The laminin β1-competing peptide YIGSR induces a hypercontractile, hypoproliferative airway smooth muscle phenotype in an animal model of allergic asthma

Abstract

Background: Fibroproliferative airway remodelling, including increased airway smooth muscle (ASM) mass and contractility, contributes to airway hyperresponsiveness in asthma. In vitro studies have shown that maturation of ASM cells to a (hyper)contractile phenotype is dependent on laminin, which can be inhibited by the laminin-competing peptide Tyr-Ile-Gly-Ser-Arg (YIGSR). The role of laminins in ASM remodelling in chronic asthma in vivo, however, has not yet been established.

Methods: Using an established guinea pig model of allergic asthma, we investigated the effects of topical treatment of the airways with YIGSR on features of airway remodelling induced by repeated allergen challenge, including ASM hyperplasia and hypercontractility, inflammation and fibrosis. Human ASM cells were used to investigate the direct effects of YIGSR on ASM proliferation in vitro.

Results: Topical administration of YIGSR attenuated allergen-induced ASM hyperplasia and pulmonary expression of the proliferative marker proliferating cell nuclear antigen (PCNA). Treatment with YIGSR also increased both the expression of sm-MHC and ASM contractility in saline- and allergen-challenged animals; this suggests that treatment with the laminin-competing peptide YIGSR mimics rather than inhibits laminin function in vivo. In addition, treatment with YIGSR increased allergen-induced fibrosis and submucosal eosinophilia. Immobilized YIGSR concentration-dependently reduced PDGF-induced proliferation of cultured ASM to a similar extent as laminin-coated culture plates. Notably, the effects of both immobilized YIGSR and laminin were antagonized by soluble YIGSR.

Conclusion: These results indicate that the laminin-competing peptide YIGSR promotes a contractile, hypoproliferative ASM phenotype in vivo, an effect that appears to be linked to the microenvironment in which the cells are exposed to the peptide.

Figure 1

Increased ASM mass after repeated allergen challenge in vivo is inhibited by topical treatment with YIGSR. To assess the role of laminins in increased ASM mass in asthma, the effects of treatment with YIGSR were evaluated in a guinea pig model of chronic allergic asthma. (A) Treatment with YIGSR fully inhibited ovalbumin-induced increase in sm-MHC positive area in cartilaginous airways. (B) Changes in ASM mass were mainly dependent on changes in ASM cell number, only a small increase in cell size was observed for the YIGSR-treated, ovalbumin-challenged animals. (C) Increased pulmonary expression of the proliferative marker PCNA after repeated ovalbumin-challenges, was almost fully reversed by YIGSR. Representative blots of PCNA and β-actin are shown. No effects of YIGSR were shown in saline-challenged animals for any of the parameters. *P < 0.05, ***P < 0.001 compared to saline-treated, saline-challenged controls. ^###P < 0.001 compared to saline-treated, ovalbumin-challenged controls. Data represent means ± SEM of 5-7 animals.

Figure 2

Topical treatment of the airways with YIGSR increases ASM contractility and contractile protein accumulation. (A) Treatment with YIGSR enhanced the maximal methacholine-induced isometric contraction of epithelium-denuded tracheal smooth muscle preparations both in saline- and in ovalbumin-challenged animals. (B) Treatment with YIGSR increased pulmonary expression of sm-MHC, both in saline- and in ovalbumin-challenged animals. Representative blots of sm-MHC and β-actin are shown. ***P < 0.001 compared to saline-treated, saline-challenged controls. ^##P < 0.01 compared to saline-treated, ovalbumin-challenged controls. Data represent means ± SEM of 5-7 animals.

Figure 3

YIGSR treatment increases allergen-induced eosinophilic inflammation in the submucosal airway compartment. (A) Ovalbumin-induced eosinophil numbers in the submucosal compartment are increased by YIGSR treatment. (B) YIGSR treatment does not affect eosinophilic cell number in the adventitial compartment. No effects of YIGSR were found in saline-challenged animals for any of the conditions. ***P < 0.001 compared to saline-treated, saline-challenged controls. ^#P < 0.05 compared to saline-treated, ovalbumin-challenged animals. Data represent means ± SEM of 5-7 animals.

Figure 4

YIGSR treatment increases allergen-induced fibrosis in the guinea pig lung. Hydroxyproline content in guinea pig lung after repeated saline- or ovalbumin-challenges in saline- and YIGSR-treated animals. ***P < 0.001 compared to saline-treated, saline-challenged controls. ^##P < 0.01 compared to saline-treated, ovalbumin-challenged animals. Data represent means ± SEM of 5-7 animals.

Figure 5

Effects of immobilized and soluble YIGSR on basal and PDGF-induced human ASM cell proliferation. (A) Culturing of human ASM cells on immobilized YIGSR matrices inhibits PDGF-induced thymidine-incorporation in a YIGSR concentration-dependent fashion. Under unstimulated (Basal) conditions, no effects of immobilized YIGSR were observed. (B) Immobilized RGDS or its negative control GRADSP did not affect basal or PDGF-induced thymidine-incorporation. (C) The inhibitory effects of immobilized laminin-111 and YIGSR matrices on PDGF-induced thymidine-incorporation were normalized by soluble YIGSR. ***P < 0.001 compared to thymidine-incorporation of unstimulated cells (basal) cultured on uncoated matrices (plastic). ^#P < 0.05 and ^##P < 0.01 compared to PDGF-induced thymidine-incorporation of cells cultured on uncoated matrices. Data represent means ± SEM of 4-5 independent experiments of 3 different donors, performed in duplicate.