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1.  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
2.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  COMPARISON OF VASCULAR ENDOTHELIAL GROWTH FACTOR AND FIBROBLAST GROWTH FACTOR-2 IN A SWINE MODEL OF ENDOTHELIAL DYSFUNCTION 
Objectives
Growth-factor based angiogenesis, with or without cell therapy, is a promising therapeutic modality for patients with coronary artery disease. We compared the relative efficacies of surgically delivered vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) in a swine model of hypercholesterolemia-induced endothelial dysfunction which captures many of the pathophysiologic abnormalities of human coronary disease.
Methods
Yucatan mini-swine (20–30 kg), fed a high cholesterol diet (total 20 weeks), underwent circumflex ameroid placement to create chronic myocardial ischemia, followed three weeks later by perivascular administration of VEGF (2 μg; n=6), FGF-2 (100 μg; n=6), or placebo (n=7) in the ischemic territory. Normocholesterolemic animals (n=7) served as controls. Four weeks later, endothelial function, collateral-dependent perfusion, as well as myocardial protein and mRNA levels of angiogenic mediators were assessed.
Results
Endothelial dysfunction was observed in all hypercholesterolemic animals as impaired microvessel relaxation in response to adenosine diphosphate and VEGF. VEGF administration improved baseline-adjusted collateral-dependent perfusion at rest(−0.03±0.05 vs. −0.12±0.04, VEGF vs. placebo, p=0.09), but FGF-2 delivery caused a significantly greater improvement in perfusion compared to either group (+0.15±0.03, p<0.05 vs. HC-placebo and HC-VEGF) at rest. Molecular analysis revealed increased eNOS expression (135% ± 8%, p=0.03 vs. placebo) in all growth factor treated animals and increased expression of FGF-2 receptor, FGFR1, (65 ± 26%, p = 0.04 vs. placebo) in FGF-2 treated animals. No significant changes were demonstrated in other angiogenic mediators including Akt, Syndecan-4.
Conclusions
In the setting of hypercholesterolemic endothelial dysfunction, FGF-2 is more effective than VEGF at enhancing collateral-dependent perfusion and thus, may be a better candidate than VEGF for angiogenic therapy in patients with end-stage CAD.
doi:10.1016/j.ejcts.2007.12.016
PMCID: PMC2329802  PMID: 18201892
Endothelial Dysfunction; Vascular Endothelial Growth Factor; Fibroblast Growth Factor; Myocardial Ischemia; Angiogenesis; Molecular Biology
16.  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
17.  The impact of hyperbaric oxygen therapy on serological values of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) 
Head & Face Medicine  2010;6:29.
Background
Hyperbaric oxygen (HBO) therapy is an effective adjunct treatment for ischemic disorders such as chronic infection or chronic wounds. It combines hyperoxic effects with the stimulating potential of post-therapeutic reactive hypoxia. As its crucial effects, stimulation of fibroblast growth, induction of collagen synthesis and the initiation of angiogenesis are discussed. Angiogenesis is a multistage process resulting in the growth of blood vessels. It includes degradation of extracellular matrix, proliferation and migration of different cell populations and finally formation of new vessel structures. This complex chain of procedures is orchestrated by different cytokines and growth factors. Crucial mediators of angiogenesis are basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF); their in-vivo function is still not fully understood.
Methods
Forty-three patients suffering from sudden sensorineural hearing loss or tinnitus were treated with HBO. The therapy included 10 sessions of 90 minutes each, one session a day. Serological levels of bFGF and VEGF were assessed by enzyme-linked immunosorbent assays performed according to the manufacturer's instructions on day 1, 2, 5 and 10 of HBO therapy and were compared to mean values of the control group, related to the patient's age and sex, and their development observed over the ten days of HBO.
Results
There was no sex- or age dependency of bFGF observed in the present study, whereas under HBO our results showed a significant mitigation of the bFGF concentration. In the present data, there was no connection between the VEGF concentration and the patients' ages. Women showed significantly higher levels of VEGF. There was no significant change of VEGF concentration or the VEGF/bFGF ratio during HBO. All scored results varied within the range of standard values as described in the current literature.
Conclusions
A significant effect of HBO on serum concentrations of bFGF and VEGF was not verified in the present study. Additional application of exogenous growth factors in conjunction with HBO was not obviously linked by a coherent cause-and-effect chain as far as wound healing is concerned.
doi:10.1186/1746-160X-6-29
PMCID: PMC3022549  PMID: 21176170
18.  The role of angiogenic factors in predicting clinical outcome in severe bacterial infection in Malawian children 
Critical Care  2010;14(3):R91.
Introduction
Severe sepsis is a disease of the microcirculation, with endothelial dysfunction playing a key role in its pathogenesis and subsequent associated mortality. Angiogenesis in damaged small vessels may ameliorate this dysfunction. The aim of the study was to determine whether the angiogenic factors (vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and angiopoietin-1 (Ang-1) and -2 (Ang-2)) are mortality indicators in Malawian children with severe bacterial infection.
Methods
In 293 children with severe bacterial infection, plasma VEGF, PDGF, FGF, and Ang-1 and Ang-2 were measured on admission; in 50 of the children with meningitis, VEGF, PDGF, and FGF were also measured in the CSF. Healthy controls comprised children from some of the villages of the index cases. Univariable and multivariable logistic regression analyses were performed to develop a prognostic model.
Results
The median age was 2.4 years, and the IQR, 0.7 to 6.0 years. There were 211 children with bacterial meningitis (72%) and 82 (28%) with pneumonia, and 154 (53%) children were HIV infected. Mean VEGF, PDGF, and FGF concentrations were higher in survivors than in nonsurvivors, but only PDGF remained significantly increased in multivariate analysis (P = 0.007). Mean Ang-1 was significantly increased, and Ang-2 was significantly decreased in survivors compared with nonsurvivors (6,000 versus 3,900 pg/ml, P = 0.03; and 7,700 versus 11,900 pg/ml, P = 0.02, respectively). With a logistic regression model and controlling for confounding factors, only female sex (OR, 3.95; 95% CI, 1.33 to 11.76) and low Ang-1 (OR, 0.23; 95% CI, 0.08 to 0.69) were significantly associated with mortality. In children with bacterial meningitis, mean CSF VEGF, PDGF, and FGF concentrations were higher than paired plasma concentrations, and mean CSF, VEGF, and FGF concentrations were higher in nonsurvivors than in survivors (P = 0.02 and 0.001, respectively).
Conclusions
Lower plasma VEGF, PDGF, FGF, and Ang-1 concentrations and higher Ang-2 concentrations are associated with an unfavorable outcome in children with severe bacterial infection. These angiogenic factors may be important in the endothelial dysregulation seen in severe bacterial infection, and they could be used as biomarkers for the early identification of patients at risk of a poor outcome.
doi:10.1186/cc9025
PMCID: PMC2911728  PMID: 20492647
19.  Elevated levels of the angiogenic cytokines basic fibroblast growth factor and vascular endothelial growth factor in sera of cancer patients. 
British Journal of Cancer  1997;76(2):238-243.
The concentration of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) was determined in the serum of 90 untreated and 42 treated metastatic cancer patients, including patients with colorectal, breast, ovarian and renal carcinomas, with an enzyme-linked immunosorbent assay (ELISA). Levels higher than the 95th percentile of the concentrations of a control group, i.e. 7.5 pg ml(-1) for bFGF and 500 pg ml(-1) for VEGF, were identified as 'elevated'. One measurement during follow-up was included into the analysis per patient. For 19 treated patients, consecutive serum samples were analysed. Fifty-seven per cent of all untreated patients had elevated serum levels of one or both angiogenic factors. The fraction of patients with elevated serum levels of bFGF and/or VEGF was similar in the different tumour types. Agreement of bFGF levels and VEGF levels, classified in relation to their respective cut-off values, was present in 67% of all patients. Fifty-eight per cent of the patients with progressive disease during treatment compared with 15% of the patients showing response to treatment (chi-squared test P < 0.05) had elevated bFGF and/or VEGF serum levels. When consecutive serum samples were analysed, two-thirds of the patients showing progressive disease had increasing serum levels of the angiogenic factors compared with less than one-tenth of the patients showing response (chi-squared test P < 0.05). The lack of association between the serum bFGF and VEGF levels and the tumour type may suggest an aspecific host reaction responsible for solid tumour-related angiogenesis. The main determinants of the serum bFGF and VEGF concentration are the progression kinetics of the metastatic carcinomas.
PMCID: PMC2223937  PMID: 9231925
20.  Lenvatinib, an angiogenesis inhibitor targeting VEGFR/FGFR, shows broad antitumor activity in human tumor xenograft models associated with microvessel density and pericyte coverage 
Vascular Cell  2014;6:18.
Background
Lenvatinib is an oral inhibitor of multiple receptor tyrosine kinases (RTKs) targeting vascular endothelial growth factor receptor (VEGFR1-3), fibroblast growth factor receptor (FGFR1-4), platelet growth factor receptor α (PDGFR α), RET and KIT. Antiangiogenesis activity of lenvatinib in VEGF- and FGF-driven angiogenesis models in both in vitro and in vivo was determined. Roles of tumor vasculature (microvessel density (MVD) and pericyte coverage) as biomarkers for lenvatinib were also examined in this study.
Method
We evaluated antiangiogenesis activity of lenvatinib against VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. Effects of lenvatinib on in vivo angiogenesis, which was enhanced by overexpressed VEGF or FGF in human pancreatic cancer KP-1 cells, were examined in the mouse dorsal air sac assay. We determined antitumor activity of lenvatinib in a broad panel of human tumor xenograft models to test if vascular score, which consisted of high MVD and low pericyte coverage, was associated with sensitivity to lenvatinib treatment. Vascular score was also analyzed using human tumor specimens with 18 different types of human primary tumors.
Result
Lenvatinib inhibited VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. In vivo angiogenesis induced by overexpressed VEGF (KP-1/VEGF transfectants) or FGF (KP-1/FGF transfectants) was significantly suppressed with oral treatments of lenvatinib. Lenvatinib showed significant antitumor activity in KP-1/VEGF and five 5 of 7 different types of human tumor xenograft models at between 1 to 100 mg/kg. We divided 19 human tumor xenograft models into lenvatinib-sensitive (tumor-shrinkage) and relatively resistant (slow-growth) subgroups based on sensitivity to lenvatinib treatments at 100 mg/kg. IHC analysis showed that vascular score was significantly higher in sensitive subgroup than relatively resistant subgroup (p < 0.0004). Among 18 types of human primary tumors, kidney cancer had the highest MVD, while liver cancer had the lowest pericyte coverage, and cancers in Kidney and Stomach had highest vascular score.
Conclusion
These results indicated that Lenvatinib inhibited VEGF- and FGF-driven angiogenesis and showed a broad spectrum of antitumor activity with a wide therapeutic window. MVD and pericyte-coverage of tumor vasculature might be biomarkers and suggest cases that would respond for lenvatinib therapy.
doi:10.1186/2045-824X-6-18
PMCID: PMC4156793  PMID: 25197551
Lenvatinib; VEGFR2 kinase inhibitor; FGFR kinase inhibitor; Pericyte coverage; Microvessel density
21.  Development of a new pre-vascularized tissue-engineered construct using pre-differentiated rADSCs, arteriovenous vascular bundle and porous nano-hydroxyapatide-polyamide 66 scaffold 
Background
Development of a pre-vascularized tissue-engineered construct with intrinsic vascular system for cell growth and tissue formation still faces many difficulties due to the complexity of the vascular network of natural bone tissue. The present study was to design and form a new vascularized tissue-engineered construct using pre-differentiated rADSCs, arteriovenous vascular bundle and porous nHA-PA 66 scaffold.
Methods
rADSCs were pre-differentiated to endothelial cells (rADSCs-Endo) and then incorporated in nHA-PA 66 scaffolds in vitro. Subsequently, in vivo experiments were carried out according to the following groups: Group A (rADSCs-Endo/nHA-PA 66 scaffold with arteriovenous vascular bundle), Group B (rADSCs/nHA-PA 66 scaffold with arteriovenous vascular bundle); Group C (nHA-PA66 scaffold with arteriovenous vascular bundle), Group D (nHA-PA 66 scaffold only). The vessel density and vessel diameter were measured based on histological and immunohistochemical evaluation, furthermore, the VEGF-C, FGF-2 and BMP-2 protein expressions were also evaluated by western blot analysis.
Results
The results of in vivo experiments showed that the vessel density and vessel diameter in group A were significantly higher than the other three groups. Between Group B and C, no statistical difference was observed at each time point. In accordance with the results, there were dramatically higher expressions of VEGF-C and FGF-2 protein in Group A than that of Group B, C and D at 2 or 4 weeks. Statistical differences were not observed in VEGF-C and FGF-2 expression between Group B and C. BMP-2 was not expressed in any group at each time point.
Conclusions
Compared with muscular wrapping method, arteriovenous vascular bundle implantation could promote vascularization of the scaffold; and the angiogenesis of the scaffold was significantly accelerated when pre-differentiated rADSCs (endothelial differentiation) were added. These positive results implicate the combination of pre-differentiated rADSCs (endothelial differentiation) and arteriovenous vascular bundle may achieve rapidly angiogenesis of biomaterial scaffold.
doi:10.1186/1471-2474-14-318
PMCID: PMC3826526  PMID: 24209783
Adipose-derived stem cells; Tissue engineering; Angiogenesis; Scaffolds; Prefabrication
22.  Differential Activation of Multiple Signaling Pathways Dictates eNOS Upregulation by FGF2 but not VEGF in Placental Artery Endothelial Cells1 
Placenta  2008;29(8):708-717.
Fibroblast growth factor (FGF2), but not vascular endothelial growth factor (VEGF), upregulates endothelial nitric oxide synthase (eNOS) protein expression, at least in part, via activation of extracellular signal-regulated kinase 2/1 (ERK2/1) in ovine fetoplacental artery endothelial (oFPAE) cells. Herein we further investigated the temporal effects of FGF2 and VEGF on other signaling pathways including members (Jun N-terminal kinase JNK1/2 and p38MAPK) of mitogen-activated protein kinases (MAPK), phosphatidylinositol 3 kinase/v-akt murine thymoma viral oncogene homolog 1 (PI3K/AKT1), and the tyrosine kinase c-SRC, and examined if either one or more of these pathways play a role in the differential regulation of eNOS by FGF2 and VEGF. We first confirmed that in oFPAE cells, FGF2, but not VEGF, increased eNOS protein. FGF2 stimulated eNOS protein in a time and concentration dependent manner, which also depended on cell density. FGF2 provoked sustained (5 min to 12 h) whereas VEGF only stimulated transient (5 min) ERK2/1 phosphorylation. FGF2 was 1.7-fold more potent in stimulating ERK2/1 phosphorylation than VEGF. FGF2 and VEGF only transiently activated JNK1/2 and AKT1 within 5 min; however, FGF2 was a stronger stimulus than VEGF. FGF2 and VEGF did not significantly activate p38MAPK at 5 min; however, VEGF stimulated p38MAPK phosphorylation at 60 min. VEGF but not FGF2 significantly stimulated c-SRC phosphorylation. Inhibitors of MEK-ERK2/1 (PD98059), JNK1/2 (SP600125) and PI3K (wortmannin), but not p38MAPK (SB203580) and SRC (PP2), decreased the FGF2-increased eNOS protein expression. Thus, the FGF2-induced eNOS protein expression requires activation of multiple signaling pathways including ERK2/1, JNK1/2 and PI3K/AKT1. Differences in intensity and temporal patterns of activation of these pathways by FGF2 and VEGF may account for their differential effects on eNOS expression in OFPAE cells.
doi:10.1016/j.placenta.2008.05.005
PMCID: PMC2596925  PMID: 18571718
FGF2; VEGF; signaling pathways; eNOS protein; endothelial cells; placenta
23.  PKCε ACTIVATION PROMOTES FGF-2 EXOCYTOSIS AND INDUCES ENDOTHELIAL CELL PROLIFERATION AND SPROUTING 
Journal of molecular and cellular cardiology  2013;63:10.1016/j.yjmcc.2013.07.006.
Protein kinase C epsilon (PKCε) activation controls fibroblast growth factor-2 (FGF-2) angiogenic signaling. Here, we examined the effect of activating PKCε on FGF-2 dependent vascular growth and endothelial activation. ψεRACK, a selective PKCε agonist induces pro-angiogenic responses in endothelial cells, including formation of capillary like structures and cell growth. These effects are mediated by FGF-2 export to the cell membrane, as documented by biotinylation and immunofluorescence, and FGF-2/FGFR1 signaling activation, as attested by ERK1/2-STAT-3 phosphorylation and de novo FGF-2 synthesis. Similarly, vascular endothelial growth factor (VEGF) activates PKCε in endothelial cells, and promotes FGF-2 export and FGF-2/FGFR1 signaling activation. ψεRACK fails to elicit responses in FGF-2−/− endothelial cells, and in cells pretreated with methylamine (MeNH2), an exocytosis inhibitor, indicating that both intracellular FGF-2 and its export toward the membrane are required for the ψεRACK activity. In vivo ψεRACK does not induce angiogenesis in the rabbit cornea. However, ψεRACK promotes VEGF angiogenic responses, an effect sustained by endothelial FGF-2 release and synthesis, since anti-FGF-2 antibody strongly attenuates VEGF responses. The results demonstrate that PKCε stimulation promotes angiogenesis and modulates VEGF activity, by inducing FGF-2 release and autocrine signaling.
doi:10.1016/j.yjmcc.2013.07.006
PMCID: PMC3812807  PMID: 23880610
Protein Kinase C ε; Endothelial cells; Fibroblast Growth Factor-2; Vascular Endothelial Growth Factor; Angiogenesis; Exocytosis
24.  Study of the association of adrenomedullin and basic-fibroblast growth factors with the peripheral arterial blood flow and endothelial dysfunction biomarkers in type 2 diabetic patients with peripheral vascular insufficiency 
Background
Progressive micro-vascular vaso-degeneration is the major factor in progression of diabetic complications. Adrenomedullin (AM) and basic-Fibroblast growth factor (b-FGF) are strongly correlated with angiogenesis in vascular diseases. This study aims to provide base line data regarding the vascular effects and correlation of AM, and b-FGF with the peripheral blood flow in diabetic patients with peripheral vascular disease (PVD), and their effect on endothelial dysfunction markers. Ninety age- and sex matched females were enrolled in the study: 30 were controls, 30 had diabetes without complications (group II) and 30 had diabetes with PVD (group III) diagnosed by ankle/ brachial index (A/BI). Plasma levels of AM, b-FGF, intercellular adhesion molecule −1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were measured by indirect enzyme immunoassay (ELISA).
Results
There was a significant increase in plasma AM, VCAM-1and ICAM-1, while a significant decrease in plasma b-FGF in diabetic patients with PVD (p < 0.05). A positive correlation was observed between plasma AM, b-FGF and A/BI and a negative correlation with VCAM −1 and ICAM in diabetic PVD. AM was not a predictor, while b-FG, VCAM-1 and ICAM-1 could be predictors for peripheral blood flow in diabetic PVD.
Conclusion
This study elucidates for the first time that AM and b-FGF are correlated and have a direct impact on the peripheral blood flow, the rise of AM in diabetic PVD may be a consecutive and compensatory vasculo-protective effect as its angiogenic and anti-inflammatory properties act to relief the endothelial insult. Down expression of b-FGF may be a predisposing factor for micro-vascular derangement. It is not clear if the rise of AM and the decline of b- FGF levels may be consequences or predisposing factors for VCAM-1 and ICAM-1 elevation as these endothelial dysfunction biomarkers could reduce peripheral blood flow and vascular integrity. It is optimistic to believe that drug intervention through AM and b-FGF administration together with reversing the endothelial inflammatory process by targeting VCAM and ICAM could reduce the prevalence of diabetic vascular complications, reduce the risk of cerebrovascular and cardiovascular morbidity in diabetes through normalizing vascular endothelium function and peripheral blood flow.
doi:10.1186/s12929-014-0094-y
PMCID: PMC4195904  PMID: 25287126
Diabetic vasculopathy; Adrenomedullin; Basic-Fibroblast growth factor
25.  Cheiradone: a vascular endothelial cell growth factor receptor antagonist 
BMC Cell Biology  2008;9:7.
Background
Angiogenesis, the growth of new blood vessels from the pre-existing vasculature is associated with physiological (for example wound healing) and pathological conditions (tumour development). Vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF) are the major angiogenic regulators. We have identified a natural product (cheiradone) isolated from a Euphorbia species which inhibited in vivo and in vitro VEGF- stimulated angiogenesis but had no effect on FGF-2 or EGF activity. Two primary cultures, bovine aortic and human dermal endothelial cells were used in in vitro (proliferation, wound healing, invasion in Matrigel and tube formation) and in vivo (the chick chorioallantoic membrane) models of angiogenesis in the presence of growth factors and cheiradone. In all cases, the concentration of cheiradone which caused 50% inhibition (IC50) was determined. The effect of cheiradone on the binding of growth factors to their receptors was also investigated.
Results
Cheiradone inhibited all stages of VEGF-induced angiogenesis with IC50 values in the range 5.20–7.50 μM but did not inhibit FGF-2 or EGF-induced angiogenesis. It also inhibited VEGF binding to VEGF receptor-1 and 2 with IC50 values of 2.9 and 0.61 μM respectively.
Conclusion
Cheiradone inhibited VEGF-induced angiogenesis by binding to VEGF receptors -1 and -2 and may be a useful investigative tool to study the specific contribution of VEGF to angiogenesis and may have therapeutic potential.
doi:10.1186/1471-2121-9-7
PMCID: PMC2248182  PMID: 18230134

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