Quantitative assessment of angiogenic responses by the directed in vivo angiogenesis assay.

One of the major problems in angiogenesis research remains the lack of suitable methods for quantifying the angiogenic response in vivo. We describe the development and application of the directed in vivo angiogenesis assay (DIVAA) and demonstrated that it is reproducible and quantitative. This assay consists of subcutaneous implantation of semiclosed silicone cylinders (angioreactors) into nude mice. Angioreactors are filled with only 18 micro l of extracellular matrix premixed with or without angiogenic factors. Vascularization within angioreactors is quantified by the intravenous injection of fluorescein isothiocyanate (FITC)-dextran before their recovery, followed by spectrofluorimetry. Angioreactors examined by immunofluorescence show cells and invading angiogenic vessels at different developmental stages. The minimally detectable angiogenic response requires 9 days after implantation and >/=50 ng/ml (P < 0.01) of either fibroblast growth factor-2 or vascular endothelial growth factor. Characterization of this assay system demonstrates that the FITC-labeled dextran quantitation is highly reproducible and that levels of FITC-dextran are not significantly influenced by vascular permeability. DIVAA allows accurate dose-response analysis and identification of effective doses of angiogenesis-modulating factors in vivo. TNP-470 potently inhibits angiogenesis (EC(50) = 88 pmol/L) induced by 500 ng/ml of fibroblast growth factor-2. This inhibition correlates with decreased endothelial cell invasion. DIVAA efficiently detects differences in anti-angiogenic potencies of thrombospondin-1 peptides (25 micro mol/L) and demonstrates a partial inhibition of angiogenesis ( approximately 40%) in a matrix metalloprotease (MMP)-2-deficient mouse compared with that in wild-type animals. Zymography of angioreactors from MMP-deficient and control animals reveals quantitative changes in MMP expression. These results support DIVAA as an assay to compare potencies of angiogenic factors or inhibitors, and for profiling molecular markers of angiogenesis in vivo.

Quantitative assessment of angiogenesis and tumor vessel architecture by computer-assisted digital image analysis: effects of VEGF-toxin conjugate on tumor microvessel density.

Tumor growth is angiogenesis dependent. As a consequence, strategies aimed at disrupting this mechanism are heavily investigated. Several angiogenesis assays are used to directly compare the efficacy of anti-angiogenic compounds. However, objective assessment of new vascular growth has been difficult to achieve. The aim of this study was to test and develop a computer-assisted image analysis method that would give an unbiased quantification of the microvessel density. Human tumors were grown in athymic mice and tumor biopsies were taken after a weeklong treatment with VEGF-toxin conjugate. Frozen tumor sections were prepared and stained with PE-conjugated anti-CD-31 antibodies and vessels were imaged with a fluorescence microscope. Vessel density was analyzed by quantifying PE-positive pixels per recorded field. In addition, images were further processed to investigate morphological differences by an automated binarization and skeletonization protocol. This procedure allowed the computer-assisted estimation of important angiogenic parameters such as total vessel number, length, and branch points. Based on these indices, differences in the angiogenic response between control tumors and those treated with VEGF-toxin conjugate were readily detected (P < 0.007 for all parameters). More importantly, computer-generated measurements correlated well with manual microvessel counts and showed significantly less variation. Our results suggest that computer-assisted image analysis represents a rapid, objective, and alternative method for the quantitative assessment of tumor angiogenesis and vessel architecture.

Physiological assessment of augmented vascularity induced by VEGF in ischemic rabbit hindlimb.

This study was designed to assess the physiological consequences of augmented vascularity induced by administration of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, in a rabbit model of hindlimb ischemia. Ten days after excision of the common and superficial femoral arteries from one hindlimb of 24 New Zealand White rabbits, VEGF (n = 15) or saline (control; n = 9) was selectively injected into the ipsilateral internal iliac artery. Limb perfusion was evaluated immediately pre-VEGF (baseline) and again at days 10 and 30. A Doppler guide wire was advanced to the internal iliac artery to record flow velocity at rest and at maximum flow velocity provoked by intra-arterial injection of papaverine. At baseline and at day 10, no differences in flow parameters were observed between the control and the VEGF-treated animals. By day 30, however, flow at rest (P < 0.05), maximum flow velocity (P < 0.001), and maximum blood flow (P < 0.001) were all significantly higher in the VEGF-treated group. These physiological findings complement previous-anatomic studies by providing evidence that a single intra-arterial bolus of VEGF augments flow, particularly maximum flow, in the rabbit ischemic hindlimb. These data thus support the notion that VEGF administration represents a potential treatment strategy for certain patients with lower extremity ischemia.

[Culture of human blood vessel endothelial cells--a simple and inexpensive culture method].

The purpose of this review is to introduce a simple and inexpensive culture method for human umbilical blood vessel endothelial cells. The medium used is MCDB-104 supplemented with 10% fetal bovine serum, 70 ng/ml endothelial cell growth factor from new-born bovine brains, 10 ng/ml murine epidermal growth factor, and 100 micrograms/ml heparin. The culture dishes are coated with gelatin. Under these conditions, endothelial cells from human vessels were grown with doubling times of 18-22 hrs and reached saturation densities of 8-12 x 10(4) cells/cm2. To determine the lifespan of the endothelial cells, the cells were serially subcultivated weekly at an inoculum size of 1,000 cells/cm2. Human endothelial cells from umbilical vein and artery were grown for 21 to 37 passages with 55 to 125 population doublings. This culture method seems to be useful for studying cell proliferation and functions of human endothelial cells.