Synthesis, storage, and release of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) by human mast cells: implications for the biological significance of VEGF206

Abstract

Mast cells have been implicated in various diseases that are accompanied by neovascularization. The exact mechanisms by which mast cells might mediate an angiogenic response, however, are unclear and therefore, we have investigated the possible expression of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in the human mast cell line HMC-1 and in human skin mast cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that mast cells constitutively express VEGF121, VEGF165, and VEGF189. After a prolonged stimulation of cells for 24 h with phorbol 12-myristate 13-acetate (PMA) and the ionophore A23187, an additional transcript representing VEGF206 was detectable, as could be verified by sequence analysis. These results were confirmed at the protein level by Western blot analysis. When the amounts of VEGF released under unstimulated and stimulated conditions were compared, a significant increase was detectable after stimulation of cells. Human microvascular endothelial cells (HMVEC) responded to the supernatant of unstimulated HMC-1 cells with a dose-dependent mitogenic effect, neutralizable up to 90% in the presence of a VEGF-specific monoclonal antibody. Flow cytometry and postembedding immunoelectron microscopy were used to detect VEGF in its cell-associated form. VEGF was exclusively detectable in the secretory granules of isolated human skin mast cells. These results show that both normal and leukemic human mast cells constitutively express bioactive VEGF. Furthermore, this study contributes to the understanding of the physiological role of the strongly heparin-binding VEGF isoforms, since these were found for the first time to be expressed in an activation-dependent manner in HMC-1 cells.

Figure 1

Expression of VEGF transcripts in HMC-1 cells. Semiquantitative RT-PCR analysis of mRNA from unstimulated HMC-1 cells (lane 3) and cells stimulated for 24 h with PMA/A23187 (lane 4). (A) The primers chosen for this amplification allowed detection of all VEGF isoforms. (B) A VEGF₂₀₆-specific primer pair was chosen (Figure 2A), and the product amplified was cut from the gel and identified by direct sequencing (Figure 2B). lane 1, size marker; lane 2, negative control (sample without cDNA). The positions of the size markers are on the left side and the sizes of the amplified products are indicated on the right side.

Figure 2

(A) Design of the PCR primers used to amplify a VEGF-specific sequence (438 bp) containing 67% of the VEGF₂₀₆-specific exon 6b. (B) The product amplified was directly sequenced using the sense primer of PCR amplification. The 3′-end of the amplicon sequenced showed the expected exon 6b-specific nucleotide sequence.

Figure 3

Immunoblot analysis of HMC-1 cell-derived supernatants. Concentrated supernatants of unstimulated cells (lane 1) and HMC-1 cells stimulated for 24 h with PMA/A23187 (lanes 2, 3, and 6) were used for SDS-PAGE analysis and blotting on nitrocellulose membranes. Precleaning of supernatants with heparin beads was necessary to minimize background staining, but during this step, VEGF₁₂₁ was lost to a large extent. Recombinant human VEGF₁₆₅ was used as positive control (lanes 4 and 5). Lanes 1–5 were probed with an VEGF-specific antibody, and lane 6 was probed with an isotype-specific control antibody.

Figure 4

Time-dependent secretion of VEGF under nonstimulating and stimulating conditions as measured by a VEGF-specific ELISA. Additionally, the stimulated and the unstimulated release of VEGF was monitored in the presence of 1 μM cycloheximide (CHX), an inhibitor of protein synthesis. The values are given as means ± SD of five independent experiments.

Figure 5

Detection of intracellular VEGF in unstimlated HMC-1 cells by flow cytometry. Before antibody staining cells were fixed in a mixture of 4% paraformaldehyde and 0.1% glutaraldehyde and permeabilized with 0.03% saponin. The dotted line of the fluorescence histogram overlay illustrates the binding of the VEGF-specific mAb, and the solid line represents the unspecific binding of an isotype-specific control mAb.

Figure 6

Proliferation of HMVEC in response to HMC-1 cell-derived supernatants. The CM of unstimulated HMC-1 cells was concentrated 10-fold by ultrafiltration, and dilutions were tested for its mitogenic activity in an HMVEC proliferation assay. A dose-dependent increase in HMVEC proliferation rate was detectable. Results are shown for one representative experiment performed in triplicate. Similar results were obtained in four independent experiments (our unpublished data).

Figure 7

Inhibition of the HMC-1- and VEGF-induced HMVEC proliferation with a neutralizing VEGF-specific antibody. (A) The mitogenic effects of VEGF (10 ng/ml) and of 100% CM were suppressed up to 100% and 90%, respectively, in the presence of a neutralizing VEGF-specific mAb or up to 75% by a preincubation of the CM with heparin-conjugated agarose beads. Data are expressed as percentage of stimulation of HMVEC proliferation in comparison to control medium. (B) This Figure demonstrates the VEGF-specific mAb-induced dose-dependent neutralization of the mitogenic effect of CM. The antibody used showed its half-maximal inhibitory effect at a concentration of 25 ng/ml. A control antibody showed no inhibitory effect. In both figures mean values ± SD from three separate experiments are shown.

Figure 8

Immunoelectron microscopic localization of VEGF in ultrathin sections of human skin mast cells embedded in LR-White resin. (A) A typical mast cell isolated from human foreskin by enzymatic dispersion and enrichment by elutriation is shown. Arrowheads indicate mast cell-specific granules. N, Nucleus. Bar, 3 μm. (B) Immunolabeling was detected exclusively in mast cell-specific granules (arrowheads). N, Nucleus. Bar, 1 μm. (C) In the presence of an isotype-matched irrelevant mAb, almost no immunoreactive structures were ascertained. N, Nucleus. Bar, 1 μm.