Daniel T. Connolly

Determination of the number of endothelial cells in culture using an acid phosphatase assay

A simple assay is described in which small numbers of endothelial cells in culture can be determined by measuring acid phosphatase activity. After removal of the growth medium from cells grown in 96-well culture plates, the cells are lysed in buffer containing the detergent Triton X-100 and the phosphatase substrate p-nitrophenyl phosphate. After 2 h at 37 degrees C, the reaction is stopped with sodium hydroxide, and color development is determined using a rapid multiwell plate reader. The assay detects 100 to 10,000 cells per well. The assay has been used to determine growth curves for endothelial cells in the presence and absence of endothelial cell growth factor from bovine hypothalamus and to monitor fractions during purification of the growth factor. Minor modifications in the assay allow it to be fully automated.

Specific binding of vascular permeability factor to endothelial cells.

Vascular permeability factor (VPF), also known as vascular endothelial cell growth factor, has recently been purified from guinea pig, human, and bovine sources. We show that various fetal or adult endothelial cell strains originating from either capillary or large vessels possess specific high affinity and saturable binding sites for guinea pig tumor-derived [125I]VPF. Two classes of sites with KDs of approximately 10 pM and 1 nM were detected for all endothelial cell types examined. Guinea pig [125I]VPF binding to endothelial cells was inhibited by human VPF (ID50 = 0.8 ng/ml) and by suramin (ID50 = 75 micrograms/ml) but not by heparin. Cross-linking experiments revealed specific [125I]VPF-receptor complexes of two types. Most of the complexes migrated very slowing in SDS-PAGE, indicating that they were of very high molecular weight and probably highly cross-linked. A portion of the molecules migrated as 270 kDa complexes, indicating that the molecular weight of the endothelial cell VPF receptor is about 230 kDa.

Enhancement of angiogenesis by bFGF in mandibular bone graft healing in the rabbit

The effectiveness of basic fibroblast growth factor (bFGF) as an in vivo angiogenic factor was evaluated in autogeneic bone grafts to the rabbit mandible. Block cortical grafts harvested from the ilium were implanted into sites in the mandibular ramus or body. The basic fibroblast growth factor was continuously introduced over a period of 14 days through subcutaneous osmotic pumps. Increased vascularity, as assessed by vessel number and depth of penetration into the grafts, was noted at 10 days postoperatively in the bFGF stimulated side as compared to contralateral control grafts. At the fourteenth postoperative day, bFGF administration was discontinued and a decrease in angiogenesis was noted over the ensuing 2 weeks so that after 1 postoperative month, little difference could be detected between the stimulated and nonstimulated grafts. Assessment of osseous healing at the 1 month postoperative interval using triple fluorochrome labeling did not reveal evidence of accelerated osteogenesis on the previously stimulated side.

Human fibroblast-derived growth factor is a mitogen and chemoattractant for endothelial cells

Human fibroblasts were found to produce a potent mitogen and chemoattractant for fetal bovine aortic endothelial cells. Homogenates from AG1523 and AG1518 foreskin, CCD18Lu lung, and CCD18Co colon fibroblasts produced half-maximal stimulation of endothelial cell growth at concentrations of 1-7 micrograms/ml. The factor was purified from large-scale cultures of the CCD18Co fibroblasts using cation exchange chromatography and heparin-Sepharose chromatography. Such preparations were mitogenic for endothelial cells in vitro at concentrations of about 5-10 ng/ml, and promoted chemotaxis at 0.1-1 ng/ml. Heparinase treatment of the cells prevented the chemotactic response. These properties suggest that the factor may be related to fibroblast growth factor.

Effects of a variety of cytokines and inducing agents on vascular permeability factor mRNA levels in U937 cells.

Vascular permeability factor (VPF) is an approximately 40-kDa disulfide-linked dimeric glycoprotein that is active in increasing blood vessel permeability, endothelial cell growth and angiogenesis. Little is known about VPF gene regulation. In this study, we investigated the effects of a variety of cytokines and inducing agents on VPF mRNA levels in the monocyte-like U937 cell line. Transforming growth factor-beta 1 caused a 1.8-fold increase in VPF mRNA levels after 4 hours, followed by a decline to basal levels by 18 hours. Phorbol 12-myristate 13-acetate, a potent inducer of the differentiation of U937 cells, caused a 12.5-fold increase in VPF mRNA levels at 24 hours, coinciding with the differentiation of these monocyte-like cells into macrophage-like cells.

Regulation of Vascular Function by Vascular Permeability Factor

The blood vessels in and around tumors can be distinguished from normal vasculature in several ways. Tumor-associated vessels are derived from surrounding host blood vessels as a result of neovascularization (Folkman, 1985) and are typically more permeable than normal vessels (Dvorak et al., 1988, Gerlowski and Jain,1983). These observations lead to the hypothesis that tumors can alter blood vessel function by producing regulator substances. A polypeptide regulator known as vascular permeability factor (VPF) or vascular endothelial growth factor (VEGF) has now been purified and found to display a surprisingly wide spectrum of vascular activities, some of which are pro-inflammatory in nature. In addition to synthesis by tumor cells, VPF has now been found to be a product of monocytes and lymphocytes. Taken together, these results suggest that VPF may be a new inflammatory cytokine.