Mast Cell Proteases 6 and 7 Stimulate Angiogenesis by Inducing Endothelial Cells to Release Angiogenic Factors.

Mast cell proteases are thought to be involved with tumor progression and neo-vascularization. However, their exact role is still unclear. The present study was undertaken to further elucidate the function of specific subtypes of recombinant mouse mast cell proteases (rmMCP-6 and 7) in neo-vascularization. SVEC4-10 cells were cultured on Geltrex® with either rmMCP-6 or 7 and tube formation was analyzed by fluorescence microscopy and scanning electron microscopy. Additionally, the capacity of these proteases to induce the release of angiogenic factors and pro and anti-angiogenic proteins was analyzed. Both rmMCP-6 and 7 were able to stimulate tube formation. Scanning electron microscopy showed that incubation with the proteases induced SVEC4-10 cells to invade the gel matrix. However, the expression and activity of metalloproteases were not altered by incubation with the mast cell proteases. Furthermore, rmMCP-6 and rmMCP-7 were able to induce the differential release of angiogenic factors from the SVEC4-10 cells. rmMCP-7 was more efficient in stimulating tube formation and release of angiogenic factors than rmMCP-6. These results suggest that the subtypes of proteases released by mast cells may influence endothelial cells during in vivo neo-vascularization

Mast Cells Interact with Endothelial Cells to Accelerate In Vitro Angiogenesis

Angiogenesis is a complex process that involves interactions between endothelial cells and various other cell types as well as the tissue microenvironment. Several previous studies have demonstrated that mast cells accumulate at angiogenic sites. In spite of the evidence suggesting a relationship between mast cells and angiogenesis, the association of mast cells and endothelial cells remains poorly understood. The present study aims to investigate the relationship between mast cells and endothelial cells during in vitro angiogenesis. When endothelial cells were co-cultured with mast cells, angiogenesis was stimulated. Furthermore, there was direct intercellular communication via gap junctions between the two cell types. In addition, the presence of mast cells stimulated endothelial cells to release angiogenic factors. Moreover, conditioned medium from the co-cultures also stimulated in vitro angiogenesis. The results from this investigation demonstrate that mast cells have both direct and indirect proangiogenic effects and provide new insights into the role of mast cells in angiogenesis

Action of differentially released angiogenic factors.

<p>Action of differentially released angiogenic factors.</p

Angiogenic factors released in the presence of rmMCP-6 or rmMCP-7.

<p>Angiogenic factors released in the presence of rmMCP-6 or rmMCP-7.</p

Mast cell tryptases induce an increase of angiogenesis in chicken chorioallantoic membrane.

<p>When compared to the control CAM (no tryptases), CAM incubated in presence of rmMCP-6 and 7 showed an increase in the number of blood vessels. Moreover, an increase in blood vessel caliber was also seen in the presence of the tryptases. The graph shows the number of vessel per field (6.5mm²) from each sample.</p

The Role of Mast Cell Specific Chymases and Tryptases in Tumor Angiogenesis

An association between mast cells and tumor angiogenesis is known to exist, but the exact role that mast cells play in this process is still unclear. It is thought that the mediators released by mast cells are important in neovascularization. However, it is not known how individual mediators are involved in this process. The major constituents of mast cell secretory granules are the mast cell specific proteases chymase, tryptase, and carboxypeptidase A3. Several previous studies aimed to understand the way in which specific mast cell granule constituents act to induce tumor angiogenesis. A body of evidence indicates that mast cell proteases are the pivotal players in inducing tumor angiogenesis. In this review, the likely mechanisms by which tryptase and chymase can act directly or indirectly to induce tumor angiogenesis are discussed. Finally, information presented here in this review indicates that mast cell proteases significantly influence angiogenesis thus affecting tumor growth and progression. This also suggests that these proteases could serve as novel therapeutic targets for the treatment of various types of cancer

In the presence of pre-formed mast cell mediators, SVEC4-10 cells form tubes and loops.

<p>The cells were cultured for 5 hours at 37°C on Geltrex^® in the presence of Mast Cell Mediators (MCM) from bone marrow derived mast cell. (A) When the SVEC4-10 cells were cultured in the presence of MCM, most of the cells were spread on the substrate, and formed both tubes (T) and loops (L). (B) When the cells were incubated in absence of mast cell mediators, the cells were spread on the substrate forming tubes (T), but there was no loop formation. (C) Quantitative analysis shown that the tube formation was higher in presence of mast cell mediators. The significance was determined by Student's <i>t</i>-test *<i>p</i> ≤ 0.05.</p

In the presence of rmMCP-6 and rmMCP-7 the endothelial cells invade the Geltrex^®.

<p>SVEC4-10 cells were cultured for 5 hours at 37°C on Geltrex^® in the presence of rmMCP-6, rmMCP-7 or in the absence of tryptases. In the absence of rmMCP-6 or -7, most of the SVEC4-10 cells remained rounded (arrows) and few cells spread on the substrate (arrowheads). In the presence of rmMCP-6 or -7 almost all endothelial cells were spread on the substrate and were organized into tubes (asterisks). Additionally, the SVEC4–10 cells invaded the Geltrex^® only in the presence of the tryptases.</p

Expression of Mast Cell Proteases Correlates with Mast Cell Maturation and Angiogenesis during Tumor Progression

Tumor cells are surrounded by infiltrating inflammatory cells, such as lymphocytes, neutrophils, macrophages, and mast cells. A body of evidence indicates that mast cells are associated with various types of tumors. Although role of mast cells can be directly related to their granule content, their function in angiogenesis and tumor progression remains obscure. This study aims to understand the role of mast cells in these processes. Tumors were chemically induced in BALB/c mice and tumor progression was divided into Phases I, II and III. Phase I tumors exhibited a large number of mast cells, which increased in phase II and remained unchanged in phase III. The expression of mouse mast cell protease (mMCP)-4, mMCP-5, mMCP-6, mMCP-7, and carboxypeptidase A were analyzed at the 3 stages. Our results show that with the exception of mMCP-4 expression of these mast cell chymase (mMCP-5), tryptases (mMCP-6 and 7), and carboxypeptidase A (mMC-CPA) increased during tumor progression. Chymase and tryptase activity increased at all stages of tumor progression whereas the number of mast cells remained constant from phase II to III. The number of new blood vessels increased significantly in phase I, while in phases II and III an enlargement of existing blood vessels occurred. In vitro, mMCP-6 and 7 are able to induce vessel formation. The present study suggests that mast cells are involved in induction of angiogenesis in the early stages of tumor development and in modulating blood vessel growth in the later stages of tumor progression

In the presence of rmMCP-6 and rmMCP-7 SVEC4-10 cells show a differential release of angiogenesis related cytokines/proteins.

<p>The graph shows the cytokines/proteins that were released exclusively by rmMCP-6 (blue) or rmMCP-7 (red). Four cytokines/proteins that were released by control cells were increased in the presence of the tryptases (green). The cells were cultured for 5 hours at 37°C on Geltrex^® in the presence of rmMCP-6, rmMCP-7 or in the absence of tryptases. After incubation, a mouse cytokine array kit was used to analyze the protein expression of different pro- and anti-angiogenic factors in the culture supernatants. The data shown is the mean spot pixel density that was quantified from the arrays using the image analysis software Adobe Photoshop CS6 V 13.0.</p