In the current study, we demonstrate that treatment with the laminin β1 chain-competing peptide YIGSR promotes the formation of a hypercontractile, hypoproliferative ASM phenotype in an animal model of chronic asthma. Topical application of YIGSR to the airways inhibited ASM hyperplasia induced by repeated allergen challenge. However, ASM contractility and contractile protein expression were increased under basal and allergen-challenged conditions. These results appear to be in contrast to previous in vitro studies, demonstrating that soluble YIGSR inhibits maturation of human ASM cells to a hypercontractile, hypoproliferative ASM phenotype [14, 15].
Accumulation of ASM in the airway wall is a characteristic feature of asthma, which may be due to an increase in cell number (hyperplasia) [37, 38] as well as an increase in cell size (hypertrophy) [37, 39]. This ASM accumulation contributes importantly to increased airway resistance and airway hyperresponsiveness [40, 41]. Switching of the ASM phenotype from a contractile to a proliferative state is thought to contribute to the increased ASM mass in asthma . In support, various mitogenic stimuli, including growth factors and ECM proteins, induce a proliferative ASM phenotype in vitro [10–12], an effect that can be inhibited by culturing the cells on immobilized laminin-111 [11, 22, 23] or endogenously produced laminin-211 . These inhibitory effects can be reversed using soluble YIGSR , a binding motif present in the laminin β1 chain . Similarly, in our study culturing human ASM cells on laminin-111 reduced PDGF-induced proliferation, an effect fully normalized by soluble YIGSR. In contrast to this effect of soluble YIGSR, we also show that immobilized YIGSR concentration-dependently inhibited growth factor-induced myocyte proliferation to the same extent as laminin-111. Interestingly, previous work has also shown a disparate effect of immobilized and soluble YIGSR, with the former promoting attachment of various cells [26, 35, 36] whereas the latter blocked attachment to laminin-111  or matrigel . The effects of immobilized YIGSR peptide are specific, as culturing on RGDS or GRADSP did not alter proliferation. Of note, addition of soluble YIGSR normalized the effects of immobilized YIGSR, an affect consistent with studies using alveolar cells and a laminin α chain peptide (Ser-Ile-Asn-Asn-Asn-Arg, or SINNNR) . Collectively, these findings suggest that the laminin-competing peptide YIGSR may either promote or inhibit ASM proliferative responses, depending on the microenvironment of the peptide. The mechanisms underlying these differential effects are unknown. However, since the anti-mitogenic effects of the peptide are only observed when the peptide is immobilized, we speculate that this may be associated with bridging of the 67 kDa laminin receptor LAMR1 - which has high affinity to the YIGSR motif  - whereas soluble YIGSR may competitively inhibit this type of interaction. Similarly, it has been established that binding of ECM proteins such as fibronectin as a monovalent or multivalent ligand to α5β1 integrin has diverse effects on focal contacts, tyrosine kinase activation and cytoskeletal dynamics . Our data indicate that future studies of the ligation of soluble and immobilized YIGSR peptides to specific cell surface receptors and resulting intracellular signaling events are needed.
In addition to ASM accumulation, increased expression of contractile proteins and ASM contractility, and ECM deposition are features of airway remodelling in asthma . In the airways of asthmatics increased expression of laminin α2 and β2 chains is observed [18, 19], and laminin γ2 chain expression inversely correlates with epithelial integrity . Laminins have not only been shown to inhibit ASM proliferation, but also to be critical in maintenance and induction of a (hyper)contractile ASM phenotype. Indeed, culturing of ASM cells on a laminin-111 matrix inhibits proliferation [11, 22, 23], maintains contractile protein expression in the presence of growth factors , and prevents induction of a hypocontractile phenotype by PDGF . Induction of a contractile ASM phenotype in serum-free culture supplemented with insulin is associated with increased expression of laminin α2, β1 and γ1 chains, all found in the laminin-211 isoform [14, 15]. Importantly, the expression of endogenous laminin is required for phenotype maturation, as soluble YIGSR prevents contractile protein accumulation and hypercontractility [14, 15]. Recently, using our guinea pig model of chronic asthma we showed that treatment with the RGD-containing RGDS peptide largely inhibits ASM hyperplasia and hypercontractility . The RGD sequence exists in several ECM proteins [24, 25], thus the specific contribution of laminins cannot be discerned from these prior studies. In the present study we found that in vivo treatment with YIGSR inhibited allergen-induced ASM hyperplasia, but increased both the expression of sm-MHC and ASM contractility. In addition, a small increase in cell size in the allergen-challenged YIGSR treated animals was observed suggesting that hypertrophy may also have played a role in the observed effects. Collectively, our results indicate that treatment with YIGSR inhibits allergen-induced ASM hyperplasia and increases ASM contractility in vivo, suggesting that YIGSR mimics and/or promotes rather than inhibits laminin function under this condition.
Eosinophils express a number of integrins, of which the α6β1 mediates adhesion to laminin, but not to collagen type I or type IV [45, 46]. Eosinophils isolated from allergic donors show higher adhesion to laminin than those isolated from healthy subjects . Migration of eosinophils through matrigel, a basement membrane extract containing laminin-111, also requires interaction with β1-integrins . These findings suggest that laminin-competing peptides could affect allergen-induced airway infiltration of inflammatory cells. To date no reports on YIGSR effects on eosinophil migration are available. In our study we noted that YIGSR increased allergen-induced eosinophil cell numbers in the submucosal compartment, without affecting eosinophil numbers in the adventitial compartment. The increased number of eosinophils in the submucosa suggests that, rather than, infiltration, retention time of the eosinophils in the compartment could be increased. Importantly, increased ECM deposition may be secondary to prolonged airway inflammation  and therefore increased allergen-induced airway fibrosis in YIGSR-treated animals could also indirectly result from increased eosinophilia. As increased and altered deposition of ECM proteins, including laminins and collagens, is a feature of remodelling in chronic asthma [33, 34] it is important that further investigation focus on understanding the effects of YIGSR and laminins on ECM deposition by fibroblasts and other structural cells.
In summary, our results indicate that the laminin-competing peptide YIGSR promotes a contractile, hypoproliferative ASM phenotype in vivo, an effect that is in striking contrast to current and previously reported evidence showing that soluble YIGSR prevents laminin-dependent phenotype maturation. It appears that the microenvironment of the peptide is a critical determinant of its effect as immobilized YIGSR does mimic the effects of laminin matrix on ASM in vitro. Our data suggest that topically applied YIGSR mimics rather than inhibits the effects of laminin in vivo, and its use is linked to increased allergen-induced fibrosis, submucosal eosinophilia, ASM hyperplasia and airway hypercontractility. These data indicate that strategies to develop capacity to use peptides that target ECM-cell interaction to treat bronchial asthma need to be developed with care, in particular with focus on understanding differences of such interventions that may exist between in vitro and in vivo systems.