The modulation of smooth muscle contractility by specialised pro-resolving mediators

Abstract

Smooth muscle contraction, tissue remodelling and chronic inflammation are features common to many lung conditions including asthma and pulmonary arterial hypertension (PAH). Three people in the UK die each day from a fatal asthma exacerbation, whilst right heart failure caused by increased resistance in the pulmonary arteries is an end-point that over half of patients with PAH can expect just five years after diagnosis. Novel relaxants to address the inappropriate smooth muscle contraction in these diseases are required. Recently-identified families of specialised pro-resolving lipid mediators (SPMs) including resolvins (Rv), lipoxins (LX), protectins (PD) and maresins actively limit the acute inflammatory response through the activation of G-protein-coupled receptors, but their roles in smooth muscle contraction are poorly understood. The aims of this thesis were: to determine whether SPMs, in particular RvD1, RvE1, LXA4 and PDX, can modulate spontaneous or growth factor-induced contraction of cell-populated collagen lattices; to use wire myography to define whether Rvs D1, D2 and E1 can modulate contractility in bronchiole and artery segments from rat and human donors; and finally to use immunohistology to localise expression of the pro-resolving receptors ChemR23, GPR32 and ALX in human pulmonary artery (HPA). None of the SPMs investigated affected spontaneous contraction of collagen lattices populated with human lung fibroblasts (HLF), human bronchial smooth muscle cells (HBSMC) or human umbilical artery smooth muscle cells (HUASMC), nor did they affect TGFβ1-enhanced lattice contraction. However, RvE1 abolished the platelet-derived growth factor (PDGF)-enhanced contraction of HBSMC lattices (p<0.05). In addition, PDX weakly inhibited contraction of lattices populated with HLF or HBSMC, while RvD1 and LXA4 had no effect. Wire myography was used to investigate the ability of RvD1, RvD2 and RvE1 to prevent or reverse contraction of bronchiole segments isolated from rat and human lung. None of the resolvins tested were able to reverse carbachol (CCh)-induced pre-constriction in rat bronchioles or leukotriene D4 (LTD4)-induced preconstriction in human bronchioles. Pre-treatment with resolvins D1 or E1 failed to prevent subsequent contraction of rat bronchioles induced by either CCh or the stable thromboxane mimetic U46619, compared to the same segments contracted previously without resolvin. Pre-treatment of rat and human bronchioles with RvD1 (10 nM) appeared to dampen LTD4-induced constriction, although complications with suspected tachyphylaxis and generally low responsiveness to LTD4 in rat bronchioles may have precluded significant outcomes. Wire myography was used to explore Rv effects in rat and human blood vessels. Rvs D1, D2 and E1 all failed to reverse phenylephrine (PE)- or U46619-induced pre-constriction in segments of rat thoracic aorta (RTA), and HPA, these resolvins did not reverse pre-constriction induced by LTD4. Pre-treatment of HPA with RvD1 or RvE1 had no effect on subsequent constriction by U46619, while their effects on LTD4-induced constriction were masked by changing baseline responses to this contractile agonist, as seen in bronchiole segments. However, in naive segments of RTA pre-treated for 1 or 24 hour with or without resolvin in parallel, RvE1 (0.1-300 nM), RvD1 and RvD2 (1-100 nM) each significantly inhibited constriction to U46619 with a bell-shaped inhibition curve, when compared to the control segments constricted by U46619 without resolvin (10 nM RvE1 p<0.0001; 10 nM RvD1 p<0.01; 10 nM RvD2 p<0.05). The significant inhibitory effect of RvE1 (1 hour) was also seen in U46619-constricted HPA segments (p<0.0001 with 10 nM RvE1), but not in RTA constricted with PE, indicating a selective effect on thromboxane receptor signalling. Immunohistochemistry of paraffin wax-embedded HPA sections indicated that the pro-resolving receptors GPR32 and ChemR23 were both expressed in the vascular smooth muscle, with GPR32 also apparent in the endothelium. This thesis provides the first evidence that acute pre-treatment with SPMs can directly impair the contractile responses of intact segments of rat aorta and human pulmonary arteries to well-known contractile agonists, and that they can also modulate the PDGF-enhanced contraction of 3D collagen matrices by human bronchial smooth muscle cells. These findings provide novel insights into SPM activity with important implications for understanding the regulation of smooth muscle contractility in chronic inflammatory diseases such as asthma, PAH and systemic hypertension.

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ORCID for Anthony Sampson: orcid.org/0009-0008-9653-8935

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