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Originally Published 25 October 2021
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Resolvin E1 Attenuates Pulmonary Hypertension by Suppressing Wnt7a/β-Catenin Signaling

Guizhu Liu, Naifu Wan, Qian Liu, Yuqin Chen https://orcid.org/0000-0002-7011-7250, Hui Cui, Yuanyang Wang, Jiaoqi Ren, Xia Shen, Wenju Lu, Ying Yu [email protected], Yujun Shen https://orcid.org/0000-0002-9266-9064 [email protected], and Jian Wang https://orcid.org/0000-0002-1278-256X [email protected]Author Info & Affiliations
Hypertension
Volume 78, Number 6
https://doi.org/10.1161/HYPERTENSIONAHA.121.17809
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Graphical Abstract

Abstract

Pulmonary arterial hypertension (PAH) is a devastating disease characterized by severe pulmonary vascular wall remodeling and perivascular inflammation. Resolvin E1 (RvE1), a proresolving lipid mediator, has protective effects against various inflammatory diseases. However, the effect of RvE1 on PAH development remains to be determined. We aimed to investigate whether RvE1 has a therapeutic effect on PAH and, if so, to elucidate the molecular mechanisms underlying its effects. A hypoxia+SU5416-induced mouse model of pulmonary hypertension (PH) and an monocrotaline-induced rat model of PH were used to test therapeutic effect of RvE1. Lung tissues and plasma samples were collected from patients with PAH and rodent models to examine RvE1 production and its receptor chemerin chemokine-like receptor 1 (ChemR23) expression. We observed that RvE1 generation was reduced in the plasma of patients with idiopathic PAH and in lungs from experimental rodent models of PH. ChemR23 expression was markedly downregulated in hypoxia-exposed mouse pulmonary artery smooth muscle cells (PASMCs) and pulmonary arteries from PH rodents and patients with idiopathic PAH. RvE1 treatment alleviated experimental PH in both male and female rodents by inhibiting PASMC proliferation. Deletion of ChemR23 in vascular SMCs abolished the protective effect of RvE1 against hypoxia+SU5416-induced PAH in mice. Mechanistically, the RvE1/ChemR23 axis suppressed hypoxia-induced PASMC proliferation by inhibiting proliferative wingless-type MMTV integration site family member 7a/β-catenin signaling. Activation of ChemR23 by RvE1 diminished wingless-type MMTV integration site family member 7a expression in PASMCs by inhibiting protein kinase A-mediated Egr2 (early growth response 2) phosphorylation at Ser349. Thus, the RvE1/ChemR23 axis represses experimental PAH by modulating wingless-type MMTV integration site family member 7a/β-catenin signaling in PASMCs and may serve as a therapeutic target for the management of PAH.

Introduction

Pulmonary arterial hypertension (PAH) is a rare but often fatal vascular disorder that is clinically defined as mean pulmonary arterial pressure >20 mm Hg, normal left atrial pressure, and pulmonary vascular resistance ≥3 Wood units.1 Pathologically, PAH is characterized by progressive remodeling and obliteration of small pulmonary arteries, leading to elevated pulmonary arterial pressure and ultimately, right heart failure. Pulmonary arterial smooth muscle cell (PASMC) hypertrophy and proliferation are the main processes in pathological pulmonary vascular remodeling in PAH.2 PASMC proliferation is triggered by endothelial dysfunction, hypoxia, inflammation, or mechanical stress and is amplified by vasoconstrictors, growth factors, chemokines, and abnormal extracellular matrix components.3 Currently available drugs, such as prostacyclin analogs, endothelin receptor antagonists, and phosphodiesterase inhibitors improve PAH symptoms but fail to reverse pulmonary vascular remodeling and decrease overall PAH mortality during long-term follow-up.4 Therefore, there is an urgent need to identify novel therapeutic targets, such as agents aimed at pulmonary vascular remodeling, to improve patient outcomes.5
Resolvins, including the resolvin E and resolvin D series, are specialized proresolving mediators derived from omega-3 fatty acids, primarily eicosapentaenoic, acid and docosahexaenoic acid. Resolvin E1 (RvE1; 5S, 12R, 18-R-trihydroxyeicosapentaenoic acid) is generated from eicosapentaenoic acid by sequential reactions in the P450 enzymatic pathway or by transcellular biosynthesis of cyclooxygenase/5-lipoxygenase.6 RvE1 has protective effects against multiple inflammatory diseases, such as peritonitis,6 asthma,7 atherosclerosis,8 myocardial infarction,9 and even cancer.10 A synthetic RvE1 analog11 is under human clinical trials for the treatment of ocular inflammatory diseases. Moreover, RvE1 has been demonstrated to inhibit thromboxane- or cytokine-induced contractility of human pulmonary artery12,13 and to attenuate injury-induced vascular neointimal formation in mice.14 Treatment of eicosapentaenoic acid, a RvE1 precursor, significantly alleviates monocrotaline-induced PAH in rats.15 However, whether RvE1 has a direct therapeutic effect on PAH remains to be determined.
At least 2 G protein-coupled receptors are involved in transducing RvE1 signals: chemerin chemokine-like receptor 1 (ChemR23) and BLT1 (leukotriene B4 receptor 1). BLT1 is also activated by leukotriene B4 and is highly expressed in granulocytes and activated T cells. ChemR23, which is also a receptor for chemerin, is abundant in monocytes and dendritic cells. RvE1 promotes resolution of inflammation via ChemR23 on macrophages and dendritic cells, whereas it attenuates polymorphonuclear neutrophil transendothelial migration by BLT1 receptor.6,16 Notably, ChemR23 is also highly expressed in vascular smooth muscle cells (VSMCs) and plays important role in maintaining VSMC functions such as proliferation and contraction.17,18 RvE1 confers vascular protection against atherosclerosis,19 aortic valve stenosis,20 and vascular calcification21 through ChemR23. However, the role of the RvE1/ChemR23 axis in hypoxia-induced pulmonary vascular remodeling remains unclear.
In this study, we analyzed RvE1 production in patients with idiopathic PAH and animal models and explore potential role of RvE1/ChemR23 axis in PAH.

Methods

The data that support the findings of this study are available from the corresponding authors on reasonable request. Detailed Methods are available in the Supplemental Material.

Mice

Eight-to-10-week-old male/female mice were used in all experiments in this study. ChemR23Flox/Flox (ChemR23F/F) mice were generated at Shanghai Model Organisms Center Inc (Shanghai, China), using the CRISPR/Cas9 strategy. ChemR23F/FSM22 (smooth muscle protein 22)Cre mice were generated by crossing ChemR23F/F mice with SM22Cre transgenic mice.22 Further details are described in the Supplemental Material.

Rodent Models of PH

A hypoxia+SU5416 (HySu)-induced mouse model of pulmonary hypertension (PH) and an monocrotaline-induced rat model of PH were used to examine the effect of RvE1 on PH development.22–25 RvE1 was administered daily by intraperitoneal injection (10 μg/kg)7,26 at the beginning of HySu exposure or at the third week after monocrotaline injection. Further details are described in the Supplemental Material.

Histological Analysis

Histological analysis was performed as previously described,23,24,27 and further details are described in the Supplemental Material.

Measurement of RvE1 Levels

RvE1 was measured by liquid chromatography-tandem mass spectrometry as previously described,28 and further details are described in the Supplemental Material.

Cell Culture

Primary mouse PASMCs and primary mouse pulmonary arterial endothelial cells (PAECs) were isolated and cultured as previously described.22,29 Human PASMCs (cat: PCS-100-023) were purchased from the American Type Culture Collection (Manassas, VA). Human PAECs were obtained from Lonza, Cleveland Clinic. Further details are described in the Supplemental Material.

RNA Sequencing and Analysis

Primary mouse PASMCs with hypoxia exposure were treated with RvE1 (4 ng/mL) or phosphate buffered saline (PBS) for 48 hours before collected. RNA sequencing analyses were performed as previously described.30–32 Further details are described in the Supplemental Material.

Electroporation

Electroporation of mouse PASMCs was performed as previously described.33 Further details are described in the Supplemental Material.

Statistics

All data are expressed as the mean±standard error of the mean (SEM). Data analysis was performed using GraphPad Prism software (version 6; GraphPad Software Inc, San Diego, CA). The normal distribution of data was examined using the Shapiro-Wilk normality test. Means of 2 groups were compared using the Mann-Whitney U test. Multiple-group comparisons were made by Kruskal-Wallis tests followed by Dunn multiple comparisons tests for post hoc analyses (95% CI). P<0.05 was considered statistically significant. Randomization and blind analyses were performed whenever possible.

Results

The RvE1/ChemR23 Axis Is Downregulated in PAs From PH Rodent Models and Patients With Idiopathic PAH

As an anti-inflammatory and proresolving mediator, RvE1 is lower in the plasma of patients with chronic inflammation diseases than in healthy volunteers, which is negatively related to disease severity.19,20,34 Accordingly, liquid chromatography-tandem mass spectrometry (Figure S1A in the Supplemental Material) revealed markedly reduced RvE1 levels in plasma from patients with idiopathic PAH as compared with plasma from healthy volunteers and in lung tissues from hypoxia-exposed mice and monocrotaline-treated rats as compared with tissues obtained from controls (Figure S1B through S1D). ChemR23, but not BLT1, was notably downregulated in both mouse and human PASMCs (Figure 1A) in response to hypoxia, as well as in pulmonary arteries (PAs) from experimental models of PH (Figure 1B and 1C). ChemR23 expression were detected in both mouse PAECs and PASMCs (Figure S2A), with relatively low in human PAECs (Figure S2B and S2C). Consistent herewith, immunofluorescence staining showed that ChemR23 mainly colocalized with α-SMA (alpha-smooth muscle actin) in the tunica media of PAs and was dramatically downregulated in PAs from experimental models of PH and patients with idiopathic PAH (Figure 1D through 1F).
Figure 1. ChemR23 is downregulated in pulmonary arteries (PAs) from pulmonary hypertension (PH) rodent models and patients with idiopathic pulmonary arterial hypertension (PAH). A, Relative mRNA levels of BLT1 and ChemR23 in cultured mouse pulmonary arterial smooth muscle cell (PASMCs; left) and human PASMCs (right) in response to hypoxia. *P<0.05 vs control (n=6). B, Relative mRNA levels of BLT1 and ChemR23 in PAs from mice subjected to hypoxia and SU5416 (HySu). *P<0.05 vs normoxia (n=6). C, Relative mRNA levels of BLT1 and ChemR23 in PAs from monocrotaline (MCT)-treated rats. *P<0.05 vs vehicle (n=6). D, Left: Representative immunofluorescence images of ChemR23 (red) and α-SMA (green) expression in PAs from HySu-treated mice. Scale bar: 50 μm; (right) Quantification of ChemR23 expression in left. *P<0.05 vs normoxia (n=6). E, Left: Representative immunofluorescence images of ChemR23 (red) and α-SMA (green) expression in PAs from MCT-treated rats. Scale bar: 50 μm; (right) Quantification of ChemR23 expression in left. *P<0.05 vs vehicle (n=6). F, Left: Representative immunofluorescence images of ChemR23 (red) and α-SMA (green) expression in PAs from patients with PAH. Scale bar: 50 μm; (right) Quantification of ChemR23 expression in (left). *P<0.05 vs normal (n=5–6). Data represent the mean±SEM. Statistical significance was evaluated using the Mann-Whitney U test. α-SMA indicates alpha-smooth muscle actin.

The RvE1/ChemR23 Axis Inhibits Hypoxia-Induced PASMC Proliferation by Arresting Cells at the G0/G1 Phase

Dose-dependent inhibition of hypoxia-induced proliferation in human PASMCs was observed by RvE1 treatment (Figure S3A), as evidenced by gradually reduced PCNA (proliferating cell nuclear antigen) staining in hypoxia-treated human PASMCs (Figure S3B). Silencing of ChemR23, but not BLT1, abrogated the inhibitory effect of RvE1 on hypoxia-induced human PASMCs proliferation (Figure S3C) and attenuated RvE1-mediated suppression of PCNA expression in hypoxia-exposed PASMCs (Figure S3D). Moreover, RvE1 inhibited hypoxia-induced acceleration of G1-to-S transition in human PASMCs accompanied by downregulation of cyclin D1 and cyclin dependent kinase 4 expression (Figure S3E). Again, knockdown of ChemR23, but not BLT1, abolished RvE1-induced G0/G1 cell cycle arrest of human PASMCs in response to hypoxia (Figure S3F). These results indicated that the RvE1/ChemR23 axis may attenuate PAH development, probably through suppressing PASMCs proliferation.

RvE1 Treatment Attenuates Experimental PH Progression in Rodents

We next assessed whether RvE1 treatment could prevent the progression of HySu-induced PH in mice. RvE1 (10 μg/kg, once per day, intraperitoneally) was administered to mice at the beginning of HySu exposure (Figure 2A). As expected, RvE1 prevented the development of PH in mice by reducing right ventricular systolic pressure and ratio of the weight of the right ventricle to the weight of the left ventricle plus septum (RV/[LV+S] ratio; Figure 2B and 2C) and suppressing pulmonary vascular remodeling, as indicated by decreased pulmonary vascular wall thickness (Figure 2D) and muscularization (Figure 2E). Moreover, RvE1 treatment markedly inhibited HySu-induced mouse PASMC proliferation by repressing PCNA expression (Figures 2F) and reduced HySu-induced perivascular infiltration of macrophages and T cells (Figure S4A through S4D). To further explore the therapeutic effect of RvE1 on established PH, RvE1 was administered to monocrotaline-treated rats once a day at the beginning of the third week after monocrotaline treatment (Figure 3A). Likewise, RvE1 exerted a therapeutic effect on PH and pulmonary vascular remodeling in monocrotaline-treated male and female rats by suppressing PASMCs proliferation (Figure 3B through 3E; Figure S5). VSMC-specific deletion of ChemR23 (ChemR23F/FSM22Cre, Figure S6) aggravated HySu-induced PH in male and female mice, with significant elevations in right ventricular systolic pressure and RV/(LV+S) and increased pulmonary vascular wall thickness, muscularization, and PASMC proliferation (Figure 4A through 4E; Figure S7). Thus, RvE1 prevents hypoxia-induced PAH and pulmonary vascular remodeling by inhibiting PASMCs proliferation via the ChemR23 receptor.
Figure 2. Resolvin E1 (RvE1) prevents the development of hypoxia+SU5416 (HySu)-induced pulmonary hypertension (PH) in mice. A, Protocol for administration of RvE1 to mice exposed to HySu. B and C, Right ventricular systolic pressure (RVSP; B) and RV/left ventricular (LV)+S ratio (C) in mice exposed to HySu with RvE1 treatment. *P<0.05 vs PBS, #P<0.05 vs control (n=8). D, Up: Representative images of H&E staining and immunofluorescence staining of α-SMA (red) in PAs from HySu-exposed mice with RvE1 treatment. Scale bars: 50 μm; (down). Quantification of the ratio of vascular medial thickness to total vessel size of pulmonary arteries (PAs) from HySu-exposed mice with RvE1 treatment in up. *P<0.05 vs PBS (n=6). E, Proportion of non (N), partially (P), and fully (F) muscularized PAs from HySu-exposed mice with RvE1 treatment #P<0.05 vs control, (n=6). F, Up: Representative images of immunofluorescence staining of α-SMA (green) and PCNA (proliferating cell nuclear antigen) (red) in PAs from HySu-exposed mice with RvE1 treatment. Scale bars: 50 μm; (down) Quantification of PCNA+α-SMA+ cells in up. *P<0.05 vs PBS (n=6). Data represent the mean±SEM. Statistical significance was evaluated using Kruskal-Wallis tests followed by Dunn test. α-SMA indicates alpha-smooth muscle actin.
Figure 3. Resolvin E1 (RvE1) alleviates monocrotaline (MCT)-induced pulmonary hypertension (PH) in rats. A, Protocol for MCT-induced PH in rats. B and C, Effect of RvE1 on right ventricular systolic pressure (RVSP; B) and RV/left ventricular (LV)+S ratio (C) in MCT-treated rats. *P<0.05 vs PBS, #P<0.05 vs vehicle (n=10). D, Up: Representative images of H&E staining and immunofluorescence staining of α-SMA (green) in pulmonary arteries (PAs) from MCT-treated rats with RvE1 treatment. Scale bars: 50 μm (down). Quantification of the ratio of vascular medial thickness to total vessel size of PAs from MCT-treated rats with RvE1 treatment in up. *P<0.05 vs PBS, #P<0.05 vs vehicle, (n=6). E, Up: Representative images of immunofluorescence staining of α-SMA (green) and PCNA (proliferating cell nuclear antigen) (red) in PAs from MCT-treated rats with RvE1 treatment. Scale bars: 50 μm; (down). Quantification of PCNA+α-SMA+ cells in up. *P<0.05 vs PBS, #P<0.05 vs vehicle (n=6). Data represent the mean±SEM. Statistical significance was evaluated using Kruskal-Wallis tests followed by Dunn test. α-SMA indicates alpha-smooth muscle actin.
Figure 4. Vascular smooth muscle cell (VSMC)-specific ChemR23 deletion abrogates the protective effect of resolvin E1 (RvE1) against hypoxia+SU5416 (HySu)-induced pulmonary hypertension (PH) in mice. A, Right ventricular systolic pressure (RVSP) in ChemR23F/FSM22Cre and ChemR23F/F mice exposed to HySu with RvE1 treatment. *P<0.05 vs PBS, #P<0.05 vs ChemR23F/F (n=10). B, RV/left ventricular (LV)+S in ChemR23F/FSM22Cre and ChemR23F/F mice exposed to HySu with RvE1 treatment. *P<0.05 vs PBS, #P<0.05 vs ChemR23F/F (n=10). C, Up: Representative images of H&E staining and immunofluorescence staining of α-SMA (red) in pulmonary arteries (PAs) from HySu-treated ChemR23F/FSM22Cre and ChemR23F/F mice with RvE1 treatment. Scale bars: 50 μm; (down). Quantification of the ratio of vascular medial thickness to total vessel size of PAs from the HySu exposure model in up. *P<0.05 vs PBS, #P<0.05 vs ChemR23F/F (n=6). D, Proportion of non (N), partially (P), and fully (F) muscularized PAs from HySu-treated ChemR23F/FSM22Cre and ChemR23F/F mice with RvE1 treatment. *P<0.05 vs ChemR23F/F, #P<0.05 vs ChemR23F/F (n=6). E, Up: Representative images of immunofluorescence staining of α-SMA (green) and PCNA (proliferating cell nuclear antigen) (red) in PAs from HySu-treated ChemR23F/FSM22Cre and ChemR23F/F mice with RvE1 treatment. Scale bars: 50 μm; (down) Quantification of PCNA+α-SMA+ cells in up. *P<0.05 vs PBS, #P<0.05 vs ChemR23F/F (n=6). Data represent the mean±SEM. Statistical significance was evaluated using Kruskal-Wallis tests followed by Dunn test. α-SMA indicates alpha-smooth muscle actin; and SM22, smooth muscle protein 22.

The RvE1/ChemR23 Axis Suppresses Hypoxia-Induced PASMCs Proliferation by Suppressing Wnt7a/β-Catenin Signaling

To understand the mechanisms by which RvE1 regulates PASMCs proliferation, we compared RNA-seq data of hypoxia-challenged mouse primary PASMCs treated or not with RvE1 and identified 85 upregulated genes and 136 downregulated genes (Figure S8A). Among known proproliferative genes, 8 top down-regulated genes were evaluated further (Figure S8B). The RvE1-induced downregulation in mRNA expression of leucine rich repeat containing 17, thrombopoietin, wingless-type MMTV integration site family member 7a (Wnt7a), and angiotensin II receptor type 2 were confirmed in mouse PASMCs exposed in hypoxia by real-time quantitative polymerase chain reaction (Figure S8C). Interestingly, knockdown of ChemR23 specifically reversed the RvE1-induced reduction in Wnt7a mRNA expression in hypoxia-treated mouse PASMCs (Figure S8D). Accordingly, RvE1 reduced Wnt7a protein expression and strongly inhibited the activity of canonical Wnt/β-catenin signaling, as evidenced by markedly increased phosphorylated β-catenin (Ser33/37/Thr41; Figure 5A) and reduced β-catenin nuclear translocation Figure S9. Knockdown of Wnt7a (Figure 5B) abolished ChemR23 silencing-promoted proliferation of RvE1-treated mouse PASMCs (Figure 5C) by increasing β-catenin phosphorylation and consequently suppressing its target genes-Cyclin D1 and PCNA (Figure 5D). Consistent herewith, RvE1 treatment reduced Wnt7a expression in PAs from monocrotaline-treated rats and HySu-exposed mice (Figures 5E), while ChemR23 deletion restored the reduced expression of Wnt7a in PAs from HySu-exposed mice (Figure 5F). Collectively, these findings indicated that the RvE1/ChemR23 axis attenuates hypoxia-induced PAH by suppressing PASMC proliferation by inhibiting Wnt7a/β-catenin signaling.
Figure 5. The resolvin E1 (RvE1)/ChemR23 axis suppresses pulmonary arterial smooth muscle cell (PASMC) proliferation in response to hypoxia via Wnt7a/β-catenin signaling. A, Protein levels of Wnt7a, phosphorylated β-catenin(p-β-catenin), and total β-catenin in hypoxia-challenged mouse PASMCs with RvE1 treatment as determined by Western blotting (n=6). B, Knockdown efficiency of Wnt7a siRNA in mouse PASMCs as assessed by real-time polymerase chain reaction. *P<0.05 vs scramble (n=6). C, Effect of Wnt7a knockdown on the proliferation of hypoxia-challenged mouse PASMCs with RvE1 treatment. *P<0.05 vs scramble (n=6). D, Western blot analysis of the effect of Wnt7a knockdown on Wnt7a/β-catenin signaling components in hypoxia-challenged mouse PASMCs with RvE1 treatment after transfection of ChemR23 siRNA (n=6). E, Up, Representative images of immunofluorescence staining of α-SMA (green) and Wnt7a (red) in pulmonary arteries (PAs) from monocrotaline (MCT)-treated rats with RvE1 treatment. Scale bars: 50 μm; (down) Quantification of Wnt7a expression in PAs in up *P<0.05 vs PBS, #P<0.05 vs vehicle (n=6). F, Up: Representative images of immunofluorescence staining of α-SMA (green) and Wnt7a (red) in PAs from HySu-exposed ChemR23F/FSM22cre and ChemR23F/F mice with RvE1 treatment. Scale bars: 50 μm (down). Quantification of Wnt7a expression in PAs in up. *P<0.05 vs RvE1, #P<0.05 vs ChemR23F/F (n=6). Data represent the mean±SEM. Statistical significance was evaluated using the Mann-Whitney U test or Kruskal-Wallis tests followed by Dunn test. α-SMA indicates alpha-smooth muscle actin; and SM22, smooth muscle protein 22.

The RvE1/ChemR23 Axis Inhibits the Transcriptional Expression of Wnt7a in Hypoxia-Treated PASMCs Through Suppressing PKA-Mediated Egr2 Phosphorylation

ChemR23 is coupled to heterotrimeric Gi-type G protein35 and triggers multiple signaling cascades, including PKA (protein kinase A),36 protein kinase C,37 and Rho-Rho associated coiled-coil containing protein kinase signaling.38 Blockade of Gi by treatment with the pertussis toxin abrogated the inhibitory effect of RvE1 on Wnt7a expression in hypoxia-challenged mouse PASMCs cells (Figure S10A). Inhibition of protein kinase C and Rho associated coiled-coil containing protein kinase activity had no significant influence on RvE1-mediated suppression of Wnt7a/β-catenin signaling (Figure S10B), while inhibition of PKA activity by H89 abolished the increase in Wnt7a expression induced by ChemR23 silencing in hypoxia-challenged mouse PASMCs (Figure S10C). We next examined the impact of RvE1 on Wnt7a transcriptional activity using promoter truncation and luciferase assays. We found that the Wnt7a promoter region at −200 to −100 bp mediated the inhibitory luciferase activity of RvE1 (Figure S10D). Several transcription factors, such as E2F Transcription Factor 6, Egr (early growth response) 1, Egr2, and Kruppel Like Factor 5, were predicted to bind to this region using the Homer software (http://homer.ucsd.edu/homer/, Table S3). Notably, mutations in the binding motif of Egr2 and Kruppel Like Factor 5 eliminated the suppressive effect of RvE1 on Wnt7a promoter activity (Figure S10D). Furthermore, knockdown of Egr2, but not Kruppel Like Factor 5 completely abolished forskolin-induced Wnt7a expression and its downstream signaling in mouse PASMCs (Figure S10E). Chromatin immunoprecipitation assay confirmed that the recruitment of Egr2 to Wnt7a promoter was dramatically decreased in hypoxia-treated mouse PASMCs upon RvE1 treatment (Figures 6A). Interestingly, a conserved PKA phosphorylation motif RRXS (arginine-arginine- any amino acid-serine) (Ser349) was identified in the Egr2 protein of different species (Figure 6B) by NetPhos 3.1 Server 3.39 Indeed, adenylate cyclase activator forskolin promoted, whereas RvE1 inhibited PKA-mediated Egr2 phosphorylation in mouse PASMCs (Figures 6C and 6D), and mutation of Ser349 to alanine (S349A) prevented forskolin-induced or RvE1-suppressed phosphorylation of Egr2 (Figures 6C and 6D). Moreover, forced expression of WT Egr2, but not the S349A mutant, drove Wnt7a promoter activity, and RvE1 markedly inhibited WT Egr2 transcriptional activity (Figure 6E). We observed reduced luciferase activity in Egr2 S349A-transfected cells after RvE1 treatment (Figure 6E), probably because of the effect of RvE1 on endogenous Egr2 expression in mouse PASMCs. Taken together, these findings indicated that RvE1/ChemR23 axis suppresses Wnt7a expression through the Gi/PKA/Egr2 pathway (Figure 6F).
Figure 6. Resolvin E1 (RvE1)/ChemR23 axis inhibits the transcription expression of Wnt7a in hypoxia-challenged mouse vascular smooth muscle cells (VSMCs) through suppressing PKA (protein kinase A)-mediated Egr (early growth response) 2 phosphorylation. A, Up: Gel electrophoresis of PCR-amplified Egr2 binding motif-containing fragments from mouse Wnt7a promoter in hypoxia-exposed mouse pulmonary arterial smooth muscle cell (PASMCs) with or without RvE1 treatment; (down). ChIP analysis of Egr2 binding to the Wnt7a promoter in RvE1-treated mouse PASMCs. *P<0.05 vs PBS, n=6. B, Amino acid sequence alignment of mouse, human, rat, dog, pig, and frog Wnt7a proteins. Ser349 is indicated by an arrowhead, the box indicates the phospho-PKA substrate motif. C, Effect of Egr2 S349A mutant on forskolin-induced Egr2 phosphorylation, detected as the levels of phospho(p)-PKA-substrate (RRXS*/T*) on flag-tagged WT and S349A mutant Egr2 in mouse PASMCs (n=6). D, Effect of Egr2 S349A mutant on RvE1-induced Egr2 phosphorylation, detected as the levels of p-PKA-substrate (RRXS*/T*) on flag-tagged WT and S349A mutant Egr2 in mouse PASMCs (n=6). E, Luciferase reporter assay of RvE1-regulated Wnt7a promoter activity in mouse PASMCs transfected with flag-tagged WT and S349A mutant Egr2 plasmids. *P<0.05 vs vector, $P<0.05 vs WT, #P<0.05 vs PBS (n=6). F, Schematic illustration of ChemR23-mediated inhibition on PASMC proliferation via suppression of Wnt7a-β-catenin signaling in a Gi/PKA/Egr2-dependent manner. Data represent the mean±SEM. Statistical significance was evaluated using Kruskal-Wallis tests followed by Dunn test.

Discussion

The RvE1/ChemR23 axis promotes inflammation resolution by modulating immune cell functions. In this study, we observed that RvE1 generation was reduced in lung tissues from PAH animals and in plasma from patients with PAH, and its receptor ChemR23 was markedly downregulated in pulmonary arteries in experimental PAH rodents. RvE1 treatment attenuated the development of experimental PH in rodents of both sexes through the suppression of pulmonary artery remodeling. VSMC-specific ablation of the ChemR23 receptor abolished the protective effect of RvE1 on PAH. Mechanistically, the RvE1/ChemR23 axis suppressed hypoxia-induced PASMC proliferation by suppressing PKA/Egr2-mediated Wnt7a/β-catenin signaling. These results suggest that the proresolving molecule RvE1 may be beneficial for the management of PAH.
Accumulating evidence demonstrates that damping inflammation attenuates experimental PH by suppressing PASMC proliferation.40,41 As a powerful anti-inflammatory agent, RvE1 has been demonstrated to have protective effects against vascular injuries in various animal models. For example, RvE1 prevents Western diet-induced atherogenesis in rabbits by reducing the intima/media ratio42 and attenuates wire injury-induced vascular neointimal formation in mice.14 In vitro, RvE1 has been shown to inhibit the enhanced proliferation in human PASMCs derived from patients with idiopathic PAH.15 RvE1 also inhibits transforming growth factor beta 1-triggered mesenchymal transition of PAECs.15 We observed reduced RvE1 generation in both PAH patients and hypoxia-exposed animals, probably due to pulmonary endothelial dysfunction in the disease status.43 Interestingly, RvE1 at 4 ng/mL effectively inhibits hypoxia-induced proliferation of human PASMCs and administration of RvE1 (10 μg/kg) significantly attenuates the development of experimental PH in rodents through suppressing pulmonary vascular remodeling. Indeed, the effect of nanogram amounts of RvE1 in vitro are comparable to those achieved by introducing microgram amounts of RvE1 in vivo as observed in many animal models of inflammatory diseases.6,7,44
RvE1 can exert its anti-inflammatory effects by acting through the ChemR23 receptor.45 Disruption of RvE1/ChemR23 signaling in hyperlipidemic mice increases atherosclerotic plaque size and necrotic core formation.8 And the RvE1/ChemR23 axis mediates the protective effect of omega-3 polyunsaturated fatty acids against aortic valve calcification in atherogenic mice.20 We14 and others46 also observed that RvE1 treatment suppresses injury-induced vascular neointimal hyperplasia by acting on ChemR23. We observed ChemR23 was required for RvE1-mediated inhibition of hypoxia-induced PASMC proliferation and deletion of ChemR23 in VSMCs abrogated the beneficial effects of RvE1 on hypoxia-induced PA remodeling in mice. In agreement with our results, treatment of ChemR23 agonist chemerin-9 suppresses the migration and proliferation of human aortic smooth muscle cells and attenuate aortic atherosclerotic lesions in Apoe–/– mice.47 On the contrary, others reported that chemerin/ChemR23 signaling stimulated proliferation in human VSMCs,48 and in conjunction with endothelin-1, also promoted proliferation and migration in rat PASMCs.17 These discrepancies might be due to the different receptor conformations of chemR23 bond by chemerin peptides and RvE1, which resulted in various levels of activation of different downstream signaling molecules.49
Both canonical and noncanonical Wnt/β-catenin pathways are involved in the pathogenesis of PAH, including heritable and idiopathic PAH.50 In culture, hypoxic exposure and inflammatory cytokines activate the Wnt/β-catenin pathway in PASMCs and then promote proliferative responses by increasing the expression of cell cycle proteins.51,52 Pharmaceutical inhibition of Wnt/β-catenin signaling ameliorates pulmonary hypertension in rodents by suppressing PASMC proliferation and pulmonary vascular remodeling.53 Indeed, Wnt proteins such as Wnt3a, Wnt5a, and Wnt4 induced by different cytokines in VSMCs facilitate VSMC proliferation in an autocrinal manner.54 Likewise, Wnt7a, another activator of Wnt/β-catenin signaling, was markedly upregulated in PASMCs in PAH mice and promoted PASMC proliferation, which suppressed the RvE1/ChemR23 axis. Consistent herewith, the proproliferation property of Wnt7a/β-catenin is also observed in neural stem cells and myogenic stem cells55 and in ovarian cancer cells.56
The Egr family members, including Egr1, 2, 3, and 4 are C(2)H(2)-type zinc-finger transcription factors that regulate a wide range of biological processes. Egr1 and Egr2 are abundantly expressed in VSMCs and can be induced in response to vascular stresses.57 Egr1 has been demonstrated to promote VSMC proliferation in multiple vascular pathological processes, including vascular injury58 and PAH.59 Here, we demonstrated that Egr2 directly drove Wnt7a expression in PASMCs and its transcriptional activity was regulated by the RvE1/ChemR23 axis in a PKA-dependent manner. Phosphorylation of Egr2 (Ser349) by PKA promoted Wnt7a transcription and hypoxia-induced proliferation of PASMCs. Likewise, Egr2 promotes adenosine A (3) receptor-induced proliferation of human coronary smooth muscle cells.60

Perspectives

PAH is a progressive, fatal pulmonary circulatory disease characterized by excessive PASMCs proliferation and hypertrophy. We found RvE1/ChemR23 axis attenuated hypoxia-induced PAH in rodents through inhibition of Wnt7a-mediated PASMCs proliferation. Given that reduced generation of RvE1 in patients with idiopathic PAH, supplementation of additional RvE1 or targeting its receptor ChemR23 may be beneficial in the management of PAH in clinic.

Novelty and Significance

What Is New?

•
Resolvin E1 (RvE1) confers protective effects against various inflammatory diseases in animal models including acute lung injury and asthma. Little is known about its effects on the development of pulmonary arterial hypertension (PAH). We provided evidences that RvE1 alleviated experimental PAH in both male and female rodents by suppression of pulmonary vascular remodeling.
•
RvE1 regulates vascular functions, the underlying mechanism is still unknown. We revealed that RvE1 suppresses hypoxia-induced pulmonary artery smooth muscle cell proliferation by chemerin chemokine-like receptor 1 (ChemR23)-mediated inhibition of Wnt signaling. Ablation of ChemR23 in vascular smooth muscle cells abrogated the beneficial effects of RvE1 on PAH.

What Is Relevant?

•
Serum RvE1 levels are markedly decreased in patients with severe idiopathic PAH. Administration of additional RvE1 or activation of ChemR23 receptor might improve clinical outcomes in patients with PAH.

Summary

RvE1/ChemR23 axis may serve as a novel therapeutic target for PAH.

Footnote

Nonstandard Abbreviations and Acronyms

Egr
early growth response
HySu
hypoxia+SU5416
PAEC
pulmonary arterial endothelial cell
PAH
pulmonary arterial hypertension
PAs
pulmonary arteries
PASMC
pulmonary arterial smooth muscle cell
PH
pulmonary hypertension
PKA
protein kinase A
RvE1
resolvin E1
VSMC
vascular smooth muscle cells

Supplemental Material

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Information

Published In

Go to Hypertension
Go to Hypertension
Hypertension
Volume 78 • Number 6 • December 2021
Pages: 1914 - 1926
PubMed: 34689593

Copyright

© 2021 American Heart Association, Inc.

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History

Received: 2 June 2021
Accepted: 19 September 2021
Published online: 25 October 2021
Published in print: December 2021

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Keywords

  1. ChemR23
  2. PASMCs
  3. pulmonary arterial hypertension
  4. Resolvin E1
  5. Wnt7a

Subjects

  1. Pulmonary Hypertension
  2. Remodeling
  3. Vascular Disease

Authors

Affiliations

Guizhu Liu*
From the State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China (G.L., Y.C., W.L., Y.Y., J.W.)
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Naifu Wan*
Department of Cardiology, Ruijin Hospital, Shanghai Jiao tong University School of Medicine, Shanghai, China (N.W.)
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Qian Liu*
Department of Pharmacology, Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China (Q.L., Y.W., Y.Y., Y.S.)
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Yuqin Chen https://orcid.org/0000-0002-7011-7250
From the State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China (G.L., Y.C., W.L., Y.Y., J.W.)
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Hui Cui
School of Life Science and Technology, Shanghai Tech University, Shanghai, China (H.C., X.S.)
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Yuanyang Wang
Department of Pharmacology, Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China (Q.L., Y.W., Y.Y., Y.S.)
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Jiaoqi Ren
Department of Geriatrics, Huashan Hospital, Fudan University, Shanghai, China (J.R.)
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Xia Shen
School of Life Science and Technology, Shanghai Tech University, Shanghai, China (H.C., X.S.)
CAS Key Laboratory of Nutrition, Metabolism and Food Safety, Shanghai Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China (X.S., Y.Y.).
View all articles by this author
Wenju Lu
From the State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China (G.L., Y.C., W.L., Y.Y., J.W.)
View all articles by this author
Ying Yu† [email protected]
From the State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China (G.L., Y.C., W.L., Y.Y., J.W.)
Department of Pharmacology, Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China (Q.L., Y.W., Y.Y., Y.S.)
CAS Key Laboratory of Nutrition, Metabolism and Food Safety, Shanghai Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China (X.S., Y.Y.).
View all articles by this author
Yujun Shen† https://orcid.org/0000-0002-9266-9064 [email protected]
Department of Pharmacology, Tianjin Key Laboratory of Inflammatory Biology, Center for Cardiovascular Diseases, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China (Q.L., Y.W., Y.Y., Y.S.)
View all articles by this author
Jian Wang† https://orcid.org/0000-0002-1278-256X [email protected]
From the State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China (G.L., Y.C., W.L., Y.Y., J.W.)
Department of Medicine, University of California, San Diego, La Jolla (J.W.)
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Notes

*
G. Liu, N. Wan, and Q. Liu contributed equally.
†
Y. Yu, Y. Shen, and J. Wang contributed equally.
The Supplemental Material is available with this article at Supplemental Material.
For Sources of Funding and Disclosures, see page 1924.
Correspondence to: Jian Wang, State Key Laboratory of Respiratory Disease, The First Affiliated Hospital, Guangzhou Medical University, 151 Yan jiang Rd, Guangzhou, Guangdong 510120, China, Email [email protected]
Yujun Shen, Department of Pharmacology, School of Basic Medical Science, Tianjin Medical University, 22 Qixiang tai Rd, Tianjin 300070, China, Email [email protected]
Ying Yu, Department of Pharmacology, School of Basic Medical Science, Tianjin Medical University, 22 Qixiang tai Rd, Tianjin 300070, China, Email [email protected]

Disclosures

None.

Sources of Funding

This work was supported by grants from the National Science Foundation of China (81900046, 82030015, 81630004, 81800061, 81970540, 31771269, 82120108001), Guangdong Department of Science and Technology Grant (2019A1515010672), Guangdong Science and Technology Foundation (2019B030316028), National Key Technology R&D Program (2018YFC1311900), and State Key Laboratory of Respiratory Disease Independent Program Young Scholar Project (SKLRD-QN-201906). Postgraduate Innovation Fund of 13th Five-Year comprehensive investment, Tianjin Medical University (YJSCX201808) and the Natural Science Foundation of Tianjin (18JCYBJC27300). Y. Yu is a fellow at the Jiangsu Collaborative Innovation Center for Cardiovascular Disease Translational Medicine.

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Resolvin E1 Attenuates Pulmonary Hypertension by Suppressing Wnt7a/β-Catenin Signaling
Hypertension
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