Quantitative Angiogenesis Research Articles

Quantitation of angiogenesis in vitro induced~by VEGF-A and FGF-2 in two different human endothelial cultures – an all-in-one assay

Angiogenic therapy is considered to be a promising tool for treatment of ischemic diseases. Many in vivo and in vitro assays have been developed to identify potential proangiogenic drugs and to investigate their mode of action. However, until now no validated system exists that would allow quantitation of angiogenesis in vitro in only one assay. Here, a previously established all-in-one in vitro assay based on staging of the angiogenic cascade was validated by quantitation of the effects of the known proangiogenic factors VEGF-A and FGF-2. Both growth factors were applied separately or in combination to human endothelial cell cultures derived from the heart and the foreskin, and angiogenesis was quantitated over 30 days of culture. Additionally, gene expression of VEGFR-1, VEGFR-2 and FGFR-1 at 3, 10, 20 or 40 days of cultivation was quantitated by RT-qPCR. In both cultures, VEGF-A as well as FGF-2 induced a run through all defined stages of angiogenesis in vitro. Application of VEGF-A only led to formation of irregular globular endothelial structures, while FGF-2 resulted in development of regular capillary-like structures. Quantitation of the angiogenic effects of VEGF-A and transcripts of VEGFR-1 and VEGFR-2 showed that a high VEGFR-1/VEGFR-2 ratio evoked deceleration of angiogenesis.

Inhibition of experimental angiogenesis of cornea by somatostatin.

This study evaluated the inhibitory activity of somatostatin 14 on the angiogenesis of cornea in vivo. Corneal neovascularization was induced with a pellet containing 90 ng of basic fibroblast growth factor (bFGF) in a rat corneal pocket model. Three kinds of pellets were made containing bFGF plus somatostatin (SST) 0 ng, 20 ng and 200 ng for the control group, group 1 and group 2, respectively. Neovascularization was observed biomicroscopically from day 4 to day 8, and the corneas were then examined for changes in histology. Quantitation of angiogenesis in the cornea was accomplished by caliper and image analysis. The 200-ng dose of SST showed significant inhibition of both length and area of neovascularization on day 7 (0.62+/-0.11 mm vs 1.29+/-0.16 mm, 0.50+/-0.16 mm2 vs 1.35+/-0.29mm2, group 2 vs control; P<0.05). The 20 ng of somatostatin did not demonstrate any significant inhibition of neovascularization compared with the control group. Our study demonstrated that SST 14 can reduce bFGF-induced corneal angiogenesis. This shows the potential value of somatostatin in the treatment of corneal neovascularization.

Quantitation of Angiogenesis by Measuring CD31 and Factor VIII Visually and by Image Cytometry in Renal Cell Carcinomas: Prognostic Significance

Quantitation of Angiogenesis by Measuring CD31 and Factor VIII Visually and by Image Cytometry in Renal Cell Carcinomas: Prognostic Significance

The Heparan Sulfate Suleparoide Inhibits Rat Corneal Angiogenesis and in vitro Neovascularization

The purpose of this study was to evaluate the inhibitory activity of the heparan sulfate suleparoide on vascular cell growth in vitro and angiogenesis in vivo.Human HUV-EC-C endothelial cell proliferation and microvessel sprouting from cultured rat aortic rings were assayed by the bioreduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. The inhibition of the neoforming capillary network in the chorioallantoic membrane of chick embryo (CAM) was evaluated by agarose disks containing suleparoide and applied on the CAM surface. AgNO3/KNO3injury was used to induce corneal neovascularization and to evaluate the therapeutic effect of topical suleparoide, while the involvement of bFGF in angiogenesis was evidenced by immunohistochemistry of corneal tissue. Quantitation of angiogenesis in the CAM and the cornea was accomplished by image analysis.Suleparoide dose-dependently inhibited HUV-EC-C cell proliferation (50% inhibitory concentration [IC50], 197.5±15.2 μg ml−1) and reduced microvessel sprouting in vitro (IC50, 351±22 μg ml−1). Likewise, suleparoide 150 μg in agarose disks produced an avascular area of 19.7±2.7% of the total area of the CAM (P<0.05 as compared to controls). bFGF levels were significantly enhanced in the cornea after AgNO3/KNO3injury, and the increase appeared to be time-dependent (25.6±1.8 and 43.2±7.4%, vs. uninjured controls after 24 hr and 48 hr, respectively,P<0.05). Suleparoide 4.8 mg eye−1day−1for six days reduced the length of blood vessels and the area of the cornea infiltrated by them (59.6±7.4% decrease vs. controls,P<0.05).These results demonstrate that suleparoide is an active agent against angiogenesis and suggest that the therapeutic effect of the drug could be of value to treat corneal neovascularization.

Angiogenesis in hepatocellular carcinoma as evaluated by CD34 immunohistochemistry.

To clarify the relationship between angiogenesis and hepatocarcinogenesis on progression of hepatocellular carcinoma (HCC), we quantitatively evaluated angiogenesis by CD34 immunohistochemistry in liver cirrhosis (LC), adenomatous hyperplasia (AH), and HCC, and proliferative activity estimated by Ki-67 immunohistochemistry. Angiogenesis was evaluated by CD34 immunohistochemistry using monoclonal antibody HPCA-2, and tumor proliferative activity was evaluated using monoclonal antibody MIB-1. We used an image analysis system to assess the microvessel density as the area percentage of the endothelial area. Angiogenesis was generally observed in HCC and there was no significant difference among all clinical stages and histological grades of HCC. On the other hand, the staining of CD34 was partly observed in sinusoids of AH, although no positive staining was seen in any sinusoids of LC. The proliferative activity was significantly correlated with the clinical stage and histological grade of HCC. Our results indicate that the quantitation of angiogenesis does not provide significant prognostic information in HCC, but that it may have diagnostic value in distinguishing HCC from non-HCC. Meanwhile, AH, which is not morphologically diagnosed as cancer, shows positive staining for CD34, suggesting that some portion of AH contains cancerous characteristics.

Angiogenesis in Neoplasia; Insights from in Situ Hybridization Studies

Abstract The formation of vascular stroma (angiogenesis) is essential for tumor growth and may also influence invasion and metastasis. Quantitation of angiogenesis may serve as a prognostic marker in certain malignancies and novel therapies targeted at inhibiting angiogenesis may prove to play an important role in control of tumor growth. In situ hybridization (ISH) studies have helped to define some of the molecular and cellular events responsible for the formation of vascular stroma in malignancy. Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a multifunctional cytokine associated with tumor angiogenesis. Vascular endothelial cells express at least two tyrosine kinase receptors for VPF, flt-1 and KDR. VPF acts as an angiogenic factor in several ways. It is an endothelial cell growth factor, alters endothelial cell gene expression, induces endothelial cell migration, and it is also a potent inducer of microvascular hyperpermeability (50,000 times as strong as histamine).