Cannabinoids reduce granuloma-associated angiogenesis in rats by controlling transcription and expression of mast cell protease-5

Abstract

Background and purpose: Chronic inflammatory conditions, such as granulomas, are associated with angiogenesis. Mast cells represent the main cell type orchestrating angiogenesis, through the release of their granule content. Therefore, compounds able to modulate mast cell behaviour may be considered as a new pharmacological approach to treat angiogenesis-dependent events. Here, we tested the effect of selective cannabinoid (CB) receptor agonists in a model of angiogenesis-dependent granuloma formation induced by lambda-carrageenin in rats.

Experimental approach: Granulomas were induced by lambda-carrageenin-soaked sponges implanted subcutaneously on the back of male Wistar rats. After 96 h, implants were removed and granuloma formation was measured (wet weight); angiogenesis was evaluated by histological analysis and by the measurement of haemoglobin content. Mast cells in the granulomas were evaluated histologically and by RT-PCR and immunoblotting analysis for mast cell-derived proteins (rat mast cell protease-5 (rMCP-5) and nerve growth factor). Selective CB1 and CB2 receptor agonists(,) ACEA and JWH-015 (0.001-0.1 mg mL(-1)), were given locally only once, at the time of implantation.

Key results: The CB1 and CB2 receptor agonists decreased the weight and vascularization of granulomas after 96 h. This treatment also reduced mast cell number and activation in granulomatous tissue. Specifically, these compounds prevented the transcription and expression of rMCP-5, a protein involved in sprouting and advance of new blood vessels.

Conclusion and implications: Modulation of mast cell function by cannabinoids reduced granuloma formation and associated angiogenesis. Therefore cannabinoid-related drugs may be useful in the management of granulomatous diseases accompanied by angiogenesis.

Figure 1

Effect of the selective CB₁ receptor agonist ACEA and the selective CB₂ receptor agonist JWH-015 on λ-carrageenin (CAR)-induced granuloma. (a) ACEA (0.5, 5, 50 μg per implant) and JWH-015 (0.5, 5, 50 μg), significantly and in a concentration-dependent manner reduced granulomatous tissue formation, evaluated as wet weight at 96 h after implantation. (b) Local administration of ACEA (50 μg) or JWH-015 (50 μg) or the non-selective CB₁/CB₂ receptor agonist, WIN 55,212-2 (WIN; 50 μg), significantly reduced angiogenesis, evaluated as haemoglobin (Hb) content of the granuloma. No effects were observed in the group treated with vehicle (10% ethanol;EtOH). Results are expressed as mean±s.e.m. of three separate experiments. ^*P<0.05, ^***P<0.001 vs saline; °°P<0.01, °°°P<0.001 vs λ-carrageenin alone.

Figure 2

Effect of the selective CB₁ receptor agonist ACEA (50 μg) and the selective CB₂ receptor agonist JWH-015 (50 μg) on λ-carrageenin (CAR)-induced mast cell activation. In panel (a) is shown a representative histological analysis of mast cells present in granulomatous tissue. Mast cells were evaluated in paraffin sections stained with 0.05% (w v⁻¹) toluidine blue and counterstained with 0.1% (w v⁻¹) nuclear fast red (magnification × 100). The square panel is an enlargement showing the differences between not degranulated (deep blue) and degranulated (light blue) mast cells. In panel (b) is shown the total number of mast cells; in panel (c) the percentage of mast cell degranulation (the ratio between not degranulated and degranulated mast cells) is shown; in panel (d) is shown the percentage of the β−hexosaminidase release, as a specific marker of mast cell degranulation. Ketotifen (KET; 50 μg) was used as a positive control. Results are expressed as mean±s.e.m. of three separate experiments. ^*P<0.05, ^**P<0.01, ^***P<0.001 vs saline; °°°P<0.001 vs λ-carrageenin alone.

Figure 3

Effects of the selective CB₁ receptor agonist ACEA and the CB₂ receptor agonist JWH-015 on rMCP-5 transcription and expression. (a) Representative Vistra green-stained agarose gel of RT-PCR products corresponding to rMCP-5 mRNA in sponges injected with saline solution (SAL) or with λ-carrageenin (CAR) or λ-carrageenin in the presence of ACEA (50 μg) or JWH-0.15 (50 μg). β-actin, a housekeeping gene, was used as a control. The gel shown corresponds to the products from 35 cycles of amplification for rMCP-5 and 15 cycles for β-actin. (b) Summary of values from three independent experiments expressed as percent, after normalization to β-actin mRNA level. Results are expressed as mean±s.e.m. of three separate experiments. ^*P<0.05, ^***P<0.001 vs saline.; °°°P<0.001 vs λ-carrageenin alone.

Figure 4

Effects of the CB₁ receptor agonist ACEA (50 μg) and the CB₂ receptor agonist JWH-015 (50 μg) on expression of proteins secreted by mast cells. In (A) rMCP-5 and (B) NGF protein expression is shown by a representative western blot (a) and relative densitometric analysis (b). Tubulin expression is shown as a control. Data are expressed as mean±s.e.mean of three separate experiments. ^*P<0.05, ^**P<0.01, ^***P<0.001 vs saline; ^□□P<0.01, ^□□□P<0.001 vs λ-carrageenin (CAR) alone.

Figure 5

Effect of the CB₁ receptor agonist ACEA (50 μg) and the CB₂ receptor agonist JWH-015 (50 μg) on angiogenesis in granulomatous tissue. Angiogenesis was evaluated by histological analysis and vessel number. Panel (a) shows a representative histological analysis of granulomatous tissue stained with hematoxylin and eosin. Fields are representative of three separate experiments. Original magnification × 100. Panel (b) shows the number of blood vessels in granulomatous tissue. Results are expressed as mean±s.e.m. of three separate experiments. ^*P<0.05, ^***P<0.001 vs saline; °°°P<0.001 vs λ-carrageenin (CAR) alone.