Inhibition of granuloma-associated angiogenesis by controlling mast cell mediator release:role of mast cell protease-5

We investigated the role of mast cells in granuloma-associated angiogenesis in rat by using: (i) a mast cell membrane stabilizer, ketotifen; (ii) a mast cell depleting agent, compound 48/80. Moreover, we focused on the role of chymases, which exhibit proinflammatory and proangiogenic properties by using: (i) chymostatin, an inhibitor of chymase activity; (ii) a specific antisense oligonucleotide (AS-ODN) designed against rat mast cell protease-5 (rMCP-5), the most abundantly expressed chymase in the skin. The formation of granuloma was evaluated, as wet weight, 96h after the subcutaneous implant of two λ-carrageenin (1%)-soaked sponges on the back of male Wistar rats. Angiogenesis was evaluated as haemoglobin content in the granulomatous tissue and as level of tumour necrosis factor-α (TNF-α) in the exudates. A single injection of ketotifen (1–5–25mgkg−1i.p.) significantly reduced granuloma formation by 31.6, 44.6 and 71.9%, and haemoglobin content by 17.0, 35.0 and 66.2%, suggesting that the release of mediator(s) from mast cells modulates the process. Chymostatin (5–10nmol−1site−1day−1) reduced granuloma-associated angiogenesis by 57.3 and 70.0%. RT–PCR analysis showed that rMCP-5 mRNA amounts were significantly reduced by rMCP-5 AS-ODN (1.25–2.5–5.0nmolsite−1) by 69.5, 72.5 and 81.8%. In parallel experiments, rMCP-5 AS-ODN (1.25, 2.5, 5.0nmolsite−1) strongly reduced granuloma weight (26.1, 45.0 and 56.3%) and haemoglobin content (22.2, 50.4, 62.03%), suggesting that the observed effect is mediated through an antisense mechanism. In conclusion, these data suggest that: (i) inhibition of mast cell mediators release may represent a novel strategy to modulate angiogenesis; (ii) among the chymase family, rMCP-5 is a key promoter of angiogenesis in the rat.