Effect of anti-IL17 and/or Rho-kinase inhibitor treatments on vascular remodeling induced by chronic allergic pulmonary inflammation

Abstract

Background and aims: Expansion and morphological dysregulation of the bronchial vascular network occurs in asthmatic airways. Interleukin (IL) -17 and Rho-kinase (ROCK) are known to act in inflammation control and remodeling. Modulation of Rho-kinase proteins and IL-17 may be a promising approach for the treatment of asthma through the control of angiogenesis. Our objective was to analyze the effects of treatment with anti-IL17 and/or Rho-kinase inhibitor on vascular changes in mice with chronic allergic pulmonary inflammation.

Methods: Sixty-four BALB/c mice, with pulmonary inflammation induced by ovalbumin were treated with anti-IL17A (7.5/µg per dose, intraperitoneal) and/or Rho-kinase inhibitor (Y-27632-10 mg/kg, intranasal), 1 h before each ovalbumin challenge (22, 24, 26, and 28/days). Control animals were made to inhale saline. At the end of the protocol, lungs were removed, and morphometric analysis was performed to quantify vascular inflammatory, remodeling, and oxidative stress responses.

Results: Anti-IL17 or Rho-kinase inhibitor reduced the number of CD4⁺, CD8⁺, dendritic cells, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, Rho-kinase 1 and 2, transforming growth factor (TGF-β), vascular endothelial growth factor (VEGF), nuclear factor (NF)-KappaB, iNOS, metalloproteinase (MMP)-9, MMP-12, metalloproteinase inhibitor-1 (TIMP-1), FOXP-3, signal transducer and activator of transcription 1 (STAT1) and phospho-STAT1-positive cells, and actin, endothelin-1, isoprostane, biglycan, decorin, fibronectin and the collagen fibers volume fraction compared with the ovalbumin group (p < 0.05). The combination treatment, when compared with anti-IL17, resulted in potentiation of decrease in the number of IL1β- and dendritic cells-positive cells. When we compared the OVA-RHO inhibitor-anti-IL17 with OVA-RHO inhibitor we found a reduction in the number of CD8⁺ and IL-17, TGF-β, and phospho-STAT1-positive cells and endothelin-1 in the vessels (p < 0.05). There was an attenuation in the number of ROCK 2-positive cells in the group with the combined treatment when compared with anti-IL17 or Rho-kinase inhibitor-treated groups (p < 0.05).

Conclusion: We observed no difference in angiogenesis after treatment with Rho-kinase inhibitor and anti-IL17. Although the treatments did not show differences in angiogenesis, they showed differences in the markers involved in the angiogenesis process contributing to inflammation control and vascular remodeling.The reviews of this paper are available via the supplemental material section.

Keywords: Rho-kinase; Y-27632; angiogenesis; asthma; interleukin-17; neutralizing antibody; vascular remodeling.

Figure 1.

Experimental protocol.

Figure 2.

In the OVA group there was an increase in angiogenesis compared with the saline group. The inhibitor of Rho-kinase and anti-IL17 has no effect on angiogenesis in chronic allergic pulmonary inflammation induced by ovalbumin in mice. To determine the difference between groups and their statistical significance, we used the unidirectional analysis of variance followed by the Holm–Sidak method for multiple comparisons. The data are presented as dot plots with standard deviations. N = 8 animals per group. IL, interleukin; OVA, animals induced to chronic allergic pulmonary inflammation by ovalbumin; OVA-anti-IL17, animals induced to chronic allergic pulmonary inflammation and use of anti-IL17; OVA-RHO inhibitor, animals induced to chronic allergic pulmonary inflammation and use of Rho-kinase inhibitor; OVA-RHO inhibitor-anti-IL17, animals induced to chronic allergic pulmonary inflammation and use of Rho-kinase inhibitor and anti-IL17; SAL, saline group.

Figure 3.

The treatments with the inhibitor of Rho-kinase and anti-IL17 attenuate the expression of ROCK1 and ROCK2 in vessel in chronic allergic pulmonary inflammation induced by ovalbumin in mice. The attenuation of ROCK2 expression in the vessel is enhanced when the treatment of the Rho-kinase inhibitor was associated with anti-IL17 compared with OVA-RHO inhibitor group and OVA-anti-IL17 group. In OVA group there was an increase compared with the saline group. The data are presented as dot plots with standard deviations. Representative photomicrographs of ROCK1 and ROCK2 for each group. IL, interleukin; ROCK, Rho-kinase; OVA, animals induced to chronic allergic pulmonary inflammation by ovalbumin; OVA-anti-IL17, animals induced to chronic allergic pulmonary inflammation and use of anti-IL17; OVA-RHO inhibitor, animals induced to chronic allergic pulmonary inflammation and use of Rho-kinase inhibitor; OVA-RHO inhibitor-anti-IL17, animals induced to chronic allergic pulmonary inflammation and use of Rho-kinase inhibitor and anti-IL17; SAL, saline group.

Figure 4.

The treatments with the inhibitor of Rho-kinase and anti-IL17 attenuate the expression of oxidative markers in vessel in chronic allergic pulmonary inflammation induced by ovalbumin in mice. In OVA group there was an increase compared with the saline group. The data are presented as dot plots with standard deviations. Representative photomicrographs of iNOS and isoprostane for each group. The red arrows indicate the marked area in immunohistochemistry. Eight animals per group. IL, interleukin; OVA, animals induced to chronic allergic inflammation by ovalbumin; OVA-anti-IL17, animals with induction of chronic allergic pulmonary inflammation and use of anti-IL17; OVA-RHO inhibitor, animals with induction of chronic allergic pulmonary inflammation and use of Rho-kinase inhibitor; OVA-RHO inhibitor-anti-IL17, animals with induction of chronic allergic pulmonary inflammation and use of Rho-kinase inhibitor and anti-IL17; SAL, saline group.

Figure 5.

The treatments with the inhibitor of Rho-kinase and anti-IL17 attenuate the expression of IL-17 in vessel in chronic allergic pulmonary inflammation induced by ovalbumin in mice. The attenuation of IL-17 expression in the vessel is enhanced when the treatment of the Rho-kinase inhibitor was associated with anti-IL17 compared with treatment of Rho-kinase inhibitor alone. In OVA group there was an increase compared with the saline group. The data are presented as dot plots with standard deviations. Representative photomicrographs of IL-17 for each group. The red arrows indicate the marked area in immunohistochemistry. IL, interleukin; OVA, animals induced to chronic allergic inflammation by ovalbumin; OVA-anti-IL17, animals with induction of chronic allergic pulmonary inflammation and use of anti-IL17; OVA-RHO inhibitor, animals with induction of chronic allergic pulmonary inflammation and use of Rho-kinase inhibitor; OVA-RHO inhibitor-anti-IL17, animals with induction of chronic allergic pulmonary inflammation and use of Rho-kinase inhibitor and anti-IL17; SAL, saline group.

Figure 6.

The treatments with the Rho-kinase and anti-IL17 attenuates the expression of remodeling markers in vessel in chronic allergic pulmonary inflammation induced by ovalbumin in mice. In OVA group there was an increase compared with the saline group in the expression of MMP-12 and TIMP-1. The data are presented as dot plots with standard deviations. Representative photomicrographs of MMP-12, TIMP-1 and TGF-β for each group. The red arrows indicate the marked area in immunohistochemistry. IL, interleukin; MMP, metalloproteinase; TIMP, metalloproteinase inhibitor; TGF-β, transforming growth factor; OVA, animals induced to chronic allergic inflammation by ovalbumin; OVA-anti-IL17, animals induced to chronic allergic pulmonary inflammation and use of anti-IL17; OVA-RHO inhibitor, animals induced to chronic allergic pulmonary inflammation and use of Rho-kinase inhibitor; OVA-RHO inhibitor-anti-IL17, animals induced to chronic allergic pulmonary inflammation and use of Rho-kinase inhibitor and anti-IL17; SAL, saline group.

Figure 7.

The treatments with Rho-kinase inhibitor and anti-IL17 have an effect on the control of mechanisms involved in chronic allergic pulmonary inflammation induced by ovalbumin. In OVA group there was an increase compared with the saline group in the expression of dendritic cells, STAT1, phospho-STAT1, NF-KappaB, and FOX-P3. The data are presented as dot plots with standard deviations. IL, interleukin; NF, nuclear factor; STAT1, signal transducer and activator of transcription 1; OVA, animals induced to chronic allergic inflammation by ovalbumin; OVA-anti-IL17, animals with induction of chronic allergic pulmonary inflammation and use anti-IL17; OVA-RHO inhibitor, animals with induction of chronic allergic pulmonary inflammation and use of Rho-kinase inhibitor; OVA-RHO inhibitor-anti-IL17, animals with induction of chronic allergic pulmonary inflammation and use of Rho-kinase inhibitor and anti-IL17; SAL, saline group.