Pulmonary vascular remodeling in Fra-2 transgenic mice is driven by type 2 inflammation and accompanied by pulmonary vascular hyperresponsiveness

Abstract

Lung vessel remodeling leads to increased pulmonary vascular resistance, causing pulmonary arterial hypertension (PAH), and consequently right ventricular hypertrophy and failure. In patients suffering from systemic sclerosis (SSc), PAH can occur and is a life-threatening complication. Dysregulation of immune processes plays a crucial role in pulmonary vascular remodeling, as has previously been shown in Fos-related antigen-2 (Fra-2) transgenic (TG) mice, a model of SSc-PAH. Here, we investigate whether vascular remodeling in the Fra-2 TG model is driven by type 2 inflammation and is associated with vascular hyperresponsiveness, an important feature of PAH pathobiology. Basal pulmonary arterial pressure and pulmonary vascular responsiveness to hypoxic ventilation and serotonin were increased in isolated, perfused, and ventilated lungs of Fra-2 TG mice compared with wild-type (WT) littermates. Similarly, contractile responses of isolated intrapulmonary arteries were elevated in Fra-2 TG mice. We also observed increased expression of contractile genes in Fra-2 overexpressing human pulmonary arterial smooth muscle cells (PASMCs) with elevated intracellular calcium levels after interleukin (IL)-13 stimulation. These findings were corroborated by transcriptomic data highlighting dysregulation of vascular smooth muscle cell contraction and type 2 inflammation in Fra-2 TG mice. In vivo, type 2-specific anti-inflammatory treatment with IL-13 neutralizing antibodies improved vascular remodeling in Fra-2 TG mice, similar to corticosteroid treatment with budesonide. Our results underscore the importance of type 2 inflammation and its potential therapeutic value in PAH-associated pulmonary vascular remodeling and hyperresponsiveness in SSc-PAH.NEW & NOTEWORTHY In patients suffering from systemic sclerosis (SSc), pulmonary arterial hypertension (PAH) is a life-threatening complication linked to immune dysregulation. Preclinical analyses in Fos-related antigen-2 (Fra-2) transgenic (TG) mice, a model of SSc-PAH, identify type 2 inflammation as a key driver of vascular remodeling. Anti-inflammatory treatment targeting type 2 inflammation via IL-13 neutralizing antibodies improved pulmonary vascular remodeling. Thus, type 2-specific anti-inflammatory treatment may be a promising therapeutic approach in SSc-PAH.

Keywords: hypoxic pulmonary vasoconstriction; pulmonary hypertension; type 2 inflammation; vascular hyperresponsiveness; vascular remodeling.

Graphical abstract

Figure 1. Fra-2 overexpression in mice leads to increased pulmonary vascular remodeling and altered pulmonary artery blood flow velocity.

A) Schematic representation of experimental setup using Fra-2 transgenic (TG) and wildtype (WT) littermate control mice. B) Collagen (Picrosirius red) and von Willebrand factor (vWF)/α-smooth muscle actin (α-SMA) double staining of pulmonary vessels in Fra-2 TG or WT control mice at the age of 8 or 16 weeks. C) Quantification of muscularization of small pulmonary vessels (25-250 μm diameter) in 8- and 16-week-old WT (n=7) and Fra-2 TG (n=7/4) mice. Non m.= non muscularized, part m.= partially muscularized, fully m.= fully muscularized. Unpaired t-tests were used to analyze the difference of non, partially and fully muscularized vessels in WT and Fra-2 TG mice. *p <0.05, **p <0.01 D) Quantification of mean vessel wall thickness in 8 (left panel) and 16 (right panel) week old Fra-2 TG and WT mice. Each datapoint represents one animal (n=4-6). E) Representative images of Doppler blood flow velocity waveforms in Fra-2 TG and WT control mice at 8 and 16 weeks of age. F) Pulmonary Acceleration Time of pulmonary arteries in Fra-2 TG and WT mice. Each datapoint represents one animal (n=6-8). One way-ANOVA with Sidak’s multiple comparison test, *p <0.05, ***p <0.001.

Figure 2. Fra-2 transgenic mice possess altered right ventricular function.

A/B) Representative images of cardiac MRI of Fra-2 transgenic (TG) and wildtype (WT) litter mate control mice at 8 and 16 weeks of age. C-G) Analysis of (C) right ventricular (RV) mass, (D) end diastolic RV volume, (E) end systolic RV volume, (F) RV stroke volume and (G) RV ejection fraction of 8- and 16-week-old Fra-2 TG and WT control mice. Each datapoint represents one animal (n=6-7). One-way ANOVA with Sidak’s multiple comparison test, *p <0.05.

Figure 3. Pulmonary arteries of Fra-2 transgenic mice exhibit stimulus-independent vascular hyperresponsiveness.

A) Transcriptome analysis of lung homogenates of 8-week-old Fra-2 TG or WT mice (n=4): Heatmap with unbiased hierarchical clustering of genes annotated to the gene ontology “vascular associated smooth muscle contraction” (GO:0014829), z-scores are shown. B) Schematic representation of isolated perfused and ventilated mouse lung setup. IPL was conducted in mice 14 to 17 weeks of age. C) Mean pulmonary arterial pressure (Ppa mean) of isolated perfused mouse lungs of wildtype (WT; n=9) or Fra-2 transgenic (Fra-2 TG; n=6) mice at baseline. Unpaired t-test, *p=0.0101. D) Representative recording of flow induced pressure changes in WT or Fra-2 TG mouse lungs under normoxic and hypoxic conditions (Ppa normalized to baseline). E) The maximum hypoxia-induced change of Ppa mean in WT (n=9) and Fra-2 TG (n=6) mouse lungs. Unpaired t-test, ***p<0.001. F) Changes of Ppa mean in response to serotonin (5-hydroxytryptamin, 5-HT) administration to the perfusion buffer in isolated perfused lungs of WT (n=6) or Fra-2 TG (n=7) mice. G) Schematic representation of pulmonary artery wire myography. H/I) Assessment of vasoreactivity of the pulmonary arteries isolated from WT (n=3) and Fra-2 TG (n=3) mice upon dose-dependent stimulation with (H) potassium chloride (KCl) and (I) thromboxane analogue U-46619. From each mouse 3-4 PA segments were measured. F-I) Two-way ANOVA with Bonferroni’s multiple comparisons test, ***p <0.001.

Figure 4. Fra-2 transgenic mice exhibit perivascular, type 2-predominant/eosinophilic inflammation.

A) Representative multi-color immunofluorescence images of endothelium (von Willebrand factor = vWF, yellow), smooth muscle cells (α-smooth muscle actin = α-SMA, green), inflammatory cells (CD45⁺, blue), collagen type I (Col1, red) and nuclei (DAPI, white) staining. B) Inflammatory cell counts in the bronchoalveolar lavage fluid (BALF) of Fra-2 TG and WT mice at the age of 8 and 16 weeks. C/D) Relative proportion of alveolar macrophages (AM), eosinophils (Eos), T-cells and B-cells in the BALF of Fra-2 TG and WT control mice at the age of C) 8 weeks and D) 16 weeks. B-D) Two-way ANOVA with Sidak’s multiple comparisons test, ***p <0.001. Each datapoint represents one animal. (E) Transcriptome analysis of lung homogenates of 8-week-old Fra-2 TG or WT mice (n=4): Heatmap with unbiased hierarchical clustering of genes annotated to the gene ontology “type 2 immune response” (GO:0042092), z-scores are shown.

Figure 5. Effects of interleukin (IL)-13 and Fra-2 overexpression on proliferation and intracellular calcium of pulmonary artery smooth muscle cells.

A) Immunohistochemical staining of IL-13 downstream signaling molecule pStat6 in the pulmonary vasculature in Fra-2 TG and WT mice. Arrows indicate intranuclear pStat6 staining in the vascular smooth muscle cell layer of Fra-2 TG mice. B) Fra-2 overexpression in PASMCs after 24, 48 and 72 hours as determined by Western blot analysis. Alpha-tubulin served as loading control. C) Proliferation of PASMCs with and without Fra-2 overexpression in basal medium (0% FCS), with IL-13 stimulation or in full medium (including 5% FCS). Each datapoint represents one measurement. In total, 6 technical replicates from 4 different donor cells were measured. D/E) Expression levels of RhoA, RhoB, ROCK1 and ROCK2 in PASMCs following Fra-2 overexpression and stimulation with IL-13. Two Way-ANOVA with Sidak’s multiple comparison test, *p <0.05, **p <0.01. Each datapoint represents one measurement. In total, 6 technical replicates from 4 different donor cells were measured. F) Results of two Way-ANOVA analysis of D and E. G) Intracellular Ca⁺⁺ in primary human PASMCs following Fra-2 overexpression and IL-13 (10 ng/ml for 24 h) treatment. Two-way ANOVA, **p <0.01.

Figure 6. Dysbalance of arginine metabolism in Fra-2 transgenic mice.

A) Transcriptome analysis of lung homogenates of 8-week-old Fra-2 TG or WT mice (n=4 animals per group): Heatmap with hierarchical clustering of genes annotated to the gene ontology “arginine metabolic process” (GO:0006525), z-scores are shown. B/C) Relative gene expression levels of endothelial nitric oxide synthase (Nos3/eNos), arginase 1 and 2 (Arg1/Arg2) in B) lung homogenates of 8- and 16-week-old Fra-2 TG and WT control mice an (C) pulmonary arteries of 16-week-old Fra-2 TG and WT mice. Unpaired t-test, *p <0.05, ** p<0.01, ***p <0.001. Each datapoint represents one animal. D) ARG1 gene expression in PASMC following Fra-2 overexpression and IL-13 stimulation (n=6 technical replicates from 4 different donor cells). E) Multi-color immunofluorescence images of smooth muscle cells (α-smooth muscle actin = α-SMA, red), Arginase 1 (green) and nuclei (DAPI, white) staining. White arrows indicate Arg1-positive cells in the vicinity of and within the vessel wall. F) NO levels measured in lung tissue homogenates of 16-week-old Fra-2 TG (n=5) and WT (n=4) mice.

Figure 7. Anti-inflammatory treatment using glucocorticosteroids or type 2-specific anti-IL-13 blocking antibodies (partially) ameliorates vascular remodeling in the Fra-2 transgenic mouse model of SSc-PAH.

A) Schematic representation of intranasal (i.n.) treatment with the glucocorticosteroid budesonide (bude) or PBS as control in Fra-2 transgenic (TG) mice. B) Collagen (Picrosirius red, SR) and von Willebrand factor (vWF)/α-smooth muscle actin (α-SMA) double staining of pulmonary vessels of Fra-2 TG mice with budesonide (n=6) or PBS (n=7) treatment. C) Quantification of muscularization of small pulmonary vessels (25-250 μm diameter). Non m.= non muscularized, part m.= partially muscularized, fully m.= fully muscularized. Two-way ANOVA with Sidak’s multiple comparison test, *p <0.05, ***p <0.001. D) Relative gene expression levels of endothelial nitric oxide synthase (Nos3/eNos), arginase 1 and 2 (Arg1/Arg2) in lung tissue of Fra-2 TG mice with (n=6) or without (n=7) budesonide treatment. Unpaired t-test, *p <0.0201. E) Schematic representation of intraperitoneal (i.p.) treatment with IL-13 neutralizing (anti-IL-13) antibodies or IgG isotype controls in Fra-2 TG mice. F) Collagen (Picrosirius red, SR) and vWF/α-SMA double staining of pulmonary vessels in Fra-2 TG mice with anti-IL-13 treatment (n=6) or IgG treatment (n=7). G) Quantification of muscularization of small pulmonary vessels (25-250 μm diameter). Non m.= non muscularized, part m.= partially muscularized, fully m.= fully muscularized. Two-way ANOVA with Sidak’s multiple comparison test, *p <0.05. H) Relative gene expression levels of endothelial nitric oxide synthase (Nos3/eNos), arginase 1 and 2 (Arg1/Arg2) in lung tissue of Fra-2 TG mice with (n=5) and without (n=4) anti-IL-13 treatment.