The small‐molecule formyl peptide receptor biased agonist, compound 17b, is a vasodilator and anti‐inflammatory in mouse precision‐cut lung slices

Pulmonary arterial hypertension (PAH), a rare fatal disorder characterised by inflammation, vascular remodelling and vasoconstriction. Current vasodilator therapies reduce pulmonary arterial pressure but not mortality. The G‐protein coupled formyl peptide receptors (FPRs) mediates vasodilatation and resolution of inflammation, actions possibly beneficial in PAH. We investigated dilator and anti‐inflammatory effects of the FPR biased agonist compound 17b in pulmonary vasculature using mouse precision‐cut lung slices (PCLS).

Pulmonary arterial hypertension (PAH) is an incurable disease defined by an increase in mean pulmonary arterial pressure (PAP) to ≥25 mmHg.PAH is associated with increased expression of inflammatory, fibrotic and contractile mediators that lead to vascular remodelling (Hensley et al., 2018;Humbert et al., 2010).The incidence of PAH is relatively rare (Strange et al., 2018).However, the estimated mortality 5 years after diagnosis is approximately 43% (Benza et al., 2012), with the leading cause of death being right ventricular failure (Kumar & Neema, 2017).
Current dilator therapies include the PGI 2 analogue iloprost, the soluble guanylate cyclase (sGC) stimulator riociguat and the phosphodiesterase type 5 (PDE5) inhibitor sildenafil.These drugs increase the synthesis or reduce the metabolism of cAMP or cGMP to cause pulmonary artery relaxation.However, these standard-of-care dilators have limited efficacy in reducing PAP and fail to target the inflammation and vascular remodelling that are the main drivers of pathogenesis and progression of PAH (Zheng et al., 2020).There is an urgent unmet need for innovative therapeutic strategies beyond vasodilation to treat PAH (Hu et al., 2022).
Formyl peptide receptors (FPRs) are a class of G-protein coupled receptors (GPCRs) that may be a novel therapeutic target for PAH.FPRs have been located in mouse aortic vascular smooth muscle cells (SMCs) (Qin et al., 2015(Qin et al., , 2022)), suggesting a potential role in regulating vascular tone.However, FPRs were originally discovered in immune cells and implicated in the regulation of inflammation (Williams et al., 1977).
Screening for synthetic, small-molecule FPR ligands has revealed different chemotypes associated with diverse signalling profiles (Cilibrizzi et al., 2009;Deora et al., 2019), with potential implications in regulation of inflammation and vascular function in PAH.FPR agonists include compound 17b, biased away from Ca 2+ signalling, and the conventional agonist compound 43 (Qin et al., 2017).We have previously shown that compound 17b, but not compound 43 elicits vasodilation in mouse aorta and reduces vascular remodelling in models of cardiovascular pathologies including myocardial infarction and diabetes (Marshall et al., 2020;Qin et al., 2017).These protective actions were attributed to compound 17b signalling bias away from pro-contractile and pro-inflammatory mechanisms downstream of

What is already known
• Vasodilator treatments for pulmonary arterial hypertension (PAH) have limited long-term efficacy.
• Current PAH therapies do not target inflammation or vascular remodelling.

What does this study add
• Mouse precision-cut lung slices were used to benchmark FPR agonists against standard-of-care vasodilators.
• FPR biased agonist compound 17b was identified as a vasodilator and anti-inflammatory in intrapulmonary arteries.

What is the clinical significance
• Compound 17b has been identified as a potential alternative or adjunct therapy for PAH.
• FPR-based pharmacotherapy may be a novel strategy to treat devastating and fatal consequences of PAH.Studies of distal pulmonary arteries, the primary site of remodelling and elevated PAP in PAH, are difficult due to their relative inaccessibility and small size.The precision-cut lung slice (PCLS) technique is an underutilised methodology for the integrated assessment of intrapulmonary artery reactivity and inflammatory mediator release from resident cells of the lung.PCLS are thin lung slices containing viable arteries, in which changes in area can be visualised under phase-contrast microscopy.Critically, PCLS preparations maintain the inter-dependency of these pulmonary arteries within the parenchyma and the diversity of pulmonary cell types, enabling assessment of cellular and functional responses.To date, studies using PCLS have focussed on contractile responses of pulmonary arteries to endogenous modulators such as 5-hydroxytryptamine (5-HT; serotonin) and acetylcholine (ACh) (Perez & Sanderson, 2005;Wright & Churg, 2008), with assessment of PAH treatments limited to endothelinreceptor antagonists (Martin et al., 2000).
Here, PCLS from naïve mice were used to investigate FPRs as a therapeutic target for PAH, by comparing the vasodilator potential of compound 17b and compound 43 with standard-of-care PAH treatments.In addition, PCLS were treated with tumour necrosis factor-α (TNF-α) or lipopolysaccharide (LPS), to assess whether dilator efficacy was maintained under simulated inflammatory conditions and to determine whether FPR agonists also inhibited cytokine release.

| Animals
All experimental procedures in animals were approved by the Animal Ethics Committee of Monash University (Monash Animal Research Platform Approval #27155) and were performed in compliance with Victorian Legislation.Animal studies are reported in compliance with the ARRIVE guidelines (Percie du Sert et al., 2020) and with the recommendations made by the British Journal of Pharmacology (Lilley et al., 2020).Eight-week-old male C57BL/6 mice were housed under a normal 12-h light:12-h dark schedule and fed a standard chow diet with free access to food and water.

| Precision-cut lung slices (PCLS) preparation
Mice were killed via intraperitoneal injection of sodium pentobarbitone (60 mgÁml À1 ) with a 26-G needle.PCLS were prepared as previously described (Perez & Sanderson, 2005).Briefly, pulmonary arteries were perfused via the right ventricle with 8% gelatin, followed by inflation of the lungs through the trachea with 2% agarose.Lungs were removed and sliced on a vibratome (Leica VT1200S, Leica Biosystems, Germany) to a thickness of 200-250 μm for vascular reactivity, 400 μm for cytokine release studies or 500 μm for immunohistochemistry.These thicknesses used for each set of experiments were optimised for visualising arteries in vascular reactivity studies and to maximise cytokine detection.

| Immunohistochemistry
PCLS were fixed in neutral buffered formalin for 18 h, embedded in paraffin and sectioned to a thickness of 4 μm.Sections were dewaxed with xylene and endogenous peroxidase activity was quenched by 3% hydrogen peroxide for 10 min.Antigen retrieval was performed by heating sections in citrate buffer for 5 min and then the samples were cooled for 25 min.Blocking was performed using BSA.Immunohistochemistry was performed using primary antibodies against FPR1 (1:300 dilution) or FPR2 (1:16,000 dilution), for 18 h.Sections were then treated with anti-rabbit secondary antibody conjugated to HRP, followed by 5 min of treatment with diaminobenzidine.Mouse kidney was used as a positive control.Images were counterstained with haematoxylin, mounted and imaged by the Monash Histology Platform.The Immuno-related procedures used comply with the recommendations made by the British Journal of Pharmacology (Alexander et al., 2018).

| Vascular reactivity
PCLS were mounted in a perfusion chamber ($100 μl volume), consisting of a microscope slide on a custom-made Perspex platform and a coverslip separated by parallel lines of silicone gel to create an open chamber for drug perfusion.Fine wire mesh was used to mount the tissue in place, with a hole cut over the artery to enable vascular responses to be visualised.Intrapulmonary arteries were distinguished from veins by their close proximity to airways and comparatively thicker smooth muscle layer.Drugs were perfused via an eightchannel gravity-fed perfusion system (Warner Instruments Inc., USA), limiting the number of different concentrations able to be tested to 6. Drugs were delivered at a constant rate, with solutions removed from the chamber under vacuum (Fluro Mechanic, Tokyo, Japan).
Arteries within PCLS were selected based on size (<150 μm in diameter) and visual inspection of an intact muscular layer connected to surrounding parenchyma.In addition, viability was established based on marked reduction in artery area to a test concentration of 3 μM of 5-HT.Arteries were visualised under phase-contrast microscopy (Nikon Eclipse Ti-U [Nikon Instruments Inc., Melville, NY, USA]; Pulnix CCD camera model TM-62EX [Jai, Miyazaki, Japan]) and images were captured every 2 s in a 744 Â 572-pixel frame using image analysis software Video Savant (IO Industries, Inc., London, ON, Canada).
Images were exported as TIFF files and lumen area was calculated on ImageJ 1.53a (National Institutes of Health, USA) via the Analyse Particles tool or manually traced for the last five frames of each treatment where sufficient contrast was not achieved.

| Statistical analysis
The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2018(Curtis et al., , 2022)).Our previous data show percentage maximum relaxation for sildenafil in mouse PCLS of 90% (SD 9%), requiring a minimum of n = 6 per group to detect a 15% difference with different drugs or treatments, based on a twosided t-test where α = 0.05.Different n values for data presented in separate figures reflect differences in the number of PCLS with functional arteries prepared from individual mice, resulting in group sizes between n = 6 and 9.The testing of different drugs in multiple PCLS prepared from each mouse was random.Analysis of changes in area was not blinded but was performed using an objective method, namely, computer-based pixel counting.The area of the artery in pixels during perfusion with buffer alone was defined as 100% area for normalisation of pre-contraction responses to 5-HT and for presentation of experimental traces.Percentage relaxation to dilators was calculated based on the percentage reduction in area from the stable pre-contraction.pEC 50 (negative log value of half the maximum effective concentration) and fitted maximum response from concentration-response experiments were generated by non-linear curve-fitting on Prism, with curve-fitting for dilator curves constrained to a maximum of 100% relaxation.All data were subjected to the Shapiro-Wilk test to confirm normality.Comparisons were performed using independent-measures or repeated-measures one-way analysis of variance (ANOVA) with Bonferroni's correction for multiple comparisons or unpaired Student's t test where applicable (α = 0.05).Post hoc tests were only performed if the F value of the one-way ANOVA achieved P < 0.05 the necessary level of statistical significance.Group sizes were all n ≥ 5, where n is equal to the number of independent values, with all data included in statistical analyses.P values less than 0.05 (P < 0.05) were considered significant.Technical replicates for duplicate ELISA samples were averaged before analysis as independent values.Graphing and statistical analysis were performed in Prism 9.4.0 (GraphPad, San Diego, USA).

| FPR1 and FPR2 are expressed in mouse lung
Immunostaining for FPR1 and FPR2 was performed on lung sections prepared from fixed PCLS.Extensive expression of both FPR1 and FPR2 was evident in the pulmonary arteries (Figure 1).In addition, FPR1 and FPR2 staining was present in the adjacent airways and in the parenchyma.Specificity of staining was confirmed using negative controls in the absence of primary antibody, while strong staining for both receptors was evident in mouse kidney, used as a positive control (Figure S1).

| FPR agonists are effective vasodilators of pulmonary arteries in mouse PCLS
To assess dilator responses in PCLS, concentration-response curves were generated in pulmonary arteries pre-contracted with 5-HT.Representative images show (1) initial lumen area during perfusion with buffer alone, (2) pre-contraction with 3 μM of 5-HT and (3) complete relaxation to 30 μM of compound 17b, the highest concentration tested (Figure 2a).An experimental trace shows the changes in artery area under these conditions (1-3), with concentration-dependent and near-complete relaxation to compound 17b (Figure 2b).This was similar in PCLS from both male and female mice (Figure 2c and Table 1).
Dilator responses to compound 17b were then compared with compound 43 and with other dilators in clinical use for PAH.Overall, the diameters of small pulmonary arteries assessed were well matched between groups (Table 1).The reduction in lumen area during 5-HT pre-contraction was greater than 10% in all arteries tested, with no difference in average pre-contraction before assessment of each dilator (Table 1) and stable for the duration of a similar protocol in the absence of dilators (data not shown).While concentration-dependent vasodilation was evident in response to both FPR agonists, the potency of compound 17b was over fivefold higher than compound 43 (Figure 2d and Table 1).Compared with standard-of-care dilator therapies for PAH, compound 17b showed sevenfold more potent dilation than sildenafil and similar potency to riociguat (Table 1).Iloprost, the PGI 2 analogue, was over 170-fold more potent than compound 17b (Table 1).Fitted maxima were not significantly different between dilators (Table 1).

| Pulmonary artery vasodilation to compound 17b is FPR1 dependent
To assess the receptor dependence of compound 17b-mediated vasodilation, PCLS were pre-treated with FPR1-(cyclosporin H) or FPR2-(quin-C7) selective antagonists.Representative images show precontraction with 5-HT, followed by the dilator response to 30 μM of compound 17b in the absence or presence of cyclosporin H, reduced from 90% to 12% relaxation (Figure 3a).The near-complete relaxation to 30 μM of compound 17b was 89 ± 7% in the vehicle control group (Figure 3b).Cyclosporin H significantly decreased the maximum dilation to compound 17b by more than 60% (to 27 ± 5%; Figure 3a,b).In contrast, the dilator response to compound 17b was maintained in the presence of quin-C7 (Figure 3b).To confirm that the inhibitory effects of cyclosporin H on compound 17b were selective, dilation to 30 μM of sildenafil was compared in the absence and presence of cyclosporin H. Cyclosporin H had no significant effect on sildenafilmediated vasodilation (Figure S2). at >85% with inhibition of sGC (with ODQ) (Figure 3c).As a positive control, complete relaxation to the sGC stimulator riociguat was significantly inhibited by $25% in the presence of ODQ (Figure S3).Relaxation to compound 17b was also maintained in the presence of inhibitors of eNOS (L-NAME) and COX (indomethacin).3.5 | Compound 17b -mediated vasodilation is independent of Ca 2+ -activated and ATP-dependent K + channels To determine whether compound 17b-mediated vasodilation was dependent on potassium channel activation, PCLS were treated with either the K + channel inhibitor TEA (Ca 2+ activated, 10 mM) or glibenclamide (ATP dependent, 10 μM).In PCLS pre-contracted with 5-HT, 30 μM of compound 17b induced complete relaxation of pulmonary arteries and this was not affected by treatment with either K + channel inhibitor (Figure 3d).

| Vasodilation to Compound 17b is maintained under inflammatory conditions in PCLS
To determine whether inflammatory conditions alter compound 17bmediated vasodilation, PCLS were cultured in the absence or presence of TNF-α or LPS for 24 h.PCLS were then perfused with increasing half-log molar concentrations of compound 17b.There were no significant differences in 5-HT-mediated pre-contraction between treatment groups (Table 2).Compound 17b potency and efficacy were maintained in both TNF-α-and LPS-treated PCLS (Figure 4a and Table 2).Although the potency of both sildenafil and iloprost was maintained in LPS-treated PCLS, their fitted maxima were reduced by 33% and 27%, respectively (Figure 4b,c and Table 2).

| DISCUSSION AND CONCLUSIONS
This study, for the first time, has identified a novel protective role for the FPR agonist compound 17b in opposing both pulmonary artery contraction and inflammation in naïve mouse PCLS.The FPR1-dependent acute vasodilator effects of the biased agonist compound 17b were shown to be greater than the conventional agonist compound 43.Of note, the potency of compound 17b was greater than that of standard-of-care for PAH, sildenafil, similar to riociguat but less than iloprost.In addition, vasodilation to compound 17b but not sildenafil or iloprost was maintained in PCLS under disease-relevant inflammatory conditions.Furthermore, compound 17b also attenuated secretion of key inflammatory cytokines associated with PAH.As such, these findings support the hypothesis that compound 17b would induce vasodilator and anti-inflammatory effects, and thus may offer T A B L E 2 Descriptive statistics of pre-contraction to 5-HT (percentage reduction in artery area) and maximum relaxation and pEC 50 from fitted individual concentration-response curves to each dilator in control, TNF-α-treated or LPS-treated PCLS.We then established the relative vasodilator potencies and efficacies of the two FPR agonists in the pulmonary vasculature using PCLS and benchmarked with standard-of-care sildenafil, riociguat and iloprost, as vasodilators that are currently in use for the treatment of PAH.This is the first time that such a comparison has been made using PCLS, an innovative method that permits visualisation of intrapulmonary artery contraction in situ.Compared with studies in isolated large pulmonary arteries, dilation in PCLS integrates the effects of surrounding pulmonary structures, similarly to an in vivo setting (Liu et al., 2019).
We have previously demonstrated that compound 17b (but not compound 43) elicited relaxation in U46619-pre-contracted mouse aortic rings (Marshall et al., 2020).However, both FPR agonists relaxed the pulmonary arteries with compound 17b being fivefold more potent than compound 43.This difference in potency may relate to differences in the signalling downstream of FPR binding.Compound 43 activates pro-contractile, pro-inflammatory intracellular calcium (Ca 2+ i ) mobilisation equipotently to each of Akt and ERK1/ERK2 phosphorylation, in engineered Chinese hamster ovary cells expressing human FPR2.In contrast, compound 17b is only a weak activator of Ca 2+ i downstream of hFPR2 (Qin et al., 2017).Both compound 17b and compound 43 are non-selective FPR1/2 agonists.Similar observations of compound 17b bias away from Ca 2+ i relative to the impact of compound 43 at hFPR1 were also observed.These data suggest that compound 17b is more likely than compound 43 to favour the vasodilator actions of FPRs.Of note is the fact that compound 43 induced concentration-dependent dilation in pulmonary arteries when measuring changes in area in situ in PCLS, but not when measuring changes in force of isolated aorta in myograph studies (Marshall et al., 2020).This discrepancy may be due to use of different agonists for precontraction (U46619 vs 5-HT) or differences between FPR expression and FPR-mediated signalling pathways between systemic and pulmonary arteries.
Further, vasodilation to compound 17b was shown to be FPR1 dependent on the basis of the inhibitory effects of the FPR1-selective antagonist cyclosporin H (Qin et al., 2013), but not the FPR2-selective quin-C7 (Stenfeldt et al., 2007).This is consistent with FPR1 (rather than FPR2) as the receptor subtype responsible for cardiac lusitropic effects of the endogenous FPR agonist Ac2-26 [annexin1-2(26)] (Qin et al., 2013).Ideally, these findings should be confirmed using additional antagonists such as the non-selective WRW4 (Seo et al., 1997) or using PCLS from FPR1 or FPR2 gene knockout mice.
We found that inhibitors of eNOS, sGC and COX did not reduce vasodilation to a single concentration of compound 17b, suggesting that the FPR agonist was not eliciting release of endogenous eNOS or COX-derived mediators such as NO or PGI 2 .According to the present study, there is little support for compound 17b-mediated vasodilation being dependent on the NO/sGC/cGMP or PGI 2 /cAMP signalling pathways.However, dilator responses in PCLS were assessed under perfused conditions, so any potential contributions of accumulated endothelium-derived relaxing factors to compound 17b-mediated vasodilation may not have been detected.Additionally, relaxation to compound 17b does not appear to be dependent on either ATPdependent or Ca 2+ -activated K + channels, based on maintained relaxation in the presence of glibenclamide or TEA.These findings are consistent with the lack of effect of glibenclamide on compound 17b-F I G U R E 6 Receptor dependence of compound 17b (17b)-mediated inhibition and effect of sildenafil (Sil) and iloprost (Ilo) on interleukin-6 (IL-6) secretion from mouse precision-cut lung slices (PCLS).PCLS were cotreated with tumour necrosis factor-α (TNF-α) (10 ngÁml À1 ) for 24 h and vehicle (Veh) control (1% dimethyl sulfoxide), 17b (10 μM), cyclosporin H (CsH) (1 μM), quin-C7 (Q-C7) (10 μM), compound 43 (43) (30 μM), Sil (10 μM) or Ilo (0.1 μM).IL-6 concentration in media from TNFα-treated PCLS was measured by ELISA (all n = 6).mediated relaxation in aorta (Marshall et al., 2020).Thus, the mechanism may be via direct inhibition of Ca 2+ -mediated contraction (Marshall et al., 2020), highlighting a potential avenue for future investigation.
Therapies for PAH include the sGC stimulator riociguat, the PDE5 inhibitor sildenafil and the potent PGI 2 analogue iloprost (Humbert et al., 2023).Although they have been shown to acutely relieve PAP, there is limited evidence of any additional anti-inflammatory actions in a clinical setting.To date, these standard-of-care dilators have not been directly compared using PCLS, which provide a platform for assessment of the reactivity of small intrapulmonary arteries and their potential contribution to PAH.Consistent with its reported effects on large pulmonary arteries (Kozlowska et al., 2013), the PGI 2 analogue iloprost, mediating cAMP-dependent vasodilation, was a potent dilator in mouse PCLS.Riociguat, which increases cGMP synthesis, was in turn more potent than sildenafil, which only inhibits cGMP metabolism.In the current study, the sGC inhibitor ODQ only partially inhibited the relaxation to a maximally effective concentration of riociguat.A higher concentration of ODQ should be tested to confirm that sGC activation is the sole mechanism of action of riociguat in this preparation.Sildenafil's dilator actions are therefore dependent on basal cGMP production and would be enhanced by diffusion of endogenous NO to the smooth muscle cells to further increase cGMP (Toxvig et al., 2019).
The finding that the PGI 2 analogue iloprost was more potent than the other dilators including the sGC activator riociguat in mouse intrapulmonary airways is consistent with previous findings in larger pulmonary arteries (Chamorro et al., 2018;Jain et al., 2014;Y. Li et al., 2012;Mondéjar-Parreño et al., 2019).However, the absolute potency for iloprost was greater in PCLS than isolated larger airways and, conversely, relatively lower for riociguat in PCLS.These discrepancies may reflect the use of different agonists to pre-contract the airways before testing the same agonist.It is acknowledged that the different methods used, whereby arteries in PCLS are not held under constant force or pressure as in a myograph but are still subject to the tethering forces of the surrounding tissue, may result in different contraction and relaxation responses.It is also possible that the expression of IP/EP receptors mediating relaxation to iloprost is higher and sGC/cGMP signalling activated by riociguat is lower in smaller airways.
Having established compound 17b as an effective dilator of mouse intrapulmonary arteries, we tested the influence of inflammation on responsiveness to compound 17b, sildenafil and iloprost.TNFα is increased in PAH and in vitro TNF-α treatment increases contractile responses of human pulmonary arteries (Hiram et al., 2015).In addition, TNF-α decreased NO production by destabilising eNOS mRNA in microvascular endothelial cells and decreased endotheliumdependent relaxation to ACh in guinea-pig intrapulmonary arteries under static conditions in PCLS (Neumann et al., 2004;Wright & Churg, 2008).LPS was used as a broad inflammatory stimulus.In human Beas2B lung epithelial cells, FPR1 expression is increased by both TNF-α and LPS (Shao et al., 2011), and LPS increases expression of FPR1 in mouse brain (Calvello et al., 2021).These studies suggest that FPR1-mediated dilation may be maintained or even increased in the presence of these inflammatory stimuli.
Both LPS and TNF-α induced cytokine release from PCLS but did not increase pre-contraction to 5-HT in PCLS.Relaxation to compound 17b, but not sildenafil or iloprost, was maintained under these inflammatory conditions.The inhibitory effects of LPS on sildenafil in mouse PCLS are consistent with impaired relaxation to other vasodilators in LPS-treated rat large pulmonary arteries (El-Awady et al., 2008).The loss of efficacy of the NO donor S-nitroso-N-acetylpenicillamine (SNAP) and the sGC activator BAY412272 after LPS treatment of pulmonary arteries was associated with increased PDE5 activity (El-Awady et al., 2008).In addition, LPS also attenuates iloprost-mediated vasodilation in mammary arteries due to increased PDE4 activity (Foudi et al., 2017).FPR agonists may therefore act as a more effective therapeutic option than sildenafil and iloprost due to maintained dilator actions in inflamed tissue, where the contribution of cAMP and cGMP to vasodilation may be compromised.
The role of FPR agonists in the resolution of inflammation is well established in cardiovascular and respiratory diseases (Filep et al., 2018).The ex vivo setting used for the current study, in which compound 17b was added to mouse PCLS at the same time as LPS or TNF-α, precluded investigation of whether compound 17b would resolve established inflammation.However, this study identified antiinflammatory effects of treatment with compound 17b, as it inhibited TNF-α-induced secretion of IL-6, KC (CXCL2; a mouse IL-8 analogue) and MCP-1 (CCL2), as well as LPS-induced secretion of TNF-α, but not IL-6, from mouse PCLS.The differences between the effects of compound 17b on IL-6 production depending on the stimulus used suggest that LPS indirectly drives IL-6 production by increasing TNF-α (sensitive to inhibition by compound 17b) and by additional direct or indirect pathway(s) (insensitive to compound 17b).Additionally, the anti-inflammatory effect of compound 17b from PCLS was FPR2 dependent, as inhibition of TNF-α-induced IL-6 secretion from PCLS by compound 17b was prevented by quin-C7, but not cyclosporin H.This novel result thus suggests that each of the established dual actions of compound 17b is dependent on different receptors, as vasodilation appears to be FPR1 dependent.
The cytokines chosen for this study are increased in PAH (Groth et al., 2014).IL-6 is one of the most important cytokines in PAH and may promote disease progression by increasing smooth muscle cell proliferation in small pulmonary arteries (Steiner et al., 2009).IL-8 and MCP-1(CCL2) also promote proliferation and enhance immune cell migration to the lung, contributing to PAH (Itoh et al., 2006;A. Li et al., 2005).These findings suggest that compound 17b may inhibit nuclear factor-κB (NF-κB)-mediated transcription of at least some of these cytokines downstream of LPS or TNF-α (Park et al., 2015), though the mechanism of compound 17b-mediated inhibition for each may differ depending on the stimulus.Future experiments should investigate the effects of compound 17b on other profibrotic and proliferative factors, such as transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) (Selimovic et al., 2009), as these factors are implicated in vascular remodelling that is resistant to current treatments for PAH.

FPR
, with biased agonist, simultaneously targets vascular function and inflammation, supporting the development of FPR-based pharmacotherapy to treat PAH.LINKED ARTICLES: This article is part of a themed issue Therapeutic Targeting of G Protein-Coupled Receptors: hot topics from the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists 2021 Virtual Annual Scientific Meeting.To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.14/issuetocK E Y W O R D S formyl peptide receptor, inflammation, precision-cut lung slice, pulmonary arterial hypertension, respiratory pharmacology, vasodilation 1 | INTRODUCTION FPR activation.The regulation of pulmonary artery contraction or inflammatory responses in the distal lung relevant to PAH by these FPR agonists have not yet been investigated.

2. 8 |
Nomenclature of targets and ligandsKey protein targets and ligands in this article are hyperlinked to corresponding entries in the IUPHAR/BPS Guide to PHARMACOLOGY http://www.guidetopharmacology.org and are permanently archived in the Concise Guide to PHARMACOLOGY 2021/22(Alexander et al., 2021).

3. 4 |
Compound 17b-mediated vasodilation is independent of sGC, eNOS and COXTo determine whether b-mediated vasodilation was dependent on sGC signalling or on the production of eNOS-or COX-derived mediators, PCLS were pre-treated with selective inhibitors of these enzymes.In arteries pre-contracted with 5-HT, compound 17b caused near-complete relaxation in the vehicle control group (Figure3c).Compound 17b-mediated vasodilation was maintained F I G U R E 1 Formyl peptide receptor 1 (FPR1) (a) and formyl peptide receptor 2 (FPR2) (b) are expressed in mouse lung.Formalin-fixed and paraffin-embedded precision-cut lung slices were stained with primary antibody for FPR1 or FPR2 and then anti-rabbit secondary antibody conjugated to horseradish peroxidase.Sections were treated with diaminobenzidine and then counterstained with haematoxylin.Scale bar represents 50 μm.Arrow indicates artery wall.F I G U R E 2 Vasodilator responses to formyl peptide receptor (FPR) agonists, compound 17b (Cmpd17b) and compound 43 (Cmpd43), and standard-of-care vasodilators sildenafil, riociguat and iloprost.Precision-cut lung slices (PCLS) from naïve 8-week-old C57BL/6 mice were precontracted for 10 min with 5-hydroxytryptamine (5-HT) (3 μM), before addition of increasing concentrations of Cmpd17b (n = 9), Cmpd43, sildenafil, riociguat and iloprost (all n = 6) every 5 min.Images were recorded every 2 s and lumen area from the last five frames of each concentration was averaged.(a) Representative images showing (1) initial lumen area during perfusion with buffer alone, (2) pre-contraction with 5-HT (3 μM) and (3) complete relaxation to Cmpd17b (30 μM).Scale bar represents 50 μm.(b) Representative trace showing concentrationdependent relaxation to Cmpd17b, with numbers corresponding to images in (a).Artery lumen area is expressed as a percentage of the initial area, calculated from phase-contrast images captured every 2 s.(c) Concentration-response curves to Cmpd17b in naïve male and female mice (n = 6).(d) Concentration-response curves to Cmpd17b, sildenafil, Cmpd43, riociguat and iloprost in male mouse PCLS.Data are expressed as percentage relaxation of the reduction in area after 5-HT precontraction.Data are presented as mean ± SEM, where each n represents results from a single PCLS from a different mouse.
greater potential benefit in PAH in comparison with current therapies that only cause vasodilatation.This study also supports the use of PCLS as a valuable tool for assessing ex vivo vasodilation and inflammatory responses, as well as GPCR-mediated responses in general.The primary aim of this study was to investigate the direct vascular effects of two small-molecule FPR agonists in the pulmonary vasculature using PCLS, namely, the conventional agonist compound 43 and the biased agonist compound 17b.First, we localised FPR1 and FPR2 receptors in vascular smooth muscle and endothelium of mouse intrapulmonary arteries using immunohistochemistry, consistent with their potential roles in regulating vascular function and inflammatory responses.
(a) Receptor dependence of 17b-mediated IL-6 inhibition and (b) effects of Sil and Ilo on IL-6 secretion.Data are presented as mean ± SEM.Repeated-measures one-way analysis of variance with Bonferroni's multiple comparisons.*P < 0.05 compared with untreated, # P < 0.05 compared with TNF-α treated, ^P < 0.05 compared with TNF-α and 17b treated.
Testing the inhibitory effects of overnight treatment with compound 17b more closely mimics the application of chronic vasodilator therapy for PAH.While we have established its anti-inflammatory effects in this setting, additional experiments are required to also assess the effects of compound 17b on the development of contraction, rather than dilation of a pre-contracted artery, to reflect potential for sustained lowering of PAP.Future studies assessing compound 17b and other FPR agonists for PAH should further investigate their mechanisms and validate vasodilator and anti-inflammatory efficacy in both human tissues and mouse models of PAH where there is increased sensitivity to constrictors associated with vascular remodelling.These findings show promising dual actions of the FPR agonist compound 17b.However, its relatively low vasodilator potency may be a barrier to clinical translation for PAH, as the high concentrations required may reach the limits of its solubility.There remains a major need to develop new synthetic FPR agonists with improved solubility compared with compound 17b, but with comparable or greater potency in vitro.This study firstly highlights the use of PCLS as an innovative technology to investigate GPCR-mediated intrapulmonary artery responses in situ.Overall, the authors have have performed the first integrated assessment of current and novel vasodilators for PAH using mouse PCLS.This research has established dual vasodilator and anti-inflammatory effects of the small-molecule biased FPR agonist compound 17b that distinguish it from standard-of-care dilators.Hence, this study provides the first preclinical evidence of benefit in targeting FPRs in the lung, supporting the further development of FPR-based pharmacotherapy for PAH.AUTHOR CONTRIBUTIONS W. R. Studley: Formal analysis; investigation; methodology; validation; visualization; writing-original draft; writing-review and editing.E. Lamanna: Investigation; methodology; resources; validation; writingoriginal draft; writing-review and editing.K. A. Martin: Investigation; methodology; validation; writing-original draft; writing-review and editing.C. A. Nold-Petry: Supervision; writing-review and editing.S. G. Royce: Investigation; methodology; validation; visualization; writing-original draft; writing-review and editing.O. L. Woodman: Funding acquisition; writing-review and editing.R. H. Ritchie: Conceptualization; funding acquisition; writing-review and editing.C. X. Qin: Conceptualization; formal analysis; funding acquisition; project administration; supervision; writing-original draft; writingreview and editing.J. E. Bourke: Conceptualization; formal analysis; funding acquisition; methodology; project administration; supervision; writing-original draft; writing-review and editing.