. However, endothelial progenitor cells contribute minimally to neovascularization. Because myeloid cells have proven to be angiogenic, and monocytes accumulate in expanding adipose tissue, they might contribute to vascularization.
Methods
The stromal vascular fraction (SVF) cells from human adipose tissue were magnetically separated according to CD45 or CD14 expression. Adipose-derived mesenchymal stromal cells (MSCs) were obtained from SVF CD45- cells. CD14+ monocytes were isolated from peripheral blood (PB) mononuclear cells and then cultured with SVF-derived MSCs. Freshly isolated or cultured cells were characterized with flow cytometry; the conditioned media were analyzed for the angiogenic growth factors, angiopoietin-2 (Ang-2), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), granulocyte colony-stimulating factor (G-CSF), and granulocyte macrophage colony-stimulating factor (GM-CSF) with Luminex Technology; their angiogenic capacity was determined in an in vivo gelatinous protein mixture (Matrigel) plug angiogenesis assay.
Results
CD45+ hematopoietic cells within the SVF contain CD14+ cells that co-express the CD34 progenitor marker and the endothelial cell antigens VEGF receptor 2 (VEGFR2/KDR), VEGFR1/Flt1, and Tie2. Co-culture experiments showed that SVF-derived MSCs promoted the acquisition of KDR and Tie-2 in PB monocytes. MSCs secreted significant amounts of Ang-2 and HGF, but minimal amounts of bFGF, G-CSF, or GM-CSF, whereas the opposite was observed for SVF CD14+ cells.
Additionally, SVF CD14+ cells secreted significantly higher levels of VEGF and bFGF than did MSCs. Culture supernatants of PB monocytes cultured with MSCs contained significantly higher concentrations of VEGF, HGF, G-CSF, and GM-CSF than did the supernatants from cultures without MSCs. Quantitative analysis of angiogenesis at 14 days after implantation demonstrated that neovascularization of the implants containing SVF CD14+ cells or PB monocytes previously co-cultured with MSCs was 3.5 or 2 times higher than that observed in the implants with SVF-derived MSCs. Moreover, immunofluorescence of Matrigel sections revealed that SVF CD14+ cells differentiated into endothelial cells and contributed to vascular endothelium.
Conclusions
The results from this study suggest that adipose tissue-resident monocytes should contribute to tissue vascularization. Because SVF CD14+ cells were more efficient in inducing angiogenesis than SVF-derived MSCs, and differentiated into vascular endothelial cells, they may constitute a new cell source for cell-based therapeutic angiogenesis.
doi:10.1186/scrt438
PMCID: PMC4055093  PMID: 24731246
6.  AB76. Adipose-derived stem cells improve erectile function through secretion of growth factor in aged rat 
Translational Andrology and Urology  2014;3(Suppl 1):AB76.
Introduction & objectives
Adipose-derived stem cells (ADSCs) have been recently considered promising therapy for erectile dysfunction (ED). However, the mechanism of ADSCs-based therapy remains to be elucidated. The aim of this study was to determine whether transplantation of ADSCs was capable of resolving aging-related ED, and to investigate its underlying mechanisms.
Materials & methods
Hepatocyte growth factor (HGF), angiopoitein-1 (ANG-1), angiopoitein-2 (ANG-2), insulin-like growth factor (IGF-1), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) secreted by ADSCs were assessed in vitro. Furthermore, the protective effects of VEGF and bFGF secreted by ADSCs were evaluated in vitro by means of neutralization of VEGF, bFGF or both using CCK-8. Sixteen 24-month-old male Sprague-Dawley rats were used for comparative analysis of 2-week treatment regiments with CM-DiI labeled ADSCs or PBS. Eight additional 5-month-old rats were used as young rats group. At 2-week post-transplantation, all the rats were analyzed for erectile function, cavernous VEGF and bFGF levels, and penile histology. The VEGF and bFGF levels of ADSCs-conditioned medium and penile tissues were determined by an enzyme-linked immunosorbent assay (ELISA). The ratio of maximal intracavernous pressure (ICP) to mean arterial blood pressure (MAP) was measured to evaluate erectile function. Immunofluorescence staining was used to evaluate the number of ADSCs, and the contents of cavernous smooth muscle and endothelium.
Results
ADSCs could secrete a large amount of VEGF and bFGF in culture medium compared to basal medium (P<0.05). Rat corpus cavernosum smooth muscle cells (CCSMCs) grew more slowly due to oxidative stress. However, conditioned DMEM of ADSCs played a protection role. Neutralization of VEGF, or both of VEGF and bFGF could significantly attenuate the effect. bFGF played a less important role in the protective effect. Compared to the young rats, the untreated aged rats showed significantly lower Max ICP/MAP (P<0.05) and ADSCs treatment significantly increased the ratio (P<0.05). Immunofluorescence staining demonstrated that there was only a small number of CM-DiI labeled ADSCs found in corpus cavernosum. The corpus cavernosum of untreated aged rats showed decreased VEGF and bFGF levels, and the contents of cavernous smooth muscle and endothelium compared to young rats (P<0.05). ADSCs treatment partially normalized these alterations (P<0.05).
Conclusions
ADSCs treatment may improve aging-related ED partially through secretion of VEGF and bFGF.
doi:10.3978/j.issn.2223-4683.2014.s076
PMCID: PMC4708429
Adipose-derived stem cells; erectile dysfunction (ED); vascular endothelial growth factor (VEGF); basic fibroblast growth factor (BFGF)
7.  Bone marrow mesenchymal stem cells overexpressing human basic fibroblast growth factor increase vasculogenesis in ischemic rats 
Administration or expression of growth factors, as well as implantation of autologous bone marrow cells, promote in vivo angiogenesis. This study investigated the angiogenic potential of combining both approaches through the allogenic transplantation of bone marrow-derived mesenchymal stem cells (MSCs) expressing human basic fibroblast growth factor (hbFGF). After establishing a hind limb ischemia model in Sprague Dawley rats, the animals were randomly divided into four treatment groups: MSCs expressing green fluorescent protein (GFP-MSC), MSCs expressing hbFGF (hbFGF-MSC), MSC controls, and phosphate-buffered saline (PBS) controls. After 2 weeks, MSC survival and differentiation, hbFGF and vascular endothelial growth factor (VEGF) expression, and microvessel density of ischemic muscles were determined. Stable hbFGF expression was observed in the hbFGF-MSC group after 2 weeks. More hbFGF-MSCs than GFP-MSCs survived and differentiated into vascular endothelial cells (P<0.001); however, their differentiation rates were similar. Moreover, allogenic transplantation of hbFGF-MSCs increased VEGF expression (P=0.008) and microvessel density (P<0.001). Transplantation of hbFGF-expressing MSCs promoted angiogenesis in an in vivo hind limb ischemia model by increasing the survival of transplanted cells that subsequently differentiated into vascular endothelial cells. This study showed the therapeutic potential of combining cell-based therapy with gene therapy to treat ischemic disease.
doi:10.1590/1414-431X20143765
PMCID: PMC4181224  PMID: 25118628
Angiogenesis; Basic fibroblast growth factor; Bone marrow mesenchymal stem cells; Gene transfection; Ischemia
8.  Activation of Multiple Signaling Pathways Is Critical for Fibroblast Growth Factor 2- and Vascular Endothelial Growth Factor-Stimulated Ovine Fetoplacental Endothelial Cell Proliferation1 
Biology of reproduction  2007;78(1):143-150.
Fibroblast growth factor-2 (FGF2) and vascular endothelial growth factor (VEGF) are two key regulators of placental angiogenesis. The potent vasodilator nitric oxide (NO) could also act as a key mediator of FGF2- and VEGF-induced angiogenesis. However, the postreceptor signaling pathways governing these FGF2- and VEGF-induced placental angiogenic responses are poorly understood. In this study, we assessed the role of endogenous NO, mitogen-activated protein kinase 3/1 (MAPK3/1), and v-akt murine thymoma viral oncogene homolog 1 (AKT1) in FGF2- and VEGF-stimulated proliferation of ovine fetoplacental endothelial (OFPAE) cells. Both FGF2 and VEGF time-dependently stimulated (P < 0.05) NO production and activated AKT1. Both FGF2- and VEGF-stimulated cell proliferation was dose-dependently inhibited (P < 0.05) by NG-monomethyl-L-arginine (L-NMMA; an NO synthase inhibitor), PD98059 (a selective MAPK3/1 kinase 1 and 2 [MAP2K1/2] inhibitor), or LY294002 (a selective phosphatidylinositol 3 kinase [PI3K] inhibitor) but not by phenyl-4,4,5,5 tetramethylimidazoline-1-oxyl 3-oxide (PTIO, a potent extracellular NO scavenger). At the maximal inhibitory dose without cytotoxicity, PD98059 and LY294002 completely inhibited VEGF-induced cell proliferation but only partially attenuated (P < 0.05) FGF2-induced cell proliferation. PD98059 and LY294002 also inhibited (P < 0.05) FGF2- and VEGF-induced phosphorylation of MAPK3/1 and AKT1, respectively. L-NMMA did not significantly affect FGF2- and VEGF-induced phosphorylation of either MAPK3/1 or AKT1. Thus, in OFPAE cells, both FGF2- and VEGF-stimulated cell proliferation is partly mediated via NO as an intracellular and downstream signal of MAPK3/1 and AKT1 activation. Moreover, activation of both MAP2K1/2/MAPK3/1 and PI3K/AKT1 pathways is critical for FGF2-stimulated cell proliferation, whereas activation of either one pathway is sufficient for mediating the VEGF-induced maximal cell proliferation, indicating that these two kinase pathways differentially mediate the FGF2- and VEGF-stimulated OFPAE cell proliferation.
doi:10.1095/biolreprod.107.064477
PMCID: PMC2441762  PMID: 17901071
AKT1; endothelial cell proliferation; FGF2; growth factors; kinases; MAPK3/1; nitric oxide; pregnancy; vascular endothelial growth factor
9.  Surgical angiogenesis with short-term immunosuppression maintains bone viability in rabbit allogenic knee joint transplantation 
Plastic and reconstructive surgery  2013;131(2):148e-157e.
Background
Vascularized Composite Allotransplantation (VCA) has potential for reconstruction of joint defects, but requires life-long immunosuppression (IS), with substantial risks. This study evaluates an alternative, using surgical angiogenesis from implanted autogenous vessels to maintain viability without long-term immunotherapy.
Methods
Vascularized knee joints were transplanted from Dutch Belted donors to New Zealand White rabbit recipients. Once positioned and revascularized microsurgically, a recipient-derived superficial inferior epigastric fascial (SIEF) flap and a saphenous AV bundle were placed within the transplanted femur and tibia, respectively, to develop a neoangiogenic, autogenous circulation. Ten transplants comprised Group 1. Group 2 (n=9) were no-angiogenesis controls with ligated flaps and AV bundles. Group 3 rabbits (n=10) were autotransplants with patent implants. Tacrolimus was used for 3 weeks to maintain nutrient flow during angiogenesis. At 16 weeks, we assessed bone healing, joint function, bone and cartilage mechanical properties and histology.
Results
Group 1 allotransplants had more robust angiogenesis, better healing, improved mechanical properties and better osteocyte viability than ligated controls (group 2). All 3 groups developed knee joint contractures and arthritic changes. Cartilage thickness and quality were poorer in allograft groups than autotransplant controls.
Conclusions
Surgical angiogenesis from implanted autogenous tissue improves bone viability, healing and material properties in rabbit allogenic knee transplants. However, joint contractures and degenerative changes occurred in all transplants, regardless of antigenicity or blood supply. Experimental studies in a larger animal model with improved methods to maintain joint mobility are needed before the merit of living joint allotransplantation can be judged.
doi:10.1097/PRS.0b013e3182789ad4
PMCID: PMC3927985  PMID: 23358010
10.  Astragalosides promote angiogenesis via vascular endothelial growth factor and basic fibroblast growth factor in a rat model of myocardial infarction 
Molecular Medicine Reports  2015;12(5):6718-6726.
The aim of the present study was to evaluate the effect of astragalosides (ASTs) on angiogenesis, as well as the expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) following myocardial infarction (MI). MI was induced in rats by ligation of the left coronary artery. Twenty-four hours after surgery, the rats were divided into low-dose, high-dose, control and sham surgery groups (n=8 per group). The low- and high-dose groups were treated with ASTs (2.5 and 10 mg/kg/day, respectively, via intraperitoneal injection), while, the control and sham surgery group rats received saline. Serum levels, and mRNA and protein expression levels of VEGF and bFGF, as well as the microvessel density (MVD) were determined four weeks post-treatment. Twenty-four hours post-surgery, VEGF and bFGF serum levels were observed to be comparable between the groups; while at four weeks, the VEGF and bFGF levels were higher in the AST-treated rats (P<0.01). Similarly, VEGF and bFGF mRNA and protein expression levels were higher following AST treatment (P<0.05). No difference in VEGF mRNA expression between the low- and high-dose groups was noted, however, an increase in the bFGF expression levels was detected in the high-dose group. Newly generated blood vessels were observed following MI, with a significant increase in MVD observed in the AST-treated groups (P<0.05). AST promotes angiogenesis of the heart and increases VEGF and bFGF expression levels. Thus, it is hypothesized that increased VEGF and bFGF levels may contribute to the AST-induced increase in angiogenesis in rat models of MI.
doi:10.3892/mmr.2015.4307
PMCID: PMC4626201  PMID: 26352430
astragaloside; myocardial infarction; vascular endothelial growth factor; basic fibroblast growth factor; angiogenesis
11.  Suppression of basic fibroblast growth factor-induced angiogenesis by a specific chymase inhibitor, BCEAB, through the chymase-angiotensin-dependent pathway in hamster sponge granulomas 
British Journal of Pharmacology  2002;137(4):554-560.
We investigated the profound involvement of mast cell chymase, an alternative angiotensin II-generating enzyme, in angiogenesis using a specific chymase inhibitor. We also studied the functional profiles of this novel inhibitor in basic fibroblast growth factor (bFGF)-induced angiogenesis.In this study, angiogenesis was induced by daily injections of bFGF (0.3 μg site−1 day−1), angiotensin I (2 nmol site−1 day−1) or angiotensin II (2 nmol site−1 day−1) into sponges implanted to male hamsters subcutaneously for 7 days. Angiogenesis in the granulation tissue surrounding sponges was evaluated by measuring the haemoglobin (Hb) content and local blood flow as the parameters for angiogenesis.A chymase inhibitor, BCEAB (4-[1-{[bis-(4-methyl-phenyl)-methyl]-carbamoyl}-3-(2-ethoxy-benzyl)-4-oxo-azetidine-2-yloxy]-benzoic acid), was simultaneously administered into the implanted sponges (2 or 5 nmol site−1 day−1, for 7 days) treated with bFGF and strongly suppressed the haemoglobin contents in sponge granulomas. In the studies using a laser doppler perfusion imager, BCEAB (5 nmol site−1 day−1) also attenuated the bFGF-induced increase of local blood flow around the implanted sponge granuloma.In bFGF-induced angiogenesis, chymase activity in sponge granulomas was substantially increased. It was also confirmed that the chymase activity increased by bFGF was significantly and dose-dependently inhibited by BCEAB (2, 5 nmol site−1 day−1).BCEAB inhibited the Hb contents and the expression of vascular endothelial growth factor (VEGF) mRNA induced by angiotensin I but not by angiotensin II.These results suggest that the significance of chymase in bFGF-induced angiogenesis was confirmed, and a novel inhibitor, BCEAB, strongly suppresses the bFGF-induced angiogenesis through the chymase-angiotensin II-VEGF dependent pathway.
doi:10.1038/sj.bjp.0704893
PMCID: PMC1573517  PMID: 12359638
Chymase; angiotensin; angiogenesis; inhibitor; VEGF; bFGF
12.  125I-labeled anti-bFGF monoclonal antibody inhibits growth of hepatocellular carcinoma 
World Journal of Gastroenterology  2016;22(21):5033-5041.
AIM: To investigate the inhibitory efficacy of 125I-labeled anti-basic fibroblast growth factor (bFGF) monoclonal antibody (mAb) in hepatocellular carcinoma (HCC).
METHODS: bFGF mAb was prepared by using the 1G9B9 hybridoma cell line with hybridization technology and extracted from ascites fluid through a Protein G Sepharose affinity column. After labeling with 125I through the chloramine-T method, bFGF mAb was further purified by a Sephadex G-25 column. Gamma radiation counter GC-1200 detected radioactivity of 125I-bFGF mAb. The murine H22 HCC xenograft model was established and randomized to interventions with control (phosphate-buffered saline), 125I-bFGF mAb, 125I plus bFGF mAb, bFGF mAb, or 125I. The ratios of tumor inhibition were then calculated. Expression of bFGF, fibroblast growth factor receptor (FGFR), platelet-derived growth factor, and vascular endothelial growth factor (VEGF) mRNA was determined by quantitative reverse transcriptase real-time polymerase chain reaction.
RESULTS: The purified bFGF mAb solution was 8.145 mg/mL with a titer of 1:2560000 and was stored at -20 °C. After coupling, 125I-bFGF mAb was used at a 1: 1280000 dilution, stored at 4 °C, and its specific radioactivity was 37 MBq/mg. The corresponding tumor weight in the control, 125I, bFGF mAb, 125I plus bFGF mAb, and 125I-bFGF mAb groups was 1.88 ± 0.25, 1.625 ± 0.21, 1.5 ± 0.18, 1.41 ± 0.16, and 0.98 ± 0.11 g, respectively. The tumor inhibition ratio in the 125I, bFGF mAb, 125I plus bFGF mAb, and 125I-bFGF mAb groups was 13.6%, 20.2%, 25.1%, and 47.9%, respectively. Growth of HCC xenografts was inhibited significantly more in the 125I-bFGF mAb group than in the other groups (P < 0.05). Expression of bFGF and FGFR mRNA in the 125I-bFGF mAb group was significantly decreased in comparison with other groups (P < 0.05). Groups under interventions revealed increased expression of VEGF mRNA (except for 125I group) compared with the control group.
CONCLUSION: 125I-bFGF mAb inhibits growth of HCC xenografts. The coupling effect of 125I-bFGF mAb is more effective than the concomitant use of 125I and bFGF mAb.
doi:10.3748/wjg.v22.i21.5033
PMCID: PMC4886378  PMID: 27275095
Basic fibroblast growth factor; 125Iodine; Monoclonal antibody; Hepatocellular carcinoma; Fibroblast growth factor receptor; Vascular endothelial growth factor
13.  Arteriogenic therapy based on simultaneous delivery of VEGF-A and FGF4 genes improves the recovery from acute limb ischemia 
Vascular Cell  2013;5:13.
Background
Gene therapy stimulating the growth of blood vessels is considered for the treatment of peripheral and myocardial ischemia. Here we aimed to achieve angiogenic synergism between vascular endothelial growth factor-A (VEGF-A, VEGF) and fibroblast growth factor 4 (FGF4) in murine normoperfused and ischemic limb muscles.
Methods
Adeno-associated viral vectors (AAVs) carrying β-galactosidase gene (AAV-LacZ), VEGF-A (AAV-VEGF-A) or two angiogenic genes (AAV-FGF4-IRES-VEGF-A) were injected into the normo-perfused adductor muscles of C57Bl/6 mice. Moreover, in a different experiment, mice were subjected to unilateral hindlimb ischemia by femoral artery ligation followed by intramuscular injections of AAV-LacZ, AAV-VEGF-A or AAV-FGF4-IRES-VEGF-A below the site of ligation. Post-ischemic blood flow recovery was assessed sequentially by color laser Doppler. Mice were monitored for 28 days.
Results
VEGF-A delivered alone (AAV-VEGF-A) or in combination with FGF4 (AAV-FGF4-IRES-VEGF-A) increased the number of capillaries in normo-perfused hindlimbs when compared to AAV-LacZ. Simultaneous overexpression of both agents (VEGF-A and FGF4) stimulated the capillary wall remodeling in the non-ischemic model. Moreover, AAV-FGF4-IRES-VEGF-A faster restored the post-ischemic foot blood flow and decreased the incidence of toe necrosis in comparison to AAV-LacZ.
Conclusions
Synergy between VEGF-A and FGF4 to produce stable and functional blood vessels may be considered a promising option in cardiovascular gene therapy.
doi:10.1186/2045-824X-5-13
PMCID: PMC3703285  PMID: 23816205
AAV; Angiogenesis; Arteriogenesis; FGF4; VEGF-A
14.  A Novel Tumor-Promoting Function Residing in the 5′ Non-coding Region of vascular endothelial growth factor mRNA 
PLoS Medicine  2008;5(5):e94.
Background
Vascular endothelial growth factor-A (VEGF) is one of the key regulators of tumor development, hence it is considered to be an important therapeutic target for cancer treatment. However, clinical trials have suggested that anti-VEGF monotherapy was less effective than standard chemotherapy. On the basis of the evidence, we hypothesized that vegf mRNA may have unrecognized function(s) in cancer cells.
Methods and Findings
Knockdown of VEGF with vegf-targeting small-interfering (si) RNAs increased susceptibility of human colon cancer cell line (HCT116) to apoptosis caused with 5-fluorouracil, etoposide, or doxorubicin. Recombinant human VEGF165 did not completely inhibit this apoptosis. Conversely, overexpression of VEGF165 increased resistance to anti-cancer drug-induced apoptosis, while an anti-VEGF165-neutralizing antibody did not completely block the resistance. We prepared plasmids encoding full-length vegf mRNA with mutation of signal sequence, vegf mRNAs lacking untranslated regions (UTRs), or mutated 5′UTRs. Using these plasmids, we revealed that the 5′UTR of vegf mRNA possessed anti-apoptotic activity. The 5′UTR-mediated activity was not affected by a protein synthesis inhibitor, cycloheximide. We established HCT116 clones stably expressing either the vegf 5′UTR or the mutated 5′UTR. The clones expressing the 5′UTR, but not the mutated one, showed increased anchorage-independent growth in vitro and formed progressive tumors when implanted in athymic nude mice. Microarray and quantitative real-time PCR analyses indicated that the vegf 5′UTR-expressing tumors had up-regulated anti-apoptotic genes, multidrug-resistant genes, and growth-promoting genes, while pro-apoptotic genes were down-regulated. Notably, expression of signal transducers and activators of transcription 1 (STAT1) was markedly repressed in the 5′UTR-expressing tumors, resulting in down-regulation of a STAT1-responsive cluster of genes (43 genes). As a result, the tumors did not respond to interferon (IFN)α therapy at all. We showed that stable silencing of endogenous vegf mRNA in HCT116 cells enhanced both STAT1 expression and IFNα responses.
Conclusions
These findings suggest that cancer cells have a survival system that is regulated by vegf mRNA and imply that both vegf mRNA and its protein may synergistically promote the malignancy of tumor cells. Therefore, combination of anti-vegf transcript strategies, such as siRNA-based gene silencing, with anti-VEGF antibody treatment may improve anti-cancer therapies that target VEGF.
Shigetada Teshima-Kondo and colleagues find that cancer cells have a survival system that is regulated by vegf mRNA and that vegf mRNA and its protein may synergistically promote the malignancy of tumor cells.
Editors' Summary
Background
Normally, throughout life, cell division (which produces new cells) and cell death are carefully balanced to keep the body in good working order. But sometimes cells acquire changes (mutations) in their genetic material that allow them to divide uncontrollably to form cancers—disorganized masses of cells. When a cancer is small, it uses the body's existing blood supply to get the oxygen and nutrients it needs for its growth and survival. But, when it gets bigger, it has to develop its own blood supply. This process is called angiogenesis. It involves the release by the cancer cells of proteins called growth factors that bind to other proteins (receptors) on the surface of endothelial cells (the cells lining blood vessels). The receptors then send signals into the endothelial cells that tell them to make new blood vessels. One important angiogenic growth factor is “vascular endothelial growth factor” (VEGF). Tumors that make large amounts of VEGF tend to be more abnormal and more aggressive than those that make less VEGF. In addition, high levels of VEGF in the blood are often associated with poor responses to chemotherapy, drug regimens designed to kill cancer cells.
Why Was This Study Done?
Because VEGF is a key regulator of tumor development, several anti-VEGF therapies—drugs that target VEGF and its receptors—have been developed. These therapies strongly suppress the growth of tumor cells in the laboratory and in animals but, when used alone, are no better at increasing the survival times of patients with cancer than standard chemotherapy. Scientists are now looking for an explanation for this disappointing result. Like all proteins, cells make VEGF by “transcribing” its DNA blueprint into an mRNA copy (vegf mRNA), the coding region of which is “translated” into the VEGF protein. Other, “noncoding” regions of vegf mRNA control when and where VEGF is made. Scientists have recently discovered that the noncoding regions of some mRNAs suppress tumor development. In this study, therefore, the researchers investigate whether vegf mRNA has an unrecognized function in tumor cells that could explain the disappointing clinical results of anti-VEGF therapeutics.
What Did the Researchers Do and Find?
The researchers first used a technique called small interfering (si) RNA knockdown to stop VEGF expression in human colon cancer cells growing in dishes. siRNAs are short RNAs that bind to and destroy specific mRNAs in cells, thereby preventing the translation of those mRNAs into proteins. The treatment of human colon cancer cells with vegf-targeting siRNAs made the cells more sensitive to chemotherapy-induced apoptosis (a type of cell death). This sensitivity was only partly reversed by adding VEGF to the cells. By contrast, cancer cells engineered to make more vegf mRNA had increased resistance to chemotherapy-induced apoptosis. Treatment of these cells with an antibody that inhibited VEGF function did not completely block this resistance. Together, these results suggest that both vegf mRNA and VEGF protein have anti-apoptotic effects. The researchers show that the anti-apoptotic activity of vegf mRNA requires a noncoding part of the mRNA called the 5′ UTR, and that whereas human colon cancer cells expressing this 5′ UTR form tumors in mice, cells expressing a mutated 5′ UTR do not. Finally, they report that the expression of several pro-apoptotic genes and of an anti-tumor pathway known as the interferon/STAT1 tumor suppression pathway is down-regulated in tumors that express the vegf 5′ UTR.
What Do These Findings Mean?
These findings suggest that some cancer cells have a survival system that is regulated by vegf mRNA and are the first to show that a 5′UTR of mRNA can promote tumor growth. They indicate that VEGF and its mRNA work together to promote their development and to increase their resistance to chemotherapy drugs. They suggest that combining therapies that prevent the production of vegf mRNA (for example, siRNA-based gene silencing) with therapies that block the function of VEGF might improve survival times for patients whose tumors overexpress VEGF.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050094.
This study is discussed further in a PLoS Medicine Perspective by Hughes and Jones
The US National Cancer Institute provides information about all aspects of cancer, including information on angiogenesis, and on bevacizumab, an anti-VEGF therapeutic (in English and Spanish)
CancerQuest, from Emory University, provides information on all aspects of cancer, including angiogenesis (in several languages)
Cancer Research UK also provides basic information about what causes cancers and how they develop, grow, and spread, including information about angiogenesis
Wikipedia has pages on VEGF and on siRNA (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.0050094
PMCID: PMC2386836  PMID: 18494554
15.  Regeneration of Dental-Pulp-like Tissue by Chemotaxis-Induced Cell Homing 
Tissue Engineering. Part A  2010;16(10):3023-3031.
Tooth infections or injuries involving dental pulp are treated routinely by root canal therapy. Endodontically treated teeth are devitalized, susceptible to re-infections, fractures, and subsequent tooth loss. Here, we report regeneration of dental-pulp-like tissue by cell homing and without cell transplantation. Upon in vivo implantation of endodontically treated real-size, native human teeth in mouse dorsum for the tested 3 weeks, delivery of basic fibroblast growth factor and/or vascular endothelial growth factor (bFGF and/or VEGF) yielded re-cellularized and revascularized connective tissue that integrated to native dentinal wall in root canals. Further, combined delivery of bFGF, VEGF, or platelet-derived growth factor (PDGF) with a basal set of nerve growth factor (NGF) and bone morphogenetic protein-7 (BMP7) generated cellularized and vascularized tissues positive of VEGF antibody staining and apparent neo-dentin formation over the surface of native dentinal wall in some, but not all, endodontically treated teeth. Newly formed dental pulp tissue appeared dense with disconnected cells surrounded by extracellular matrix. Erythrocyte-filled blood vessels were present with endothelial-like cell lining. Reconstructed, multiple microscopic images showed complete fill of dental-pulp-like tissue in the entire root canal from root apex to pulp chamber with tissue integration to dentinal wall upon delivery of bFGF, VEGF, or PDGF with a basal set of NGF and BMP7. Quantitative ELISA showed that combinatory delivery of bFGF, VEGF, or PDGF with basal NGF and BMP7 elaborated von Willerbrand factor, dentin sialoprotein, and NGF. These findings represent the first demonstration of regenerated dental-pulp-like tissue in endodontically treated root canals of real-size, native human teeth. The present chemotaxis-based approach has potent cell homing effects for re-cellularization and revascularization in endodontically treated root canals in vivo, although in an ectopic model. Regeneration of dental pulp by cell homing, rather than cell delivery, may accelerate clinical translation.
doi:10.1089/ten.tea.2010.0181
PMCID: PMC2947424  PMID: 20486799
16.  Suppression of VEGF-induced angiogenesis by the protein tyrosine kinase inhibitor, lavendustin A. 
British Journal of Pharmacology  1995;114(2):262-268.
1. Vascular endothelial growth factor (VEGF) is a heparin-binding angiogenic factor which specifically acts on endothelial cells via distinct membrane-spanning tyrosine kinase receptors. Here we used the rat sponge implant model to test the hypothesis that the angiogenic activity of VEGF can be suppressed by protein tyrosine kinase (PTK) inhibitors. 2. Neovascular responses in subcutaneous sponge implants were determined by measurements of relative sponge blood flow by use of a 133Xe clearance technique, and confirmed by histological studies and morphometric analysis. 3. Daily local administration of 250 ng VEGF165 accelerated the rate of 133Xe clearance from the sponges and induced an intense neovascularisation. This VEGF165-induced angiogenesis was inhibited by daily co-administration of the selective PTK inhibitor, lavendustin A (10 micrograms), but not its negative control, lavendustin B (10 micrograms). Blood flow measurements and morphometric analysis of 8-day-old sponges showed that lavendustin A reduced the 133Xe clearance of VEGF165-treated sponges from 32.9 +/- 1.5% to 20.9 +/- 1.6% and the total fibrovascular growth area from 62.4 +/- 6.1% to 21.6 +/- 6.8% (n = 12, P < 0.05). 4. Co-injection of suramin (3 mg), an inhibitor of heparin-binding growth factors, also suppressed the VEGF165-elicited neovascular response. In contrast, neither lavendustin A nor suramin produced any effect on the basal sponge-induced angiogenesis. 5. When given alone, low doses of VEGF165 (25 ng) or basic fibroblast growth factor (bFGF; 10 ng) did not modify the basal sponge-induced neovascularisation.(ABSTRACT TRUNCATED AT 250 WORDS)
Images
PMCID: PMC1510224  PMID: 7533611
17.  Targeting Angiogenesis in Cancer Therapy: Moving Beyond Vascular Endothelial Growth Factor 
The Oncologist  2015;20(6):660-673.
It is increasingly clear that there are many interconnected and compensatory pathways that can overcome vascular endothelial growth factor-targeted inhibition of angiogenesis. Maximizing the potential of antiangiogenic therapy is likely to require a broader therapeutic approach using a new generation of multitargeted antiangiogenic agents.
Angiogenesis, or the formation of new capillary blood vessels, occurs primarily during human development and reproduction; however, aberrant regulation of angiogenesis is also a fundamental process found in several pathologic conditions, including cancer. As a process required for invasion and metastasis, tumor angiogenesis constitutes an important point of control of cancer progression. Although not yet completely understood, the complex process of tumor angiogenesis involves highly regulated orchestration of multiple signaling pathways. The proangiogenic signaling molecule vascular endothelial growth factor (VEGF) and its cognate receptor (VEGF receptor 2 [VEGFR-2]) play a central role in angiogenesis and often are highly expressed in human cancers, and initial clinical efforts to develop antiangiogenic treatments focused largely on inhibiting VEGF/VEGFR signaling. Such approaches, however, often lead to transient responses and further disease progression because angiogenesis is regulated by multiple pathways that are able to compensate for each other when single pathways are inhibited. The platelet-derived growth factor (PDGF) and PDGF receptor (PDGFR) and fibroblast growth factor (FGF) and FGF receptor (FGFR) pathways, for example, provide potential escape mechanisms from anti-VEGF/VEGFR therapy that could facilitate resumption of tumor growth. Accordingly, more recent treatments have focused on inhibiting multiple signaling pathways simultaneously. This comprehensive review discusses the limitations of inhibiting VEGF signaling alone as an antiangiogenic strategy, the importance of other angiogenic pathways including PDGF/PDGFR and FGF/FGFR, and the novel current and emerging agents that target multiple angiogenic pathways for the treatment of advanced solid tumors.
Implications for Practice:
Significant advances in cancer treatment have been achieved with the development of antiangiogenic agents, the majority of which have focused on inhibition of the vascular endothelial growth factor (VEGF) pathway. VEGF targeting alone, however, has not proven to be as efficacious as originally hoped, and it is increasingly clear that there are many interconnected and compensatory pathways that can overcome VEGF-targeted inhibition of angiogenesis. Maximizing the potential of antiangiogenic therapy is likely to require a broader therapeutic approach using a new generation of multitargeted antiangiogenic agents.
doi:10.1634/theoncologist.2014-0465
PMCID: PMC4571783  PMID: 26001391
Angiogenesis inhibitors; Antibodies, monoclonal, humanized; Molecular targeted therapy; Receptors; Fibroblast growth factor; Platelet-derived growth factor; Vascular endothelial growth factor
18.  Clinical Role of Bone Marrow Angiogenesis in Childhood Acute Lymphocytic Leukemia 
Yonsei Medical Journal  2007;48(2):171-175.
Purpose
Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are associated with increased angiogenesis, growth, and metastasis in solid tumors. But, until today, the importance of theses factors on leukemia, especially childhood acute lymphocytic leukemia (ALL) has received limited attention. Therefore, this study examined the bone marrow plasma VEGF and bFGF levels in ALL patients and normal controls.
Patients and Methods
Bone marrow plasmas at diagnosis from 33 ALL patients (median age 5.9 years; range 1.8-13.9 years) were used for analysis. The bone marrow levels of bFGF and VEGF were determined by enzyme-linked immunosorbent assay (R&D Systems) and compared with the bone marrow levels of 7 healthy control subjects (median age 11.98 years; 6 months -13.6 years).
Results
Average VEGF was higher in relapse ALL (N=7, 216.6±79.9pg/mL) compared to standard (N=9, 36.8±12.1pg/mL) (p=0.013) or high risk ALL (N=17, 80.0±12.2pg/mL) (p=0.023). bFGF levels were also significantly higher in relapse than standard-, or high-risk ALL patients (relapse ALL; 48.6±15.4pg/mL, standard risk ALL; 18.9±5.5pg/mL, high risk ALL; 19.0±3.5pg/mL, normal control; 18.6±4.0pg/mL) (p=0.003). Three patients with refractory relapse and death had much higher VEGF and bFGF values (VEGF; 420.0±81.6pg/mL, bFGF; 85.6±3.2pg/mL).
Conclusion
Our data suggest that the increased levels of VEGF and bFGF in bone marrow may play an important role in prognosis of childhood ALL.
doi:10.3349/ymj.2007.48.2.171
PMCID: PMC2628125  PMID: 17461513
Angiogenesis factor; child; acute lymphocytic leukemia
19.  S100A13 is a new angiogenic marker in human melanoma 
Angiogenesis is critical in melanoma progression and metastasis and relies on the synthesis and release of proangiogenic molecules such as vascular endothelial growth factor (VEGF)-A and fibroblast growth factors (FGFs). S100A13 is a small calcium-binding protein that facilitates the release of FGF-1, the prototype of the FGF family. S100A13 is upregulated in astrocytic gliomas, in which it correlates with VEGF-A expression, microvessel density and tumor grading, and promotes a more aggressive, invasive phenotype in lung cancer-derived cell lines. To investigate the involvement of S100A13 in human cutaneous melanoma, we analyzed a series of 87 cutaneous melanocytic lesions: 14 common acquired melanocytic nevi, 14 atypical, so-called `dysplastic' nevi, 45 melanomas (17 radial growth phase and 28 vertical growth phase) and 14 melanoma metastases. Main clinical and pathological features, including histotype, Breslow thickness, Clark's level and outcome were recorded. Microvessel density was determined with CD105/endoglin staining. Semiquantitative determination of S100A13, FGF-1 and VEGF-A protein expression was obtained by immunostaining. Quantification of S100A13 mRNA was achieved by real-time PCR. We found that S100A13 was expressed in melanocytic lesions; compared with benign nevi, S100A13 protein expression was significantly upregulated in melanomas (P=0.024), in which it correlated positively with the intensity of VEGF-A staining (P=0.041) and microvessel density (P=0.007). The level of expression of S100A13 mRNA also significantly increased with progression of disease, from radial growth phase (0.7±0.7) to vertical growth phase (3.6±3.1) to metastases (7.0±7.0) (P<0.001). Furthermore, S100A13 mRNA correlated positively with VEGF-A (P=0.023), TNM stage (P=0.05), risk of relapse (P=0.014) and status at follow-up (P=0.024). In conclusion, S100A13 is expressed in melanocytic lesions when the angiogenic switch occurs and it may cooperate with VEGF-A in supporting the formation of new blood vessels, favoring the shift from radial to vertical tumor growth. Therefore, S100A13 may represent a new angiogenic and prognostic marker in melanoma.
doi:10.1038/modpathol.2010.54
PMCID: PMC2882157  PMID: 20208480
melanoma; tumor angiogenesis; S100A13; FGF-1; immunohistochemistry; real-time PCR
20.  Plasma VEGF levels in breast cancer patients with and without metastases 
Oncology Letters  2010;1(4):739-741.
Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis since it stimulates the formation of new blood vessels. Basic fibroblast growth factor (bFGF) is related to the promotion of endothelial cells into tube-like structures, and it is therefore expected to promote angiogenesis with a greater potency than VEGF. VEGF and bFGF are considered to be biomarkers that predict treatment effectiveness. Elevated plasma VEGF and bFGF levels have been reported in a variety of different malignant tumors, and patients with metastatic disease have also been reported to present with higher serum VEGF and bFGF levels. Other studies have documented controversial results with respect to the prognostic and predictive value of the aforementioned biomarkers. This study aimed to determine the plasma VEGF and bFGF levels in breast cancer patients without metastatic disease compared with breast cancer patients with advanced metastatic disease. The study included 93 patients with breast cancer, 46 without recurrent disease (group A) and 47 with metastatic disease (group B), as well as 21 healthy individuals. The median age was 58 years (range 34–78) for group A and 59 years (range 37–75) for group B. All 93 patients underwent chemotherapy, adjuvant for group A, and adjuvant plus chemotherapy for group B patients with advanced disease. Plasma VEGF and bFGF levels were determined using a quantitative sandwich immunoassay, and samples were tested in triplicate (ELISA). The plasma levels of VEGF and bFGF varied greatly, i.e., from extremely low to extremely high in the two groups, as well as in the healthy individuals. No statistically significant difference was found between the two groups or between the patients and healthy individuals. Data of the present study therefore showed that VEGF and bFGF levels are not valuable biomarkers for predicting treatment outcome.
doi:10.3892/ol_00000129
PMCID: PMC3436353  PMID: 22966372
vascular endothelial growth factor; breast cancer
21.  Effects on Proliferation and Differentiation of Multipotent Bone Marrow Stromal Cells Engineered to Express Growth Factors for Combined Cell and Gene Therapy 
Stem cells (Dayton, Ohio)  2011;29(11):1727-1737.
A key mechanism for mesenchymal stem cells/bone marrow stromal cells (MSCs) to promote tissue repair is by secretion of soluble growth factors (GFs). Therefore, clinical application could be optimized by a combination of cell and gene therapies, where MSCs are genetically modified to express higher levels of a specific factor. However, it remains unknown how this overexpression may alter the fate of the MSCs. Here, we show effects of overexpressing the growth factors, such as basic fibroblast growth factor (bFGF), platelet derived growth factor B (PDGF-BB), transforming growth factor β1 (TGF-β1), and vascular endothelial growth factor (VEGF), in human bone marrow-derived MSCs. Ectopic expression of bFGF or PDGF-B lead to highly proliferating MSCs and lead to a robust increase in osteogenesis. In contrast, adipogenesis was strongly inhibited in MSCs overexpressing PDGF-B and only mildly affected in MSCs overexpressing bFGF. Overexpression of TGF-β1 blocked both osteogenic and adipogenic differentiation while inducing the formation of stress fibers and increasing the expression of the smooth muscle marker calponin-1 and the chondrogenic marker collagen type II. In contrast, MSCs overexpressing VEGF did not vary from control MSCs in any parameters, likely due to the lack of VEGF receptor expression on MSCs. MSCs engineered to overexpress VEGF strongly induced the migration of endothelial cells and enhanced blood flow restoration in a xenograft model of hind limb ischemia. These data support the rationale for genetically modifying MSCs to enhance their therapeutically relevant trophic signals, when safety and efficacy can be demonstrated, and when it can be shown that there are no unwanted effects on their proliferation and differentiation.
doi:10.1002/stem.720
PMCID: PMC3784258  PMID: 21898687
Growth factors; Mesenchymal stem cells; Bone marrow stromal cells; Angiogenesis
22.  Suppression of Protein Phosphatase 2 Differentially Modulates VEGF- and FGF2-Induced Signaling in Ovine Fetoplacental Artery Endothelial Cells 
Placenta  2009;30(10):907-913.
Vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) elicit cellular responses via activation of protein kinases and phosphatases. We have reported that the MEK1/2/ERK1/2 and PI3K/AKT1 pathways are critical for VEGF- and FGF2-stimulated ovine fetoplacental endothelial (OFPAE) cell proliferation. We have also shown that protein phosphatase 3 (PPP3) differentially modulates VEGF- and FGF2-stimulated cell proliferation and activation of ERK1/2 and AKT1 in OFPAE cells. Herein, we investigated if protein phosphatase 2 (PPP2) modulated VEGF- and FGF2-induced ERK1/2, AKT1, and p38 MAPK activation and VEGF- and FGF2-stimulated cell proliferation in OFPAE cells. Small interfering RNA (siRNA) specifically targeting human PPP2 catalytic subunit α (PPP2CA) was used to suppress PPP2CA expression in OFPAE cells. When compared with scrambled siRNA, PPP2CA siRNA decreased (p < 0.05) PPP2CA protein levels (∼ 70%) and activity (∼ 50%) without altering protein levels of PPP3 catalytic subunit α (PPP3CA), nitric oxide (NO) synthase 3 (NOS3), ERK1/2, AKT1, and p38 MAPK. FGF2, but not VEGF rapidly (≤ 5 min) induced p38 MAPK phosphorylation. Suppression of PPP2CA enhanced (p < 0.05) VEGF-induced AKT1, but not ERK1/2 phosphorylation, whereas inhibited (p < 0.05) FGF2-induced ERK1/2 and p38 MAPK and slightly attenuated FGF2-induced AKT1 phosphorylation. Suppression of PPP2CA did not significantly affect VEGF- and FGF2-stimulated OFPAE cell proliferation. Thus, suppression of PPP2CA alone differentially modulated VEGF- and FGF2-induced ERK1/2, AKT1, and p38 MAPK activation, without altering VEGF- and FGF2-stimulated cell proliferation in OFPAE cells. These data also suggest that signaling molecules other than ERK1/2, AKT1, and p38 MAPK are important mediators for VEGF- and FGF2-stimulated OFPAE cell proliferation after PPP2CA suppression.
doi:10.1016/j.placenta.2009.07.003
PMCID: PMC2748137  PMID: 19692121
Endothelial cell; signaling transduction; placenta
23.  Hypoxic induction of endothelial cell growth factors in retinal cells: identification and characterization of vascular endothelial growth factor (VEGF) as the mitogen. 
Molecular Medicine  1995;1(2):182-193.
BACKGROUND: New vessel growth is often associated with ischemia, and hypoxic tissue has been identified as a potential source of angiogenic factors. In particular, ischemia is associated with the development of neovascularization in a number of ocular pathologies. For this reason, we have studied the induction of endothelial cell mitogens by hypoxia in retinal cells. MATERIALS AND METHODS: Human retinal pigment epithelium (hRPE) were grown under normoxic and hypoxic conditions and examined for the production of endothelial mitogens. Northern analysis, biosynthetic labeling and immunoprecipitation, and ELISA were used to assess the levels of vascular endothelial growth factor/vascular permeability factor (VEGF) and basic fibroblast growth factor (bFGF), two endothelial cell mitogens and potent angiogenic factors. Soluble receptors for VEGF were employed as competitive inhibitors to determine the contribution of the growth factor to the hypoxia-stimulated mitogen production. RESULTS: Following 6-24 hr of hypoxia, confluent and growing cultures of hRPE increase their levels of VEGF mRNA and protein synthesis. Biosynthetic labeling studies and RT-PCR analysis indicate that the cells secrete VEGF121 and VEGF165, the soluble forms of the angiogenic factor. In contrast, hRPE cultured under hypoxic conditions show reduced steady-state levels of basic fibroblast growth factor (bFGF) mRNA and decreased bFGF protein synthesis. Unlike VEGF, bFGF is not found in conditioned media of hRPE following 24 hr of hypoxia. Using a soluble high-affinity VEGF receptor as a competitive inhibitor of VEGF, we demonstrate that a VEGF-like activity is the sole hypoxia-inducible endothelial mitogen produced by cultured hRPE. CONCLUSIONS: From this comparison we conclude that hRPE do not respond to hypoxia with a general, nonspecific increase in the overall levels of growth factors, as is seen during cell wounding responses or serum stimulation. The physiological relevance of data from this in vitro model are affirmed by separate studies in an animal model of retinal ischemia-induced ocular neovascularization (1) in which retina-derived VEGF levels have been shown to correlate spatio-temporally with the onset of angiogenesis. Taken together, these data support the hypothesis that the induction of VEGF by hypoxia mediates the rapid, initial angiogenic response to retinal ischemia.
Images
PMCID: PMC2229943  PMID: 8529097
24.  Comparative Evaluation of TRAIL, FGF-2 and VEGF-A-Induced Angiogenesis In Vitro and In Vivo 
Tumor necrosis-factor-related apoptosis-inducing ligand (TRAIL) has been implicated in angiogenesis; the growth of new blood vessels from an existing vessel bed. Our aim was to compare pro-angiogenic responses of TRAIL, vascular endothelial growth-factor-A (VEGF-A) and fibroblast growth-factor-2 (FGF-2) either separately (10 ng/mL) or in combination, followed by the assessment of proliferation, migration and tubule formation using human microvascular endothelial-1 (HMEC-1) cells in vitro. Angiogenesis was also measured in vivo using the Matrigel plug assay. TRAIL and FGF-2 significantly augmented HMEC-1 cell proliferation and migration, with combination treatment having an enhanced effect on cell migration only. In contrast, VEGF-A did not stimulate HMEC-1 migration at 10 ng/mL. Tubule formation was induced by all three factors, with TRAIL more effective compared to VEGF-A, but not FGF-2. TRAIL at 400 ng/mL, but not VEGF-A, promoted CD31-positive staining into the Matrigel plug. However, FGF-2 was superior, stimulating cell infiltration and angiogenesis better than TRAIL and VEGF-A in vivo. These findings demonstrate that each growth factor is more effective at different processes of angiogenesis in vitro and in vivo. Understanding how these molecules stimulate different processes relating to angiogenesis may help identify new strategies and treatments aimed at inhibiting or promoting dysregulated angiogenesis in people.
doi:10.3390/ijms17122025
PMCID: PMC5187825  PMID: 27918462
angiogenesis; proliferation; scratch assay; tubule formation; Matrigel plug; TRAIL; VEGF-A; FGF-2
25.  Hypoxia Enhances FGF2- and VEGF-Stimulated Human Placental Artery Endothelial Cell Proliferation: Roles of MEK1/2/ERK1/2 and PI3K/AKT1 Pathways 
Placenta  2009;30(12):1045-1051.
Placental development occurs under a low oxygen (2–8% O2) environment, which is critical for placental development and angiogenesis. In this study, we examined if hypoxia affected fibroblast growth factor 2 (FGF2)- and vascular endothelial growth factor (VEGF)-stimulated cell proliferation via the mitogen-activated protein kinase kinase 1/2 (MEK1/2)/extracellular signal-regulated kinases 1/2 (ERK1/2) and phosphatidylinositol-3 kinase (PI3K)/v-akt murine thymomaviral oncogene homologue (AKT1) pathways in human placental artery endothelial (HPAE) cells. We observed that under normoxia (~20% O2), FGF2 and VEGF dose-dependently stimulated cell proliferation. Hypoxia (3% O2) significantly promoted FGF2- and VEGF-stimulated cell proliferation as compared to normoxia. Under both normoxia and hypoxia, FGF2 rapidly induced ERK1/2 and AKT1 phosphorylation, while VEGF induced ERK1/2, but not AKT1 phosphorylation. However, hypoxia did not significantly alter FGF2- and VEGF-induced ERK1/2 and AKT1 phosphorylation as compared to normoxia. PD98059 (a MEK1/2 inhibitor) at 20 μM and LY294002 (a PI3K inhibitor) at 5 μM attenuated FGF2- and VEGF-induced phosphorylation of ERK1/2 and AKT1, respectively. PD98059, even at doses that drastically inhibited FGF2-induced ERK1/2 phosphorylation (20 μM) and caused cell loss (40 μM), did not affect FGF2-stimulated cell proliferation, which was confirmed by U0126 (another potent MEK1/2 inhibitor). PD98059, however, dose-dependently inhibited VEGF-stimulated cell proliferation. Conversely, LY294002 dose-dependently inhibited FGF2-, but not VEGF-stimulated cell proliferation. These data suggest that in the MEK1/2/ERK1/2 and PI3K/AKT1 pathways differentially mediate FGF2- and VEGF-stimulated HPAE cell proliferation. These results also indicate that hypoxia promotes FGF2- and VEGF-stimulated cell proliferation without further activation of the PI3K/AKT1 and MEK1/2/ERK1/2, respectively.
doi:10.1016/j.placenta.2009.10.007
PMCID: PMC2788063  PMID: 19892399
hypoxia; placenta; endothelial cells; kinases; cell proliferation

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