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

Abstract

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 pro-angiogenic 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, 96 h after the subcutaneous implant of two lambda-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-alpha (TNF-alpha) in the exudates. A single injection of ketotifen (1-5-25 mg kg(-1) i.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 (5-10 nmol(-1) site(-1) day(-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 (1.25-2.5-5.0 nmol site(-1)) by 69.5, 72.5 and 81.8%. In parallel experiments, rMCP-5 AS-ODN (1.25, 2.5, 5.0 nmol site(-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.

Figure 1

Granuloma formation and mast cell degranulation in λ-carrageenin-soaked sponge implants in rat. (a) Granuloma was induced by implantation of two polyether sponges soaked with 1% (w/v) λ-carrageenin (0.5 ml sponge⁻¹) or saline (0.5 ml sponge⁻¹) as control. Granuloma formation around the sponge was evaluated 96 h after implantation and expressed as wet weight. (b) A representative histological analysis of rat granulomatous tissue around (1%) λ-carrageenin-soaked sponge implants and saline-soaked sponge implants in rats at 96 h. Mast cell degranulation was evaluated on microscopically visible connective mast cells stained with 0.05% (w/v) toluidine blue and counterstained with 0.1% (w/v) nuclear fast red (magnification × 100). (c) A differentiation between not degranulated (deep blue-black arrow) and degranulated (light blue-blue arrow) mast cells, in both saline and 1% λ-carrageenin, was performed. Results are expressed as percentage of degranulation (%). ^**P<0.01 and ^***P<0.001 vs saline.

Figure 2

Effect of ketotifen and chlorpheniramine on granuloma formation. Granuloma was induced by (1%) λ-carrageenin- or saline-soaked sponge implants in rats. Ketotifen fumarate and chlorpheniramine maleate (1–5–25 mg kg⁻¹) were given intraperitoneally at time 0, that is, the same day of sponge implantation. Granuloma formation was evaluated 96 h after implantation and expressed as wet weight. Bars represent the mean±s.e. of n=6 experiments in duplicate. ^***P<0.001 vs saline; °°°P<0.001 and °°P<0.01 vs carrageenin alone.

Figure 3

Effect of ketotifen on granuloma formation in compound 48/80 pretreated rats. Granuloma was induced by (1%) λ-carrageenin- or saline-soaked sponge implants in rats (no pretreated animals). In order to confirm the importance of mast cell-released mediators in granuloma formation, in some experiments rats were previously depleted of mast cell granule content by using compound 48/80 (48/80 pretreated animals) according to the protocol described in the methods. Ketotifen fumarate (1–5–25 mg kg⁻¹) was given intraperitoneally at time 0, that is, the same day of sponge implantation to 48/80 pretreated animals. Granuloma formation was evaluated 96 h after implantation and expressed as wet weight. Bars represent the mean±s.e. of n=4 experiments in duplicate. ^**P<0.01 vs no pretreated animals.

Figure 4

Effect of ketotifen on angiogenesis. Granuloma was induced by (1%) λ-carrageenin- or saline-soaked sponge implants in rats. Ketotifen fumarate (1–5–25 mg kg⁻¹) was given intraperitoneally at time 0, that is, the same day of sponge implantation. Angiogenesis was evaluated at 96 h after implantation as (a) haemoglobin content in the tissue and (b) TNF-α levels in the exudates. Bars represent the mean±s.e. of n=4 experiments in duplicate. ^***P<0.001 vs saline; °P<0.05 and °°°P<0.001 vs carrageenin alone.

Figure 5

Effect of chymostatin on granuloma formation and angiogenesis. Granuloma was induced by (1%) λ-carrageenin- or saline-soaked sponge implants in rats. Chymostatin (5–10 nmol site⁻¹) was given daily into the sponge. (a) Granuloma formation was evaluated 96 h after implantation and expressed as wet weight. Angiogenesis was evaluated at 96 h as (b) haemoglobin content in the tissue and (c) TNF-α levels in the exudates. Bars represent the mean±s.e. of n=6 experiments in duplicate. ^***P<0.001 vs saline; °P<0.05 °°P<0.01 and °°°P<0.001 vs carrageenin alone.

Figure 6

Expression of rMCP-5 in granulomatous tissue. (a) RT–PCR assay of rMCP-5 mRNA levels in animals injected with saline solution or with (1%) λ-carrageenin. The expression of β-actin, a housekeeping gene, was used as a control of cDNA quantity. PCR was allowed to proceed for 30, 35, and 40 cycles using rMCP5 primers and for 10, 15, and 20 cycles using β-actin primers. The results demonstrate a linear relationship along the cycles. (b) Quantitative analysis of the results of three independent experiments expressed in arbitrary units after normalization to β-actin mRNA levels.

Figure 7

Reduction in rMCP-5 mRNA expression following treatment with phosphorothioate AS-ODN. (a) RT–PCR assay of rMCP-5 mRNA levels in animals injected with (1%) λ-carrageenin in the absence or in presence of rMCP-5 AS-ODN (1.25–2.5–5 nmol site⁻¹) or S-ODN (5 nmol site⁻¹). PCR was allowed to proceed as described in the legend to Figure 6a. (b) Quantitative analysis of the results of three independent experiments after normalization to β-actin mRNA levels calibrated to the signal of animals injected with (1%) λ-carrageenin (100%). (c) Western blot analysis of chymase protein levels in animals injected with (1%) λ-carrageenin in the absence or in presence of increasing amounts of AS-ODN.

Figure 8

Effect of AS-ODN against rMCP-5 on granuloma formation and related angiogenesis. Granuloma was induced by (1%) λ-carrageenin- or saline-soaked sponge implants in rats. rMCP-5 AS-ODN (1.25–2.5–5 nmol site⁻¹) and S-ODN (5 nmol site⁻¹) were given into the sponge at time 0, that is, the same day of sponge implantation. (a) Granuloma formation was evaluated after 96 h from implantation and expressed as wet weight. (b) Granuloma-associated angiogenesis was evaluated as haemoglobin content in the tissue. Bars represent the mean±s.e. of n=6 experiments in duplicate. ^***P<0.001 vs saline; °P<0.05, °°P<0.01 and °°°P<0.001 vs carrageenin alone.