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.

Mastocytosis represents a mast cell proliferative disease that generally runs a benign clinical course, with spontaneous remissions mostly by puberty in childhood-onset disease, although rare forms, particularly in adult-onset disease, can be associated with (pre)malignant hematologic disorders and very rarely present as mast cell leukemia or malignant mastocytosis. Reasons for this divergent clinical behavior of childhood- versus adult-onset disease are unknown. Recently, two activating mutations in the intracellular domain of the proto-oncogene c-kit, which encodes a tyrosine kinase receptor for the mast cell growth factor stem cell factor, have been detected in the human leukemic mast cell line HMC-1. We have therefore studied lesional skin biopsies from patients with adult- and childhood-onset indolent mastocytosis for the presence of these codon 560 and 816 mutations. C-kit coding DNA sequences were amplified and analyzed by mutation-specific restriction analyses, and mutated polymerase chain reaction products were additionally cloned and sequenced. The codon 816 mutation was found in all six samples from adult patients, but not in any of the 11 specimens from children. In addition, the codon 560 mutation could be demonstrated for the first time in indolent mastocytosis, namely in two of four specimens from adult patients, but not in those from two children. These data thus provide a possible explanation for the divergent clinical behavior of adult- versus childhood-onset indolent mastocytosis, with the first being associated with an activating mutation, possibly as part of a neoplastic process, and the latter representing most likely a reactive process of an as yet unknown pathogenesis.

Although the TGF-beta family of growth factors probably regulates skin and hair follicle development, its exact role is still quite ill-defined. Here, we characterize the correlative expression pattern of the interdependent high affinity receptor proteins for TGF-beta1 and TGF-beta3, TGF-beta receptor type I (TGF-betaRI) and TGF-beta receptor type II (TGF-betaRII), during hair follicle development and cycling in C57BL/6 mice. During neonatal follicle development, TGF-betaRII immunoreactivity is confined to epithelial cells. Focal epidermal TGF-betaRII expression is seen even before actual hair placode formation. In contrast to the TGF-betaRII immunoreactivity in the outer root sheath, precortical hair matrix and inner root sheath cells were TGF-betaRII negative during hair bulb morphogenesis. TGF-betaRI (Alk-5) immunoreactivity largely overlapped the TGF-betaRII expression pattern, but was more widespread. During hair follicle cycling in adolescent mice, TGF-betaRII immunoreactivity was restricted to follicles, and was strikingly hair cycle dependent (maximal immunoreactivity: anagen VI and early catagen). Again, TGF-betaRI (Alk-5) immunoreactivity co-localized with TGF-betaRII immunoreactivity, but was more extensive. Reverse transcriptase polymerase chain reaction analysis of TGF-betaRII mRNA confirmed peak transcript levels in back skin with most hair follicles in the anagen VI-catagen transformation. mRNA levels of TGF-betaRI (Alk-5) did not vary significantly during the hair cycle, whereas those of TGF-betaRI (threonine-serine kinase 7 L) declined during early anagen, and were maximal during the anagen-catagen transition. This provides a basis for defining the choreography of TGF-beta-related signalling during hair follicle morphogenesis and cycling, introduces intraepidermal TGF-betaRII immunoreactivity as a marker for imminent follicle development, and supports the concept that both TGF-betaRII and TGF-betaRI stimulation is involved in, but not restricted to, the control of catagen induction.