The Role of Chemokines and Chemokine Receptors in Pulmonary Arterial Hypertension

Received: 17 May 2019 Revised: 25 July 2019 Accepted: 26 July 2019 DOI: 10.1111/bph.14826

REVIEW ARTICLE THEMED ISSUE BJP

The role of chemokines and chemokine receptors in pulmonary arterial hypertension

Argen Mamazhakypov1* | Gayathri Viswanathan2* | Allan Lawrie3 | Ralph Theo Schermuly1 | Sudarshan Rajagopal2

1 Department of Internal Medicine, Member of the German Center for Lung Research (DZL), Pulmonary arterial hypertension (PAH) is characterized by progressive pulmonary Justus Liebig University of Giessen, Giessen, artery remodelling leading to increased right ventricular pressure overload, which Germany 2 Division of Cardiology, Department of results in right heart failure and premature death. Inflammation plays a central role Medicine, Duke University Medical Center, in the development of PAH, and the recruitment and function of immune cells are Durham, NC, USA tightly regulated by chemotactic cytokines called chemokines. A number of studies 3 Department of Infection, Immunity & Cardiovascular Disease, University of Sheffield, have shown that the development and progression of PAH are associated with the Sheffield, UK dysregulated expression of several chemokines and chemokine receptors in the

Correspondence pulmonary vasculature. Moreover, some chemokines are differentially regulated in Sudarshan Rajagopal, Division of Cardiology, the pressure‐overloaded right ventricle. Recent studies have tested the efficacy of Department of Medicine, Duke University Medical Center, Box 3126, Durham, NC pharmacological agents targeting several chemokines and chemokine receptors for 27710, USA. their effects on the development of PAH, suggesting that these receptors could serve Email: [email protected] as useful therapeutic targets. In this review, we provide recent insights into the role of Funding information chemokines and chemokine receptors in PAH and RV remodelling and the opportuni- Deutsche Forschungsgemeinschaft, Grant/ Award Number: SFB1213; British Heart Foun- ties and roadblocks in targeting them. dation, Grant/Award Number: FS/18/52/ 33808; American Heart Association, Grant/ Award Number: 33670458

Abbreviations: 5‐LOX, 5‐lipoxygenase; 6MWD, six‐minute walking distance; AC, adenylate cyclase; ACKR, atypical chemokine receptor family; ADAM10, a disintegrin and metalloproteinase domain‐containing protein 10; ADAM17, ADAM metallopeptidase domain 17; AECA, anti‐endothelial cell antibodies; ALK, activin receptor–like kinase; AMD3100, AnorMeD CXCR4 antagonist; APAH, associated PAH; BMP, bone morphogenetic protein; BMPR2, bone morphogenetic protein receptor type II; BMSCs, bone marrow stromal cells; CD, cluster of differentiation; CHD‐PAH, pulmonary arterial hypertension due to congenital heart disease; CI, cardiac index; CO, cardiac output; COPD, chronic obstructive pulmonary diseases; COPD‐PH, pulmonary hypertension associated with COPD; CRP, C‐reactive protein; CSE, cigarette smoke extract; CTEPH, chronic thromboembolic pulmonary hypertension; DCs, dendritic cells; EC, endothelial cells; ECM, extracellular matrix; EPAC, guanine nucleotide exchange factor activated by cAMP; ET‐1, endothelin‐1; F1, CX3CR1 antagonist; GAGs, glycosaminoglycans; GATA‐6,

GATA‐binding protein 6; GRK, G protein receptor kinases; Gαgust, α‐gustducin; Gαt, α‐transducin; HIMF, HOX mitogenic factor; HLMCs, human lung mast cells; HMGB1 high mobility group box protein 1HOX, hypoxia; ICAM‐1, intercellular adhesion molecule 1; IP3, inositol 1,4,5‐trisphosphate; IPAH, idiopathic pulmonary arterial hypertension; IPF‐PH, pulmonary hypertension due to idiopathic pulmonary fibrosis; LCR1, C‐X‐C motif chemokine receptor 4; LV, left ventricle; MCT, monocrotaline; MCT/PE, MCT/pneumonectomy; mPAP, mean pulmonary artery pressure; NOX5, NADPH oxidase 5; NT‐proBNP, N‐terminal pro‐brain natriuretic peptide; PA, pulmonary artery; PAB, pulmonary artery banding; PAECs, pulmonary artery endothelial cells; PAFB, PA fibroblasts; PAH, pulmonary arterial hypertension;; PAP, pulmonary artery pressure; PASMC, pulmonary artery smooth muscle cells; PBSF, pre‐B‐cell‐growth‐stimulating factor; PE, pulmonary embolism; PEA, pulmonary endarterectomy; PH, pulmonary hypertension; PHD2, prolyl hydroxylase domain‐containing protein 2; PIP2, phosphatidylinositol 4,5‐bisphosphate; PMNs, polymorphonuclear leukocytes; PP2A, protein phosphatase 2; PPAH, primary pulmonary arterial hypertension; PTX, Pertussis toxin; PVR, pulmonary vascular resistance; RAP, Ras‐related proteins; ROCK, Rho‐associated PK; RV, right ventricle; RVEDD, right ventricular end diastolic dimension; RVEF, right ventricular ejection fraction; RVSP, right ventricular systolic pressure; S, septum; SB 225002, selective non‐peptide inhibitor of CXCR2; SCH527123, selective CXCR2 antagonist; SDF, stromal cell‐derived factor; SDF‐1α NP, stromal cell‐derived factor‐1α nanoparticle; shRNA, small hairpin RNA; siRNA, small interfering RNA; SMAD1/5 and SMAD2/3, SMAD family member; sPAH, secondary pulmonary arterial hypertension; sPAP, systolic pulmonary artery pressure; SpO2, oxygen saturation; SSc, systemic sclerosis; SSc‐PAH, pulmonary arterial hypertension associated with SSc; SuHx, Sugen/hypoxia‐exposed; SvO2, saturation of mixed venous oxygen; TAPSE, tricuspid annulus plane systolic excursion; TNFR1, TNF receptor 1; VSMC, vascular smooth muscle cell; WHO FC, World Health Organization functional classification; WT, wild type; XCL, X‐C motif chemokine ligand; XCR, X‐C motif chemokine receptor

*Argen Mamazhakypov and Gayathri Viswanathan have contributed equally to this work.

1 | INTRODUCTION monomers, they can also form homodimers, tetramers, or polymers, with further oligomerization in the presence of glycosaminoglycans Pulmonary arterial hypertension (PAH) is a disease of the pulmonary (GAGs), which may increase their activity (Lacalle et al., 2017). In vasculature characterized by excessive proliferation of pulmonary addition, heterodimers may form between two members of the same arterial (PA) cells, increased extracellular matrix deposition and chemokine family or different chemokine families (CC and CXC accumulation of inflammatory cells within the PA wall resulting in chemokines; Lacalle et al., 2017). In addition, chemokines may act as increased pulmonary vascular resistance (Schermuly, Ghofrani, Wilkins, agonists of some receptors while also acting as antagonists at other & Grimminger, 2011). Despite extensive studies, the exact aetiological receptors (Loetscher et al., 2001; Weng et al., 1998). All chemokines factors and mechanisms leading to disease development and progres- are secreted soluble proteins, except CXCL16 and CX3CL1, which sion are multifactorial and many remain unidentified (Humbert et al., are integral membrane proteins (Lacalle et al., 2017). After secretion, 2019). A number of dysregulated signalling pathways have been chemokines can bind to negatively charged GAGs attached to the observed to contribute to the pathogenesis of PAH and have led to endothelial cell surface or extracellular matrix (ECM), resulting in clinical therapies that target NO signalling and the endothelin and immobilization and concentration at the apical side of endothelial cells. prostacyclin receptor pathways (Galie et al., 2016). Other signalling This can result in a chemokine concentration gradient that promotes pathways have been targeted with pharmacological agents in preclini- immune and inflammatory cell recruitment to the endothelial cells lin- cal and early clinical trials to test their ability to reverse PA remodelling ing vessels and capillaries around the tissue‐source of chemokine syn- and improve RV adaptation to pressure overload (Sitbon et al., 2019). thesis (Lacalle et al., 2017). Recent studies have implicated dysregulated chemokine signalling as one mechanism underlying disease progression in several lung diseases 2.2 | Chemokine signal transduction including chronic obstructive pulmonary disease (COPD), asthma, and lung fibrosis (Tomankova, Kriegova, & Liu, 2015). Similarly, the link The major signalling pathways of chemokine receptors are presented between chemokine signalling and PAH is currently an area of exten- in Figure 1. Chemokine receptor signalling is primarily mediated by sive investigation. However, the exact mechanisms by which altered three groups of proteins that interact with the receptor: heterotrimeric chemokine signalling contribute to the pathogenesis of PAH remains G proteins, G protein receptor kinases (GRKs), and β‐arrestin adapter to be defined. In this review, we provide recent insights into the role proteins. Upon stimulation by a chemokine, the receptors activate of disturbed chemokine signalling in PAH and RV remodelling and heterotrimeric G proteins by catalysing the exchange of GDP for the opportunities and roadblocks in targeting them. GTP on the Gα subunit of the heterotrimeric G‐protein. This, in turn, leads to the dissociation of the heterotrimeric complex into Gα‐GTP and Gβγ subunits. The dissociated subunits promote signalling through 2 | CHEMOKINE SIGNALLING AND different pathways, with the Gα subunit regulating levels of a range of BIOLOGY second messengers. Based on sequence similarity and functions executed, the 16 2.1 | Chemokine biology Gα protein subunits are divided into four families: Gαs, Gαi, Gαq,

and Gα12. Each Gα subunit employs diverse signalling pathways

Chemokines, also known as chemotactic cytokines, are a group of over (Figure 1). Gαi family members include Gαi1,Gαi2,Gαi3, Go, transducin

40 small proteins that regulate cell migration and function. Despite (Gαt), and gustducin (Gαgust), which activate various signalling enzymes significant sequence diversity between chemokines, they display a such as phospholipases and PDEs, as well as mediating the opening of highly similar tertiary structure comprised of a three‐stranded β‐sheet several ion channels. The majority of chemokine receptors is coupled and a C‐terminal α‐helix. Chemokines are categorized by the pattern to the Gαi subfamily, which inhibit adenylate cyclase (AC) activity of cysteine residues in their N‐termini into four families: CXC (α and limit intracellular cAMP levels (Figure 1). Increased levels of chemokines), CC (β chemokines), XC (γ chemokines), and CX3C (δ intracellular cAMP activate several other effectors including PKA, chemokines). In addition, based on the expression status and functions guanine nucleotide exchange factor activated by cAMP (EPAC), and executed in healthy and disease states, chemokines are further classi- cyclic nucleotide‐gated channels. PKA is a crucial enzyme involved in fied into three groups: homeostatic chemokines with constitutive cell metabolism, cellular secretion, membrane permeability, and tran- expression, inflammatory chemokines with inducible expression, and scription of specific genes. Activated EPAC activates the small GTPase dual‐type chemokines having both features (homeostatic/inflamma- RAP proteins, which participate in cell growth and motility. All mem- tory or constitutive/inducible). Chemokines bind to over 20 chemo- bers of Gαi family can be irreversibly uncoupled from their receptors kine receptors, which are GPCRs. Chemokine receptors are largely by Pertussis toxin (PTX) and the fact that PTX causes strong impair- categorized into five families based on their activating chemokine ment of lymphocyte migration suggests that Gαi is mostly involved ligand (CXCR, CCR, CX3CR, or XCRs), with an additional group of in cell mobility processes (Lacalle et al., 2017). atypical chemokine receptors (ACKRs) that bind chemokines of Chemokine receptors can also couple to other Gα subunits. Gαs different families but primarily act as scavenger or decoy receptors. increases AC catalytic activity to produce cAMP by direct binding. Although chemokines may activate their specific receptors as Elevated levels of intracellular cAMP induces activation of cAMP‐ 3 MAMAZHAKYPOV ET AL. BJP

FIGURE 1 Chemokine signalling pathways. Chemokines bind to their cognate chemokine receptors expressed on different cell types. Upon receptor activation, the G protein dissociates into Gα and Gβγ subunits. Depending on the specific Gα subunit(s) to which the chemokine receptor is coupled, various downstream signalling pathways are regulated. Gαs protein activates adenylate cyclase (AC) to produce cAMP, which activates cAMP‐dependent PKA. Gαi/o inactivates AC limiting cAMP levels and PKA activity. Gαi/o proteins activate PI3Ks, which converts phosphatidylinositol 4,5‐bisphosphate (PIP2) to phosphatidylinositol (3,4,5)‐trisphosphate (PIP3), which in turn activates PKB (Akt). Gαi/o and Gα12 proteins activate Ras, which further activates various MAPKs. Gαi/o and Gαq proteins activate PLCβ, which catalyses PIP2 to IP3) and DAG. IP3 further regulates intracellular free calcium (Ca2+) levels, while DAG activates PKC. Gαq and Gα12 proteins also activates Rho family of GTPases (Rho), which further activates Rho‐associated PK (ROCK). GPCR kinases (GRK) phosphorylate GPCRs, which enable β‐arrestins to bind and internalize GPCRs, which can result in receptor recycling, if receptor phosphorylation is reversed by protein phosphatase 2 (PP2A) or degradation in lysosomes. β‐arrestin bound to GPCRs can also activate MAPK pathways such as JNK and ERKs 1 and 2 (ERK1/2). Such downstream signalling pathways regulate various cellular functions such as cell growth and differentiation, survival, apoptosis, migration, and chemotaxis. The figure was created using BioRender.com dependent kinases (PKA), which are involved in diverse biological func- signalling response to repeated stimulation over seconds to minutes, tions (Figure 1). The Gα12 subfamily consists of two α subunits, Gα12 and receptor internalization (Smith & Rajagopal, 2016). In addition, β‐ and Gα13.Gα12/13 is expressed constitutively in almost all tissues of arrestins also act as scaffolds for numerous signalling mediators the organism. The effects of Gα12/13 protein are primarily mediated including kinases and transcription factors (Smith & Rajagopal, through small GTP‐binding proteins of the Rho family, which con- 2016). These pathways can be both spatially and temporally tribute to diverse cellular functions (Ellis, 2004; Figure 1). Gαq family distinct from activation of those same pathways by heterotrimeric members consist of four subfamilies (Gq,G11,G14, and G15/16). G proteins. Gαq‐coupled receptors activate PLCβ, which converts phosphatidylinositol‐4,5,‐bisphosphate (PIP2) into inositol‐1,4,5‐trisphosphate (IP3) and DAG. These, in turn, increase intracellular free 3 | CHEMOKINES AS BIOMARKERS IN PAH calcium (Ca2+) levels and activate a number of PKs, including PKC, which phosphorylates several downstream effectors such as calmodu- The pulmonary circulation receives the entire cardiac output and pro- lin that regulate calcium‐induced signalling pathways. Gαq‐coupled vides gas exchange through its huge surface area contacting with the receptors can also activate MAPK in a PKC‐dependent and bloodstream. In addition, cells residing within the pulmonary vessels Ras‐independent manner depending on the cell type and the receptor produce various factors to maintain local homoeostasis by the expression levels. release of soluble factors that can be detected in the circulation. In After receptor activation, the receptor is phosphorylated by GRKs pathologies of the pulmonary vasculature, pulmonary arterial fibro- and other kinases on its cytoplasmic loops and C‐terminus, which blasts, endothelial and smooth muscle cells may display dysregulated enhance β‐arrestin binding to the receptor. β‐arrestins mediate synthesis of those factors, which can be detected in the circulation. receptor desensitization (Smith & Rajagopal, 2016), limiting the Accordingly, any changes to the levels of those factors may reflect 4 BJP MAMAZHAKYPOV ET AL. pathology of the pulmonary vasculature and disease severity. Based 4 | CHEMOKINE REGULATION OF on this concept, scientists and clinicians have been attempting to PULMONARY VASCULAR REMODELLING IN establish the best biomarkers to potentially diagnose, monitor, and PAH provide prognostic information on diseases such as PAH. However, so far, no biomarkers have been identified that provide information A number of chemokines and chemokine receptors have been shown on all those categories, and only a few biomarkers provide partial to play roles in inflammation and pulmonary vascular remodelling in information in PAH. Among other various circulating targets, PAH (Figure 2). chemokines also have emerged as potential biomarkers in PAH. For example, circulating levels of several chemokines are elevated in 4.1 | CXCL8/CXCR1/CXCR2 patients with different forms of pulmonary hypertension including idiopathic PAH (IPAH), PAH due to systemic sclerosis, PH due to The chemokine CXCL8, also known as IL8, is produced by various cell COPD and lung fibrosis, and chronic thromboembolic PAH (CTEPH) types including macrophages, epithelial cells, and airway smooth mus- (see below). cle cells. Several chemokine receptors are capable of binding CXCL8 Chemokine levels have been found to be correlated with disease including CXCR1 and CXCR2. In lung tissues of IPF patients with idio- severity and indices of pulmonary haemodynamics and right ventricu- pathic pulmonary fibrosis (IPF) and PH, CXCL8 is up‐regulated along lar function in multiple studies. For example, CCL2 and CXCL10 levels with CXCL1, compared to those without PH and controls (Bryant are associated with World Health Organization functional et al., 2018). IL‐4 induces CXCL8 expression in HPAECs via the classification (WHO FC; Hashimoto et al., 2004; Yang et al., 2014); ERK1/2 signalling pathway (Yang et al., 2011). CXCL8 production is CXCL10 levels are associated with NT‐proBNP (N‐terminal pro brain increased in both pulmonary artery smooth muscle cells (PASMCs) natriuretic peptide) levels (George et al., 2014; Yang et al., 2014); and pulmonary artery endothelial cells (PAECs) in response to TNF‐α CXCL10 levels are associated with six‐minute walking distance and IL‐1 stimulation (Lukacs et al., 1995). IL‐33 increases CXCL8 (6MWD; George et al., 2014; Zabini et al., 2014); CCL2, CXCL8, expression in HPAECs (Yagami et al., 2010). Hypoxia induces CXCL8 CXCL10, and CXCL13 levels are correlated with pulmonary vascular expression in PASMCs (Li et al., 2017), while only the combination of resistance (PVR; Damas et al., 2004; George et al., 2014; Kimura hypoxia and expression of high mobility group box protein 1 (HMGB1) et al., 2001; Olsson et al., 2016); CXCL10, CXCL12, and CXCL13 increases CXCL8 expression in PAECs (Li et al., 2017). PASMCs levels are associated with right atrial pressure (Olsson et al., 2016; increase CXCL8 expression in response to mechanical stress (Costa Yang et al., 2014); CXCL10, CXCL12 and CXCL16 have correlations et al., 2018). Broiler chickens, which develop spontaneous PAH with with tricuspid annulus plane systolic excursion (TAPSE; Yang et al., plexiform‐like lesions, display decreased expression of CXCL8 com- 2014); CXCL10 and CXCL12 levels are correlated with right ventricu- pared to layer chickens which are resistant to PAH (Tan, Shao, Fan, lar end‐diastolic diameter (RVEDD; Yang et al., 2014); CXCL13 levels & Ying, 2018). Consistent with a link to inflammation, NF‐κB induces are correlated with cardiac output (CO; Olsson et al., 2016); CXCL10 CXCL8 expression in PAECs via specifically binding to its promoter and CXCL12 correlated with cardiac index (CI; George et al., 2014; (Mumby et al., 2017). Endothelial‐to‐mesenchymal transition Olsson et al., 2016; Yang et al., 2014); CXCL10, CXCL12, and CXCL16 (EndoMT) of PAECs plays an important role in the pathogenesis of are correlated with right ventricular ejection fraction (RVEF; Yang PAH and EndoMT cells display increased expression of CXCL8 (Good et al., 2014); and CCL2, CCL21, and CXCL12 levels are associated et al., 2015). TLR3 agonist increases CXCL8 expression along with with adverse outcome/mortality (Duncan et al., 2012; Hoffmann‐Vold endothelin‐1 (ET‐1) in PASMCs (George et al., 2012) suggesting similar et al., 2018; Kazimierczyk et al., 2018; McCullagh et al., 2015). Some contribution of CXCL8 along with ET‐1 to the pathology of PAH. Stim- of these chemokines may increase as part of an adaptive mechanism; ulation of PAECs with hypoxia in combination with IL‐1α or TNF‐α for example, elevated levels of CXCL10 are associated with improved increases CXCL8 expression in parallel with the decrease of eNOS survival of PAH patients (Heresi, Aytekin, Newman, & Dweik, 2010). expression (Ziesche et al., 1996). Treatment with CXCR1‐ and In addition, some chemokines could serve as biomarkers for monitor- CXCR2‐overexpressing endothelial cells into rats treated with mono- ing disease progression or the effects of the treatment. For example, crotaline (MCT) attenuates the induced PAH via blocking inflammatory the beneficial effects of treatment with epoprostenol on functional cell recruitment to the pulmonary artery wall (Fu et al., 2014). and haemodynamic status in patients with PAH were associated with increased levels of CCL2 (Damas et al., 2004). In CTEPH patients, 4.2 | CXCL10/CXCR3 haemodynamic instability after pulmonary endarterectomy (PEA) is associated with a postoperative increase in CXCL8 levels (Lindner The chemokine CXCL10, also known as IFN‐γ‐induced protein 10 (IP‐ et al., 2009). Moreover, variants of chemokines gene may also be 10), causes a number of cellular effects through binding to its receptor associated with PAH. For example, increased frequencies of the 249I CXCR3. Both CXCL10 and CXCR3 are important for inflammatory cell and 280 M CX3CR1 alleles in systemic sclerosis (SSc) patients are asso- trafficking and homing to injured tissues as well as the exaggeration of ciated with the development of PAH (Marasini et al., 2005), suggesting inflammation, which leads to tissue damage (Hughes & Nibbs, 2018). In the importance of CX3CL1 in the development of PAH in those addition, CXCL10 is known to inhibit bone marrow colony formation patients. and angiogenesis. CXCR3 binds additional chemokines, including 5 MAMAZHAKYPOV ET AL. BJP

FIGURE 2 Chemokines drive pulmonary artery remodelling. In the healthy pulmonary artery (healthy state), local vascular homeostasis is characterized with a low level of inflammatory chemokines. Various factors including hypoxia, shear stress, toxins, and genetic predisposition promote the expression of various chemokines such as CCL2, CCL3, CCL5, CCL10, CCL21, CXCL8, CXCL10, CXCL12, CXCL13, CXCL16, and CX3CL1 in pulmonary artery endothelial cells, smooth muscle cells, and fibroblasts (disease initiation state). The same factors induce the expression of several chemokine receptors including CCR1, CCR2, CCR5, CCR7, CXCR1, CXCR2, CXCR3, CXCR4, and CX3CR1 in PA cells. In turn, these chemokines cause pulmonary artery cell proliferation and immune cell mobilization to the pulmonary artery wall. Accumulated chemokines further exaggerate and perpetuate pathological processes in the remodelled pulmonary arteries (disease state). Application of several chemokine antagonists including the CCR5 antagonist (maraviroc), CXCR7 antagonist (CCX771), CCR1/2 antagonist (SCH527123, SB 225002), CXCR4 antagonist (AMD3100), CX3CR1 antagonist (F1), and anti‐CXCL12 antibody reverse pulmonary artery remodelling in animal models. The figure was created using BioRender.com

CXCL4, CXCL9, and CXCL11, and has two splice variants with differ- 4.3 | CXCL12/CXCR4/ACKR3 ent ligand binding and signalling properties (Lacalle et al., 2017). Dysregulation of CXCL4 and CXCL10 and their receptor CXCR3 in CXCL12 (also known as pre‐B‐cell‐growth‐stimulating factor [PBSF] or PAECs contribute to the impaired angiogenesis and loss of recanaliza- stromal cell‐derived factor [SDF‐1]) belongs to the CXC subfamily of tion of blocked vessels in CTEPH (Zabini et al., 2012). In addition, chemokines. CXCL12 is expressed in different cell types in various adventitial fibroblasts isolated from pulmonary hypertensive calves tissues/organs including lung, liver, lymph nodes, bone marrow, fibro- also display increased expression of CXCR4 along with CXCL12 blasts, and endothelial cells (Lacalle et al., 2017). Global CXCL12‐ and CCR7 (Li et al., 2011). It has been shown that interferon‐α and deficient mice are not viable and display embryonic defects in different ‐γ (IFN‐α and IFN‐γ) induce CXCL10 expression in PAECs, PASMCs, tissues including haematopoiesis, neurogenesis, and the development and PAFBs (George et al., 2014). Similarly, different forms of IFN‐α of cardiac ventricular septa (Janssens, Struyf, & Proost, 2018). CXCL12 could increase CXCL10 expression in both PASMCs and PAECs signals through its cognate receptors, CXCR4 and ACKR3 (Janssens along with ET‐1 (Badiger et al., 2012). In PAH, adventitial et al., 2018). CXCL12 is the only specific ligand for CXCR4, whereas fibroblasts display increased expression of CXCR4 along with other in addition to CXCL12, ACKR3 can also bind CXCL11 (Janssens chemokines/chemokine receptors including CCL2, CCL5, CXCL12, et al., 2018). CXCR4 is widely expressed on haematopoietic cells and CCR7, which mediate monocyte and macrophage migration (Li including T lymphocytes, B lymphocytes, monocytes, macrophages, et al., 2011). PASMCs increase CXCL10 expression in response to neutrophils, and eosinophils (Hughes & Nibbs, 2018). In addition, the mechanical stress (Costa et al., 2018). The TLR3 agonist Poly(I:C) CXCR4 is also expressed in different tissues including brain, lung, increases CXCL10 expression along with ET‐1 in PASMCs (George intestine, heart, kidney, and liver (Hughes & Nibbs, 2018). Like et al., 2012). Similarly, Poly(I:C) increases CXCL10 production in PAECs CXCL12‐deficient mice, CXCR4‐deficient mice also die perinatally (Farkas et al., 2019). and show defects in haematopoiesis, neurogenesis, angiogenesis, and 6 BJP MAMAZHAKYPOV ET AL. cardiac development (Janssens et al., 2018). The CXCL12‐CXCR4 ACKR3 expression is increased in human lung microvascular endo- axis is involved in many homeostatic and pathological processes in thelial cells upon exposure to hypoxia (Costello et al., 2008). ACKR3 different tissues (Janssens et al., 2018). expression was also noted to be elevated in human PAH lung explants Pulmonary up‐regulation of CXCL12 is documented in and also played a role in endothelial cell regeneration, repair, and Sugen5416/hypoxia‐exposed (SuHx) rats (Farkas et al., 2014), proliferation (Costello et al., 2012). However, treatment of HOX mice hypoxia‐exposed (HOX) mice (Dahal et al., 2011; Huang et al., 2017), with the ACKR3 antagonist CCX771 did not significantly change right MCT rats (Kishimoto et al., 2015), and HOX bovine (Li et al., 2011), ventricular systolic pressure (RVSP), RV hypertrophy, or vascular in mice with spontaneous PAH due to endothelial cell specific deletion remodelling, while the CXCR4 antagonist AMD3100 did prevent vas- of the prolyl hydroxylase PHD2 (Dai, Li, Wharton, Zhu, & Zhao, 2016) cular remodelling (Gambaryan et al., 2011b). However, in newborn and in mice with von Hippel–Lindau gene mutation (Hickey et al., mice exposed to hypoxia, treatment with CCX771 markedly decreased 2010). In PAH, various pulmonary cells display augmented CXCL12 RVSP, RV hypertrophy, and pulmonary vascular remodelling (Sartina expression including adventitial fibroblasts (Li et al., 2011) and alveolar et al., 2012), suggesting that signalling through ACKR3 in the develop- macrophages (Hashimoto‐Kataoka et al., 2015). The relevance of this ment of PAH can be context‐dependent. chemokine pathway to the pathogenesis of PAH has been shown in several in vitro studies. For example, CXCL12 or CXCR4 siRNA or 4.4 | CCL2/CCR2 treatment with AMD3100 (a CXCR4 antagonist) decrease PASMC proliferation via blocking cell cycle progression via PI3K/Akt pathway The chemokine CCL2, also referred to as monocyte chemoattractant under hypoxia (Wei et al., 2015). Moreover, hypoxia‐induced PASMC protein 1 (MCP1), is constitutively expressed by several cell types proliferation is mediated by adenosine A2A receptor‐induced activa- including endothelial cells, epithelial cells, immune cells, smooth tion of the CXCL12‐CXCR4 axis (Wang et al., 2018). The CXCL12‐ muscle cells, and fibroblasts and is a potent chemoattractant for mono- CXCR4 axis protects PAECs from thrombin‐induced cell permeability cytes, basophils, T lymphocytes, and NK cells (Hughes & Nibbs, 2018). (Cheng, Eby, LaPorte, Volkman, & Majetschak, 2017). Deletion of CCR2 and CCR4 serve as cell surface receptors for CCL2. However, PHD2 in endothelial cells leads to dramatic up‐regulation of CXCL12 CCL2 preferentially binds to CCR2, which is expressed on monocytes, in those cells that further increases PASMC proliferation (Dai et al., T lymphocytes, DC cells, basophils, and NK cells (Hughes & Nibbs, 2016), and mice with endothelial cell‐specific deletion of PHD2 2018). CCR2 also binds several other chemokines including CCL8, develop spontaneous PAH with plexiform‐like lesions due to CXCL12 CCL7, and CCL13 based on their structural similarity to CCL2 (Hughes up‐regulation (Dai et al., 2016). In addition, application of anti‐CXCL12 & Nibbs, 2018). antibody prevents hypoxia‐induced PAH in neonatal mice (Young CCL2 is up‐regulated in the lungs in human PAH and several et al., 2009). In contrast, intratracheal instillation of nano‐particulated rodent models of PAH (Kishimoto et al., 2015; Otsuki et al., 2015; CXCL12 could prevent MCT induced PAH development in rats by Sanchez et al., 2007; Zhang, Kawaguchi, Hayama, Furutani, & recruiting circulating progenitor cells to repair injured lung tissue (Yin Nakanishi, 2018). PAECs, PA fibroblasts (PAFBs), infiltrating cells, and et al., 2013). macrophages display augmented expression of CCL2 within PAH lung CXCR4 expression is also increased in PAH lung tissue (Dahal, tissue (Le Hiress et al., 2015; Li et al., 2011; Sanchez et al., 2007; Heuchel, et al., 2011). Within the lungs, CXCR4 is primarily expressed Wang et al., 2014). In the setting of PAH, shear stress on endothelial in PASMCs (Farkas et al., 2014; Huang et al., 2017; Wei et al., 2015) cells increases CCL2 expression in PAECs (Li, Scott, Shandas, and PAECs (Farkas et al., 2014). Similarly, lung CXCR4 expression is Stenmark, & Tan, 2009). Similarly, mechanical stress imposed on endo- also increased in various animal models of PAH including SuHx rats thelial cells with increased flow pulsatility causes endothelial cell up‐ (Farkas et al., 2014), HOX mice (Dahal, Heuchel, et al., 2011; Huang regulation of CCL2 along with ICAM‐1 and E‐selectin, resulting in et al., 2017), and HOX rats (Wei et al., 2015). A number of animal stud- increased monocyte adhesion and recruitment (Li et al., 2009). More- ies have demonstrated a role for CXCR4 in the pathogenesis of PAH. over, mechanical stress exerted on pulmonary artery endothelial cells For example, transplantation of CXCR4‐targeting shRNA transfected in left ventricular failure may drive pulmonary venous hypertension bone marrow cells into rats prevented hypoxia exposure‐induced via endothelial cell specific up‐regulation of CCL2 (Park et al., 2014). development of PAH due to attenuated bone marrow cell recruitment Like endothelial cells, PASMCs also respond to the mechanical stress to the lungs (Yu & Hales, 2011). Bone marrow‐derived CXCR4+/ with CCL2 overexpression (Costa et al., 2018). In addition, the CCL2‐ PDGFβ+ progenitor cells contribute to hypoxia‐induced PA remodel- CCR2 axis promotes mast cell recruitment into to lung tissue during ling by differentiating into SMCs via myocardin (Jie et al., 2010). In lung diseases (Collington et al., 2010) and may be responsible for the addition, AMD3100 attenuates PAH development in several animal recruitment and accumulation of mast cells into remodelled PAs in models (Farkas et al., 2014; Gambaryan et al., 2011a; Young et al., PAH (Dahal et al., 2011). 2009; Yu & Hales, 2011). The CXCR4 axis is also involved in other PAH is characterized by the up‐regulation of several inflammatory pulmonary vascular pathologies. For instance, treatment with mediators, many of which directly modulate CCL2 expression. For AMD3100 reduces c‐kit+ cells recruitment and abnormal angiogenesis example, C‐reactive protein (CRP) and IL‐1β alone or in combination of pulmonary arteries in rat models of the hepatopulmonary syndrome, with IL‐12 induce CCL2 expression in PASMCs (Larsen et al., 2011; following common bile duct ligation (Shen et al., 2018). Li et al., 2010). In addition, macrophage migration inhibitory factor 7 MAMAZHAKYPOV ET AL. BJP

(MIF), TNF‐α, soluble CD40L, and IL‐33 also increase CCL2 expression et al., 2000). Furthermore, the contribution of CCL5 to the pathogen- in the PAECs (Damas et al., 2004; Le Hiress et al., 2015; Maus et al., esis of PAH was also confirmed in several animal models. For example, 2002; Sawada et al., 2014; Yagami et al., 2010). TNF‐α‐induced CCL5‐deficient mice are resistant to SuHx‐induced PAH (Nie et al., adhesion of monocytes to the endothelial cells are mediated by the 2018). Presence of anti‐endothelial cell antibodies (AECA) in patients CCL‐CCR2 axis (Maus et al., 2002) and is inhibited by anti‐CCL2 anti- with SSc is associated with an increased frequency of PAH, which body, anti‐CCL2 siRNA, anti‐CCR2 antibody, or CCR2 antagonist may be explained by the increased CCL5 expression in endothelial (Maus et al., 2002). In turn, CCL2 induces ET‐1 expression in endothe- cells in response to AECA stimulation (Liu et al., 2014). lial cells (Molet, Furukawa, Maghazechi, Hamid, & Giaid, 2000). Further Like CCL5, CCR5 content is also increased in PAH in several cell mechanistic insights into the role of CCL2 in PAH have been gained in types that display up‐regulated CCR5 expression, including PASMCs, several in vivo studies. For example, delivery of N‐terminus deleted PAECs, and macrophages (Amsellem et al., 2014; Nie et al., 2018). Sim- mutant gene of CCL2 into MCT rats partly attenuated PAH in those ilarly, in several animal models of PAH, disease induction is accompa- animals due to down‐regulation of CCL2 (Ikeda et al., 2002). However, nied with CCR5 up‐regulation in lung tissue including HOX mice and the results of studies with CCL2‐deficient mice are somewhat contra- CCR5‐deficient mice that develop attenuated PAH (Amsellem et al., dictory: Mice deficient for CCL2 alone or double deficient for both 2014). Mechanistically, it was shown that knock‐in mice expressing CX3CR1 and CCL2 develop similar degree of PAH, compared to WT human CCR5 display an attenuation in HOX PAH with the CCR5 mice (Amsellem et al., 2017), although CCL2‐deficient mice develop antagonist maraviroc (Amsellem et al., 2014), confirming a pathogenic mild spontaneous PAH at normoxia with exaggerated RV hypertrophy role of CCR5 in PAH. The mechanistic contribution of the CCL5‐CCR5 with hypoxia (Yu, Mao, Piantadosi, & Gunn, 2013). axis to the pathogenesis of PAH has also been demonstrated in CCR2 expression is also increased in PAH lung tissues (Amsellem in vitro studies. For example, macrophages induce PASMC prolifera- et al., 2017). Similarly, pulmonary up‐regulation of CCR2 has been tion through the CCL5‐CCR5 axis, which is blocked with CCR5 antag- demonstrated in HOX mice (Amsellem et al., 2017). Both PAECs and onist maraviroc (Amsellem et al., 2014). PASMCs display increased expression of CCR2 in PAH (Sanchez An alternative receptor for CCL5 is CCR1, whose other ligands et al., 2007). CCR2‐deficient mice have been reported to develop include also CCL3, CCL7, and CCL23. SSc patients with PAH display spontaneous PAH at normoxia and display exaggerated PAH upon increased expression of CCR1 in peripheral CD14+ mononuclear cells exposure to hypoxia due to activated Notch signalling (Kim et al., compared to healthy controls (Christmann et al., 2011). Interestingly, 2013), although a different study found that CCR2‐deficient mice studies have showed that CD14+ cells are accumulated around the had attenuated PA remodelling in response to hypoxia exposure with plexiform lesions in IPAH lung tissues (Savai et al., 2012), and are likely a similar RVSP compared to WT counterparts (Florentin et al., 2018). to play an important role in the process of PA remodelling. Taken together, the CCL2:CCR2 axis is clearly involved in several pathological processes at the cellular level that contribute to PAH, although 4.6 | CX3CL1/CX3CR1 the conflicting findings of in vivo studies with CCL2‐ and CCR2‐ deficient mice will need to be addressed in the future. The chemokine CX3CL1, also known as fractalkine, and its receptor CX3CR1 are expressed in several immune/inflammatory cells and res- 4.5 | CCL5/CCR5/CCR1 ident cells of different tissues (Lacalle et al., 2017). CX3CL1 is the only ligand of CX3CR1 and exists in both membrane‐bound and soluble The chemokine CCL5, also known as RANTES, is produced in different forms. Membrane CX3CL1 serves as an adhesion molecule, while sol- tissues by various cells including platelets, macrophages, eosinophils, uble CX3CL1 serves as a chemoattractant molecule for CX3CR1+ cells fibroblasts, endothelium, and epithelial cells (Hughes & Nibbs, 2018). (Liu et al., 2016). The membrane form of CX3CL1 is abundantly pro- CCL5 is known to bind to several receptors including CCR1, CCR3, duced by endothelial cells of different tissues/organs (Lacalle et al., and CCR5 to exert its effects on target cells (Hughes & Nibbs, 2018). 2017) and can be cleaved by metalloprotease ADAM10 resulting in CCL5 promotes migration and recruitment of T cells, dendritic cells, formation of various soluble forms (Hundhausen et al., 2003). During eosinophils, NK cells, mast cells, and basophils (Lacalle et al., 2017). inflammation, ADAM17 also participates in shedding membrane Several groups have shown the involvement of CCL5 in the patho- CX3CL1 (Garton et al., 2001). Several immune/inflammatory cells genesis of PAH. For example, CCL5 expression is increased in PAH express CX3CR1 including monocytes, macrophages, T lymphocytes, lung tissue (Dorfmuller et al., 2002; Nie et al., 2018; Price et al., natural killer (NK) cell, and dendritic cells (DC; Hughes & Nibbs, 2018). 2013). In PAH, several pulmonary cells may be the source of CCL5 The development of PAH is associated with the pulmonary up‐ including endothelial cells (Dorfmuller et al., 2002) and perivascular regulation of CX3CL1 (Perros et al., 2007). Within PAH lung tissue, dif- fibroblasts (Li et al., 2011). Similarly, pulmonary up‐regulation of ferent cell types have been found to display augmented expression of CCL5 is observed in several animal models including HOX mice CX3CL1, including perivascular inflammatory cells (Dorfmüller et al., (Amsellem et al., 2014), SuHx rats (Otsuki et al., 2015), 2011; Perros et al., 2007) and endothelial cells (Balabanian et al., MCT/pneumonectomy (MCT/PE) rats (Dorfmüller et al., 2011), and 2002; Zhang et al., 2012). Similarly, CX3CL1 is also up‐regulated in HOX mitogenic factor (HIMF)‐induced PAH mice (Yamaji‐Kegan the lungs of different animal models including MCT rats (Dorfmüller et al., 2014). CCL5 induces ET‐1 expression in endothelial cells (Molet et al., 2011; Perros et al., 2007), MCT/PE rats (Dorfmüller et al., 8 BJP MAMAZHAKYPOV ET AL.

2011), and HOX mice (Florentin et al., 2018; Zhang, Hu, et al., 2012). attenuated PAH upon 18‐day (Amsellem et al., 2017) or 4 week hyp- In contrast, another study showed CX3CL1 levels were down‐ oxia exposure (Hutchinson et al., 2016; Zhang, Hu, et al., 2012). In con- regulated in MCT rat lungs (Wang et al., 2011). In addition, circulating trast, another study observed that CX3CR1‐deficient mice display a CX3CL1+ T lymphocytes (CD4+ and CD8+) are also increased in similar degree of pulmonary hypertension (RVSP), compared to WT patients with PAH (Balabanian et al., 2002). Similarly, circulating endo- mice upon 21‐day exposure to hypoxia despite attenuated PA remod- thelial cell like cells isolated from patients with PAH also display elling (Florentin et al., 2018). Similarly, studies have yielded contrasting increased CX3CL1 expression (Zhang, Hu, et al., 2012). At a cellular results with CX3CR1‐deficient mice exposed to hypoxia, with one level, CX3CL1 expression is increased in response to different stimuli showing an increase in lung tissue monocyte and macrophage counts which contribute to the development of PAH. For example, CX3CL1 (Amsellem et al., 2017) or a decrease (Florentin et al., 2018). However, induces PASMC proliferation and expansion but not migration (Perros all of these studies demonstrated that the absence of CX3CR1 et al., 2007; Zhang, Hu, et al., 2012). Hypoxia‐exposed endothelial cells prevented PA remodelling in these rodent models of PAH. also display increased secretion of CX3CL1 (Hutchinson, Hu, & Patel, 2016; Zhang, Hu, et al., 2012), which, in turn, induces cell proliferation 4.7 | Other chemokine systems of pericytes in the PA adventitial layers (Hutchinson et al., 2016). ET‐1, which is implicated in the pathogenesis of PAH, also induces CX3CL1 A number of other chemokines and chemokine receptors have been expression in endothelial cells overexpressing ET receptors (Zhang, B shown to be dysregulated in PAH. The expression of CCR7 in the Yang, Hu, Wu, & Fallon, 2014), suggesting that CX3CL1 may be par- peripheral mononuclear cells is decreased in SSc patients with PAH tially responsible for the detrimental effects of ET‐1 in PAH. In addi- compared to those without PAH (Risbano et al., 2010). CCR7‐deficient tion, cigarette smoke extract (CSE) induces CX3CL1 expression in mice spontaneously develop PAH due to the accumulation of inflam- endothelial cells via activation of TNF‐α and NOX5 resulting in matory cells in the lung tissue (Larsen et al., 2011). In the ovalbumin CX3CR1+ monocyte adhesion to the ECs (Rius et al., 2013), which model of asthma, adiponectin‐deficient mice develop severe PA may be relevant to the development of PH due to COPD. Mice lacking remodelling associated with inflammatory cell accumulation and lung CX3CL1 develop attenuated PAH upon 4‐week exposure to hypoxia expression of several chemokines including CCL2, CCL7, and CCL11 (Zhang, Hu, et al., 2012). CX3CL1 is also increased in both the plasma (Medoff et al., 2009). CCL3 and CCL20 expressions are increased in and lung tissues of acute pulmonary embolism (PE) rats (Wang et al., the lung tissue of MCT rats (Kiss et al., 2014). Expression of CCL19, 2014) suggesting that acute haemodynamic stress imposed on the pul- CCL20, CCL21, and CXCL13 are increased in IPAH lung tissue and is monary vascular cells in acute PE may drive CX3CL1 expression in localized around plexiform lesions, which may explain decreased circu- these cells. The transcription factor GATA‐6 serves as a negative lating CCR6+ and CXCR5+ T cells in those patients (Perros et al., 2012). regulator of CX3CL1 expression in PAECs and mice with endothelial CCL2, CCL4, and CCL5 expression are up‐regulated in the pulmonary cell‐specific GATA‐6 deficiency develop spontaneous PAH and an artery of HOX rats (Yuan et al., 2014). In peripheral blood mononuclear exaggerated phenotype upon exposure to hypoxia, which is associated cells (PBMCs) of PE patients, CXCL1, CXCL2, CXCL6, CXCL13 and with the up‐regulation of CX3CL1 (Ghatnekar et al., 2013). CCR7‐ CXCL14 mRNAs were significantly up‐regulated; however, CXCL10 deficient mice develop spontaneous PAH accompanied with the pul- mRNA was significantly down‐regulated (Lv et al., 2013). PASMCs dis- monary up‐regulation of CX3CL1 and CX3CR1 along with an exagger- play augmented expression of CCL4 in response to mechanical stress ated accumulation of inflammatory cells (Larsen et al., 2011). In (Costa et al., 2018). Cigarette smoke extract induces CXCL16 up‐ addition, up‐regulation of the CX3CL1/CX3CR1 axis in pulmonary regulation in endothelial cells resulting in increased CXCR6+ platelet artery endothelial cells and circulating monocytes contributes to and monocyte adhesion to these cells (Marques et al., 2017). CCL3 inflammatory cell accumulation and abnormal angiogenesis in the lung induces ET‐1 expression in endothelial cells (Molet et al., 2000). tissue in the common bile duct ligation model of hepatopulmonary syndrome (Zhang et al., 2012). CX3CR1 is up‐regulated in PAH lung tissue (Amsellem et al., 2017; 5 | CHEMOKINES AND DNA DAMAGE Perros et al., 2007). In PAH, the up‐regulation of CX3CR1 has been noted in different cell types including smooth muscle cells, perivascular Current evidence suggests that in PAH, pulmonary artery cells display cells (Amsellem et al., 2017; Perros et al., 2007), endothelial cells phenotypes similar to those seen in cancer cells including excessive (Hutchinson et al., 2016), and pericytes (Hutchinson et al., 2016). In proliferation, migration, and apoptosis resistance, which are driven by addition, up‐regulation of CX3CR1 is observed in different rodent similarly dysregulated signalling pathways (Boucherat et al., 2017). models of PAH including HOX mice (Amsellem et al., 2017) and MCT Known pathological processes observed in PAH, including inflamma- rats (Perros et al., 2007; Wang et al., 2011). CX3CR1 is up‐regulated tion and oxidative stress, contribute to the PA remodelling at least in in circulating T lymphocytes (CD4+ and CD8+; Balabanian et al., part due to increased DNA damage (Ranchoux et al., 2016). Damaged 2002) and monocytes (Florentin et al., 2018) isolated from PAH DNA can be repaired by a signalling pathway named DNA‐damage patients. Several studies have attempted to establish the mechanistic response (DDR). In the lung tissues of IPAH patients and several ani- role for CX3CR1 in PAH in animal models by employing genetically mal models of PAH, various regulators of DDR signalling pathways modified mice. For example, CX3CR1‐deficient mice develop are up‐regulated including bromodomain‐containing protein 4 9 MAMAZHAKYPOV ET AL. BJP

(BRD4; Meloche et al., 2015; Van der Feen et al., 2019) and check- cells resulting in remodelling of the PAs and the development of point kinase 1 (CHK1; Bourgeois et al., 2019). In animal models of PAH (Song et al., 2008). PAH, BRD4 inhibition by JQ1 (Meloche et al., 2015) and RVX208 CCL5‐deficient mice exhibit increased expression of BMPR2 in (Van der Feen et al., 2019) have been shown to be effective. Inter- lung tissue compared to WT mice (Nie et al., 2018), which may estingly, RVX208 was also able to inhibit the expression of several explain the attenuated development of PAH in those mice in chemokines including CCL2, CCL5, and CXCL8 in TNF‐α stimulated response to SuHx exposure. In addition, decreased expression of PAECs, endothelial cells isolated from PAH patients, and lung BMPR2 in PAECs isolated from PAH patients was completed recov- tissues of SuHx rats (Van der Feen et al., 2019). The DDR signalling ered with siRNA targeting CCL5 (Nie et al., 2018). Supporting this pathway is also associated with the increased activity of the cyclin‐ notion, application of recombinant human CCL5 (rhCCL5) decreases dependent kinases (CDKs; Chen et al., 2016), which are also implicated BMPR2 expression in PAECs by up‐regulating and activating CCR5, in the PAH pathogenesis (Weiss et al., 2019). Cyclin D1, which is a which was restored with CCR5 antagonist maraviroc (Nie et al., main target of CDKs, promotes the synthesis of several chemokines 2018). In addition, in BMP2‐deficient mice that develop spontaneous including CCL2 CCL7, CCL11 CXCL5, CXCL9, CXCL12 GM‐CSF, and PAH at normoxia, PAH after SuHx exposure was completely CXCL1, suggesting a potential mechanism by which chemokines prevented in mice with knockouts for both BMP2 and CCL5 (Nie may promote the progression of breast cancers (Pestell et al., 2017). et al., 2018). BMP‐9 increases CXCL12 expression with a parallel Similarly, DNA damage may induce the expression of those decrease in CXCR4 in human microvascular endothelial cells (Young chemokines, which may further exert effects on the pulmonary artery et al., 2012). Moreover, hypoxia potentiates BMP‐9 induced CXCL12 cells. Taken together, DNA damage and activated DDR system may expression in PAECs (Young et al., 2012). Treatment with anti‐ contribute to the pathogenesis of PAH at least in part due to altered CXCL12 antibody decreases BMP‐9‐induced endothelial cell migra- chemokine signalling. tion (Young et al., 2012). Wild‐type mice transplanted with bone marrow cells from BMPR2 mutation donors develop PAH, which is associated with the up‐regulation of CXCL1 in the lung tissue (Yan ‐ 6 | CHEMOKINE CROSSTALK WITH BMPR2 et al., 2016). LPS treated mice heterozygous for BMPR2 develop SIGNALLING exaggerated systemic inflammation with increased levels of circulating and pulmonary CXCL8 and IL‐6, contributing to the development ‐ Mutations in the gene encoding the bone morphogenetic protein of PAH (Soon et al., 2015). In addition, BMP 9 can increase CXCL8 ‐ receptor type II (BMPR2) are associated with the development of expression in PAECs by employing both BMPR2 and activin like PAH (Morrell et al., 2019). BMPR2 mutations are found in more than kinase receptor 1 (ALK1; Upton, Davies, Trembath, & Morrell, ‐β 70% of heritable PAH and up to 25% of apparently sporadic cases of 2009). In response to TGF stimulation, PASMCs isolated from idiopathic PAH (Morrell et al., 2019). PAH patients with BMPR2 muta- PAH patients harbouring BMPR2 mutation display augmented tions have worse long‐term outcome compared to those without expression of CXCL8 (Davies et al., 2012). Taken together, dysregu- BMPR2 mutations (Evans et al., 2016). Moreover, even PAH patients lation of chemokine and BMPR2 signalling may contribute to the without BMPR2 mutations display decreased BMPR2 expression in pathogenesis of PAH (Figure 3). the lung. Similarly, pulmonary BMPR2 expression is down‐regulated in various animal models of PAH. To understand the mechanisms behind PAH development due to BMPR2 mutation, studies using 7 | CHEMOKINES IN RIGHT VENTRICULAR BMPR2 mutation mice or pulmonary artery cells deficient for BMPR2 REMODELLING have revealed that dysregulated chemokine signalling may partly contribute to disease development. For example, mice expressing domi- Accumulating evidence indicates that right ventricular remodelling in nant negative BMPR2 have increased expression of CCL2 in the lung response to the pressure overload is associated with the up‐regulation tissue (Hagen et al., 2007). In addition, endothelial cell‐specific of inflammatory mediators along with increased content/accumulation BMPR2‐deficient mice develop spontaneous PAH through the of immune cells in the myocardium (Sydykov et al., 2018). These increased recruitment of inflammatory cells to the pulmonary artery inflammatory mediators expressed by different cell types of the myo- wall, which is partially mediated by CXCR1/2 (Burton et al., 2011). cardium may further enter the circulation. Consequently, the levels Interestingly, bone morphogenetic protein‐2 (BMP‐2) decreases of these mediators may reflect changes happening in the pressure‐ CCL2 expression in PASMCs (Hagen et al., 2007). Knock‐down of overloaded myocardium. As discussed above, circulating levels of sev- BMPR2 with siRNA in PAECs exaggerates CXCL8 expression in eral chemokines in PAH patients are associated with parameters of the response to TNF‐α stimulation (Sawada et al., 2014). Combination of RV remodelling and dysfunction such as TAPSE, RV ejection fraction MCT treatment and viral‐mediated delivery of 5‐lipoxygenase (5‐ (RVEF), CO, CI and RV dimensions. Based on the evidence that LOX) into the lungs of heterozygous BMPR2‐deficient mice leads to patients with pulmonary stenosis also display increased levels of circu- PAH development associated with the up‐regulation of CCL2, CCL3, lating CXCL16 (Waehre et al., 2012), it is reasonable to speculate that and CX3CR1 in the lung tissue of those mice (Song et al., 2008). These some of the chemokines elevated in the circulation of patients with PH chemokines mediate the accumulation of perivascular inflammatory that correlate with RV parameters may originate from the right 10 BJP MAMAZHAKYPOV ET AL.

FIGURE 3 Crosstalk between chemokine and BMPR2 signalling. Binding of bone morphogenetic protein 2 (BMP2) dimer to its receptor consisting of bone morphogenetic protein receptor type 2 (BMPR2) dimer and dimers of either activin receptor–like kinase 1 or 2 or 6 (ALK1, 2, and 6) initiates the activation of SMAD1/5 signalling, which leads to the transcriptional suppression of several chemokines such as CCL2, CCL5, CXCL1, and CXCL8 and inhibition of NF‐κB signalling mediated CXCL8 transcription activated by TNF‐α through its receptor TNF receptor 1 (TNFR1). CCL5 activates its receptor CCR5 that further down‐regulates BMPR2 expression. TGF‐β activates its receptors (TβRI, TβRII), leading to CXCL8 transcription through the SMAD2/3 signalling pathway. The figure was created using BioRender.com

ventricle. The concept that isolated pressure overload drives the & Gomer, 2015; Waehre et al., 2012). However, there is little data on expression of several chemokines in different models of RV remodel- the regulation of RV fibrosis by chemokines. Only a few studies have ling has been shown in several rodent models of RV remodelling. For demonstrated that chemokine receptors are up‐regulated in the example, in a mouse model of PAB‐induced RV remodelling, several remodelled RV myocardium, such as CCR1, CCR2, and CXCR4 in chemokines were up‐regulated in the RV myocardium, including models of RV remodelling including HOX mice (Baandrup et al., CCL2, CCL5, CXCL16, CXCL10, and CX3CL1 (Waehre et al., 2012). 2011), PAB mice (Waehre et al., 2012), and PE rats (Zagorski et al., Moreover, acute RV remodelling in pulmonary embolism models in rats 2008). Chemokines in the RV may participate in both adaptive and are also associated the up‐regulation of several chemokines including maladaptive processes. For example, several chemokines, including CC‐chemokines (CCL2, 3, 4, 7, 9, 17, 20, 27) and CXC‐chemokine CX3CL1, CCL5, and CXCL16, up‐regulate the expressions of proteo- genes (CXCL1, 2, 9, 10, 16; Watts, Zagorski, Gellar, Stevinson, & Kline, glycans including decorin, lumican, fibromodulin, and biglycan in 2006; Zagorski, Gellar, Obraztsova, Kline, & Watts, 2007; Zagorski, cardiac fibroblasts (Waehre et al., 2012), while CXCL10 decreases pro- Sanapareddy, Gellar, Kline, & Watts, 2008). Similarly, in acute RV teoglycan synthesis in these cells (Waehre et al., 2012) to potentially remodelling in PAB pigs, CCL2, CXCL6, and CXCL2 chemokines are modulate extracellular matrix structure in the RV. Down‐regulated up‐regulated (Vikholm, Schiller, & Hellgren, 2014). However, other CCL2 in the RV myocardium of Fischer rats, compared to Sprague– chemokines such as XCL1 and CXCL12 are down‐regulated in the Dawley rats, in response to SuHx exposure may be partly acutely remodelled RV myocardium (Zagorski et al., 2008). Of note, responsible for the maladaptive RV remodelling and increased mortal- CXCL12 expression is increased in the RV of HOX mice (Young ity in SuHx Fischer rats (Suen, Chaudhary, Deng, Jiang, & Stewart, et al., 2009). 2018). However, all of the studies mentioned here demonstrated The detrimental effects of chemokine up‐regulation on the RV may only associations between chemokine levels and RV remodelling and be due to their contribution to cardiac fibrosis mediated by the expres- not a causal link between chemokines and RV failure. Further sion of several proteoglycans by cardiac fibroblasts (Pilling, Vakil, Cox, studies with chemokine‐specific genetically modified animals and with MAMAZHAKYPOV TABLE 1 Summary of selected studies evaluating the effects of agents targeting chemokines and chemokine receptors in animal models of PAH

Mechanism Agent of action Disease model Agent application details Main effects of pharmacological agents Reference

AMD3100 CXCR4 HOX mice (35 days) Injection (i.p.), 10 mg·kg−1 ·day−1 (Days 21–35) ‐Prevented PAH development (↓RVSP) Gambaryan antagonist ‐Prevented PA remodelling (↓PA medial et al. (2011a) AL ET

wall thickness) . ‐Prevented RV hypertrophy (↓RV/ (LV + S)) − − AMD3100 CXCR4 HOX rats (14 days) Osmotic mini pump, 10 mg·kg 1 ·day 1 (Days 1–14) ‐Prevented PAH development (↓mPAP) Yu and Hales antagonist ‐Prevented PA remodelling (↓PA medial (2011) wall thickness) ‐Prevented RV hypertrophy (↓RV/ (LV + S)) − − AMD3100 CXCR4 SuHx rats (21 days) Injection (i.p.), 5 mg·kg 1 ·day 1 (Days 1–21) ‐Prevented PAH development (↓RVSP) Farkas et al. antagonist ‐Prevented PA remodelling (↓PA (2014) muscularization) ‐Prevented RV hypertrophy (↓RV/ (LV + S)) − − AMD3100 CXCR4 Neonatal HOX mice Injection (i.p.), 7.5 mg·kg 1 ·day 1 (Days 1–7 in prevention ‐Prevented/reversed increased PAP Young et al. antagonist (7 or 14 days) strategy; Days 7–14 in curative strategy) (↓RVSP) (2009) ‐Prevented/reversed increased PAP (↓PA medial wall thickness) ‐Prevented/reversed RV hypertrophy (↓RV/(LV + S) SCH527123 CXCR1/2 Spontaneous PAH in PAEC‐specific BMPR‐II‐ Oral gavage, 10 mg·kg−1 ·day−1 (Days 1–21) ‐Prevented spontaneous PAH Burton et al. antagonist deficient mice (21 days) development (↓RVSP) (2011) ‐Prevented PA remodelling (↓PA muscularization) ‐Prevented cardiac function decline (↑CO) − − SB 225002 CXCR2 Bleomycin‐clodronate liposomes treated mice Injection (i.p.), 1.5 mg·kg 1 ·day 1 (Days 1–33) ‐Prevented PAP increase (↓sPAP) Bryant et al. antagonist (33 days) ‐Prevented PA remodelling (↓PA medial (2018) wall thickness) − Maraviroc CCR5 HOX CCR5 knock‐in mice expressing human Oral gavage, 200 mg·kg 1 daily ‐Prevented PAH development (↓RVSP) Amsellem et al. antagonist CCR5 ‐Prevented PA remodelling (↓PA (2014) BJP muscularization) ‐Prevented RV hypertrophy (↓RV/ (LV + S)) F1 CX3CR1 HOX mice Injection (i.p.), 50 μg/three times a week ‐Prevented PAH development (↓RVSP) Amsellem et al. antagonist ‐Prevented PA remodelling (↓PA (2017) muscularization) ‐Prevented RV hypertrophy (↓RV/ (LV + S))

(Continues) 11 12 BJP MAMAZHAKYPOV ET AL.

chemokine receptor antagonists and agonists in PAB models are urgently required. et al. (2011a) (2009) (2012) 8 | CHEMOKINES AND CHEMOKINE Young et al. RECEPTORS AS DRUG TARGETS PA ↓ Based on the evidence that PAH is associated with the up‐regulation number of RV/(LV + S) of several chemokines and chemokine receptors, some studies have ↓ ↓ RVSP) Sartina et al. RVSP) Yin et al. (2013) PA medial wall ↓ ↓ ↓ specifically targeted chemokine receptors with pharmacological agents in different models of PAH. The majority of the tested agents has been found to be beneficial in preventing or reversing the experimental PAH (Table 1). Chemokine receptors targeted in animal models of PAH include CXCR4 (with AMD3100) in HOX mice (Gambaryan et al., 2011a; Young et al., 2009), HOX rats (Yu & Hales, RVSP) RV/(LV + S) ↓ ↓ remodelled PA) ( medial wall thickness) ( thickness) 2011), and SuHx rats (Farkas et al., 2014); CXCR1/2 (with Prevented PAP increase ( Prevented PA remodelling ( No effect on PAH Gambaryan Prevented/reversed increased PAP Prevented/reversed RV hypertrophy Prevented/reversed increased PAP ( Reversed increased PAP ( Reversed increased PAP ( Reversed RV hypertrophy ( ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ cle; RVSP, right ventricular systolic pressure; S, septum; SuHx, SCH527123; navarixin) in BMPR2‐deficient mice with spontaneous PAH (Burton et al., 2011); CXCR2 (with SB 225002) in bleomycin‐ clodronate liposomes treated mice (Bryant et al., 2018); CCR5 (with maraviroc) in HOX mice (Amsellem et al., 2014); CX3CR1 (F1) in NP on Day 3 α

1 HOX mice (Amsellem et al., 2017); CXCR7 (with CCX771) in HOX ‐ mice (Gambaryan et al., 2011a; Sartina et al., 2012); and CXCL12 35) 14) – g SDF 7)

– ‐

μ (with anti CXCL12 antibody) in HOX (Young et al., 2009). Despite – ‐ positive findings in a number of these studies, none of these agents (Days 7 (Days 21

(Days 1 has moved forward to clinical trials. 1 1 1 − − − ·day ·day ·day 1 1 1 − − − 9 | CONCLUSIONS g·kg μ Levels of several chemokines and chemokine receptors are altered in patients with PAH, and some of them are correlated with the disease severity and prognosis. Apart from their conventional role in mobilization of immune cells to the site of inflammation, chemokines also mediate several cellular processes that are specific to the PA remodelling such as promoting PA cell proliferation, migration, and cytokine expression. Moreover, a plethora of factors such as hypoxia, shear stress, and inflammatory cytokines induce expressions of several chemokines in pulmonary artery cells. In addition, chemokines are also actively involved in BMPR2 upstream and downstream signalling pathways leading to the disease progression. Genetic manipulations of several chemokines and chemokine receptors in rodents enabled them to be protective or susceptible to the development of PAH. Moreover, employing pharmacological agents targeting chemokine receptors successfully prevented or reversed the disease in several models of PAH (Table 1). Considering the number of chemokines Neonatal HOX mice (7 days) Injection (i.p.), 25 HOX mice (35 days) Injection (s.c.), 10 mg·kg Neonatal HOX mice (14 days) Injection (s.c.), 30 mg·kg and their receptors, only a few members of this system have been studied in the context of PAH. In addition, when chemokines are studied, their tissue specific roles/effects (pulmonary vasculature vs. CXCL12 antagonist antagonist right ventricle) should be considered and evaluated in tissue‐specific Blocking Mechanism of action Disease model Agent application detailsmodels (PAH Mainand effects of pharmacological agents PAB), Reference since chemokines may exert tissue‐specific effects. Taken together, the current evidence shows that chemokines (Continued) NP CXCL12 MCT rats (21 days) Intratracheal instillation, single dose 15

α and chemokine receptors are involved in the pathogenesis of PAH 1 CXCL12 ‐ ‐

antibody and RV remodelling and pharmacological agents targeting this system SDF Anti CCX771 CXCR7 Agent CCX771 CXCR7 Abbreviations: HOX, hypoxia; LV, left ventricle; MCT, monocrotaline; mPAP, mean pulmonary artery pressure; PA, pulmonary artery; RV, right ventri Sugen plus hypoxia. TABLE 1 may be a promising strategy to treat PAH. 13 MAMAZHAKYPOV ET AL. BJP

9.1 | Nomenclature of targets and ligands preparations on IP10 and ET‐1 release from human lung cells. PLoS ONE, 7, e46779. https://doi.org/10.1371/journal.pone.0046779 ‐ Key protein targets and ligands in this article are hyperlinked to Balabanian, K., Foussat, A., Dorfmüller, P., Durand Gasselin, I., Capel, F., Bouchet‐Delbos, L., … Humbert, M. (2002). CX3C chemokine corresponding entries in http://www.guidetopharmacology.org, the fractalkine in pulmonary arterial hypertension. American Journal of common portal for data from the IUPHAR/BPS Guide to PHARMA- Respiratory and Critical Care Medicine, 165, 1419–1425. https://doi. COLOGY (Harding et al., 2018), and are permanently archived in the org/10.1164/rccm.2106007 Concise Guide to PHARMACOLOGY 2017/18 (Alexander, Boucherat, O., Vitry, G., Trinh, I., Paulin, R., Provencher, S., & Bonnet, S. (2017). Christopoulos et al., 2017 ; Alexander, Fabbro, et al., 2017a). The cancer theory of pulmonary arterial hypertension. Pulmonary Circulation, 7,285–299. https://doi.org/10.1177/2045893217701438 Bourgeois, A., Bonnet, S., Breuils‐Bonnet, S., Habbout, K., Paradis, R., ACKNOWLEDGEMENTS Tremblay, E., … Boucherat, O. (2019). Inhibition of CHK1 (checkpoint kinase 1) elicits therapeutic effects in pulmonary arterial hypertension. Arteriosclerosis, Thrombosis, and Vascular Biology, 39, 1667–1681. This work was supported by a Burroughs Wellcome Career Award for https://doi.org/10.1161/ATVBAHA.119.312537 Medical Sciences and American Heart Association (grant 33670458; Bryant, A. J., Shenoy, V., Fu, C., Marek, G., Lorentsen, K. J., Herzog, E. L., … to S.R.). A.L. is a British Heart Foundation Senior Basic Science Research Scott, E. W. (2018). Myeloid‐derived suppressor cells are necessary for Fellows (FS/18/52/33808). This work was funded by the Deutsche development of pulmonary hypertension. American Journal of Respira- Forschungsgemeinschaft (DFG) SFB1213, project A08 and B04 (to R.S.). tory Cell and Molecular Biology, 58, 170–180. https://doi.org/10.1165/ rcmb.2017‐0214OC

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