Activated Endothelial TGFβ1 Signaling Promotes Venous Thrombus Nonresolution in Mice Via Endothelin-1: Potential Role for Chronic Thromboembolic Pulmonary Hypertension

Abstract

Rationale: Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by defective thrombus resolution, pulmonary artery obstruction, and vasculopathy. TGFβ (transforming growth factor-β) signaling mutations have been implicated in pulmonary arterial hypertension, whereas the role of TGFβ in the pathophysiology of CTEPH is unknown.

Objective: To determine whether defective TGFβ signaling in endothelial cells contributes to thrombus nonresolution and fibrosis.

Methods and results: Venous thrombosis was induced by inferior vena cava ligation in mice with genetic deletion of TGFβ1 in platelets (Plt.TGFβ-KO) or TGFβ type II receptors in endothelial cells (End.TGFβRII-KO). Pulmonary endarterectomy specimens from CTEPH patients were analyzed using immunohistochemistry. Primary human and mouse endothelial cells were studied using confocal microscopy, quantitative polymerase chain reaction, and Western blot. Absence of TGFβ1 in platelets did not alter platelet number or function but was associated with faster venous thrombus resolution, whereas endothelial TGFβRII deletion resulted in larger, more fibrotic and higher vascularized venous thrombi. Increased circulating active TGFβ1 levels, endothelial TGFβRI/ALK1 (activin receptor-like kinase), and TGFβRI/ALK5 expression were detected in End.TGFβRII-KO mice, and activated TGFβ signaling was present in vessel-rich areas of CTEPH specimens. CTEPH-endothelial cells and murine endothelial cells lacking TGFβRII simultaneously expressed endothelial and mesenchymal markers and transcription factors regulating endothelial-to-mesenchymal transition, similar to TGFβ1-stimulated endothelial cells. Mechanistically, increased endothelin-1 levels were detected in TGFβRII-KO endothelial cells, murine venous thrombi, or endarterectomy specimens and plasma of CTEPH patients, and endothelin-1 overexpression was prevented by inhibition of ALK5, and to a lesser extent of ALK1. ALK5 inhibition and endothelin receptor antagonization inhibited mesenchymal lineage conversion in TGFβ1-exposed human and murine endothelial cells and improved venous thrombus resolution and pulmonary vaso-occlusions in End.TGFβRII-KO mice.

Conclusions: Endothelial TGFβ1 signaling via type I receptors and endothelin-1 contribute to mesenchymal lineage transition and thrombofibrosis, which were prevented by blocking endothelin receptors. Our findings may have relevant implications for the prevention and management of CTEPH.

Keywords: endarterectomy; endothelial cells; endothelin-1; pulmonary hypertension; thrombosis.

Figure 1.. Platelet-specific deletion of TGFβ1 and venous thrombus resolution.

The percentage of mice that formed a venous thrombus on day 1 after IVC ligation is shown (A). Thrombus resolution (B) and thrombus area at day 21 after IVC ligation (C, D) were measured by ultrasound in Plt.TGFβ-WT (n=10) and Plt.TGFβ-KO (n=9) mice. In panel B, thrombus area at day 21 is expressed relative to thrombus area at day 1 after surgery. In panel C, representative ultrasound snapshots of thrombosed IVC segments in Plt.TGFβ-WT and Plt.TGFβ-KO mice at day 21 after IVC ligation are shown (with 3-dimensional thrombus reconstruction as insert). Representative pictures of longitudinal cross-sections through the thrombosed IVC at day 21 after ligation following Carstairs’ staining (E) and quantification of thrombofibrosis (blue signal) (n=10 Plt.TGFβ-WT and n=9 Plt.TGFβ-KO mice; F). Representative pictures after Sirius red stain (G) and quantification of interstitial collagen (red signal) (n=15 for Plt.TGFβ-WT and n=9 for Plt.TGFβ-KO mice; H). Exact p-values, as determined by χ² test in (A), Two-Way ANOVA followed by Bonferroni’s multiple comparisons test (3 comparisons per genotype) in (B; non-significant p-values are not shown), Mann-Whitney test in (D) and Student’s t-test in (F) and (H), are shown in the graphs. Scale bars in (E) and (G) represent 200 μm.

Figure 2.. Endothelial-specific deletion of TGFβRII and venous thrombus remodeling.

The percentage of mice that formed a venous thrombus on day 1 after IVC ligation is shown (A). Thrombus resolution (B) and thrombus area (C, D) measured by ultrasound at day 21 after IVC ligation in End.TGFβRII-WT and End.TGFβRII-KO mice (n=11 mice per group). In panel B, thrombus area at day 14 and 21 is expressed relative to thrombus area at day 1. In panel C, representative ultrasound snapshots of thrombosed IVC segments in End.TGFβRII-WT and End.TGFβRII-KO mice at day 21 after IVC ligation shown (with 3-dimensional thrombus reconstruction as insert). Representative pictures of longitudinal cross-sections through the thrombosed IVC at day 21 after ligation following Carstairs’ staining (E) and quantification of thrombofibrosis (blue signal) (n=7 End.TGFβRII-WT and n=9 End.TGFβRII-KO mice; F). Representative pictures after Sirius red stain (G) and quantification of interstitial collagen (red signal) (n=7 mice per group; H). Exact p-values, as determined by χ² test in (A), Two-Way ANOVA followed by Bonferroni’s multiple comparisons test (3 comparisons per genotype) in (B; non-significant p-values are not shown), and Mann-Whitney test in (D, F and H) are shown in the graphs. Scale bars represent 200 μm.

Figure 3.. Detection of pulmonary vascular occlusions following venous thrombosis.

Representative images after analysis of pulmonary artery obstructions in PBS- and formalin-infused paraffin-embedded mouse lungs from End.TGFβRII-WT and End.TGFβRII-KO mice at day 21 after IVC ligation using Carstairs’ staining (A; arrows) or immunostaining of fibrinogen (B). Mice that developed a venous thrombus at day 1 and mice without a thrombus were examined separately to distinguish pulmonary embolization and in situ thrombosis (n=7 mice per group). End.TGFβRII-KO mice treated with bosentan over 3 weeks were also examined (marked in green; n=5 End.TGFβRII-WT and n=7 End.TGFβRII-KO mice). Scale bars represent 10 μm. The results of the quantitative analysis of red, fibrin-rich material (arrows in A) in the pulmonary parenchyma are shown (C). Exact p-values, as determined by One-Way ANOVA followed by Bonferroni’s multiple comparisons test (7 comparisons) are shown in panel C. Non-significant p-values are not shown. Representative images of lungs from End.TGFβRII-WT and End.TGFβRII-KO mice at day 21 after IVC ligation showing results after immunostaining for the platelet marker CD41 and TGFβ1. Scale bar represent 10 μm.

Figure 4.. Presence of myofibroblasts and endothelial cells in chronic venous thrombi of End.TGFβRII mice.

Representative images and quantitative analysis of the SMA-immunopositive area (A, C) and vascular channels lined by CD31-immunopositive cells (A, D) in 21-days-old venous thrombi of End.TGFβRII-WT (n=6 in C and n=7 in D) and End.TGFβRII-KO mice (n=6). Data shown in panel D represent the area lined by CD31-positive channels per total thrombus area (%). Scale bars represent 10 μm. Cells expressing endothelial and myofibroblast markers are highlighted by black arrows. Representative images of SMA- and CD31-immunopositive cells in vessel-rich regions of PEA tissue specimens from patients with CTEPH are shown in (B). Scale bars represent 100 μm. Representative flow cytometry dot blots after analysis of 21-days-old venous thrombi for the number of cells double-positive for CD31 and FSP1 (n=4 mice per group; E, F) or CDH5 and SMA (n=4 mice per group; G, H). Exact p-values, as determined by Mann-Whitney test, are shown in panels C, D, F and H.

Figure 5.. Expression of TGFβ1, its receptors and downstream signaling mediators in pulmonary endarterectomy specimens of patients with CTEPH.

Representative images in regions primarily containing fresh or organized thrombus, myofibroblasts, vessels or fibrosis (A) and the results of the quantitative analysis after immunohistochemical analysis of the expression of TGFβ1 (B), TGFβRII (C), TGFβRI/ALK1 (D), TGFβRI/ALK5 (E), p-SMAD2/3 (F) and p-SMAD5 (G) in PEA samples from n=9 CTEPH patients are shown. Scale bars represent 100 μm. Exact p-values, as determined by One-Way ANOVA followed by Bonferroni’s multiple comparisons test (10 comparisons), are shown in panels B-G. Non-significant p-values are not shown.

Figure 6.. Expression of endothelial and mesenchymal markers in primary human pulmonary endothelial cells from patients with CTEPH: comparison to TGFβ1 stimulation.

Representative pictures after immunofluorescence confocal microscopy for VE-cadherin (CDH5) and SMA in CTEPH tissue specimens, endothelial cells outgrown from CTEPH tissue (CTEPH-ECs) and HPAECs (A). Representative confocal pictures for CDH5 and SMA in HPAECs and HUVECs with and without TGFβ1 stimulation (10 ng/ml for 7 days) to induce EndMT (B). Quantitative analysis of CDH5 (n=5 biological replicates; C), SMA (ACTA2; n=4 for CTEPH and n=6 for all others; D), SNAIL (n=3; E), ZEB1 (n=3; F), TWIST (n=5; G) and FGFR1 (n=4 for CTEPH and n=5 for all others; H) mRNA expression in HPAECs with and without TGFβ1 stimulation (10 ng/mL for 7 days) and CTEPH-ECs. Scale bars represent 10 μm. Exact p-values, as determined by One-Way ANOVA followed by Bonferroni’s multiple comparisons test (3 comparisons) and by Mann-Whitney test for the comparison of HPAECs with and without TGFβ1 stimulation, are shown in panels C-H. Non-significant p-values are not shown.

Figure 7.. Endothelin-1 expression in primary human pulmonary endothelial cells and in patients with CTEPH: effect of TGFβ1.

Quantitative real time PCR (n=4 biological replicates; A) and western blot analysis (n=3 biological replicates; C) of endothelin-1 (EDN1) mRNA or protein expression in HPAECs treated with TGFβ1 (10 ng/mL for 7 days). A representative western blot membrane is shown in (D). In some experiments, SB431542 (10 μM) was added to block ALK5 or K02288 (10 μM) to block ALK1 dependent signaling. Exact p-values, as determined by One-Way ANOVA followed by Bonferroni’s multiple comparisons test (4 comparisons), are shown in panels A and C. Data are normalized to HPRT1 and are expressed as -fold change vs. HPAECs (set at 1). Representative confocal pictures of HPAECs treated with TGFβ1 after immunodetection of endothelin-1 (ET-1; B). Scale bars represent 10 μm. Analysis of ET-1 expression in human n=6 PEA specimens primarily containing fresh or organized thrombus, myofibroblasts, vessels or fibrosis. Representative pictures (E) and the results of the quantitative analysis (F) are shown. Scale bar represents 100 μm. Exact p-values, as determined by One-Way ANOVA followed by Bonferroni’s multiple comparisons test (10 comparisons), are shown in panel F. Plasma levels of ET-1 in patients with CTEPH (n=19) or PAH (n=26) and healthy individuals (n=5; G). Patients who received endothelin receptor antagonists are marked in green, patients who received soluble guanylate cyclase stimulators are marked in red. Exact p-values, as determined by Kruskall-Wallis test followed by Dunn’s multiple comparisons test (3 comparisons), are shown in panel G. Non-significant p-values are not shown.

Figure 8.. Endothelin-1 expression in mouse pulmonary endothelial cells or venous thrombi and effects of endothelin receptor antagonization on EndMT.

Quantitative real time PCR analysis (A) and representative confocal pictures (B) after analysis of endothelin-1 (EDN1) mRNA or protein expression in mPECs isolated from End.TGFβRII-WT and End.TGFβRII-KO mice and cultured in MV2 medium. Exact p-values, as determined by Mann-Whitney test are shown (n=6 biological replicates). Data are normalized to 18S and are expressed as -fold change vs. End.TGFβRII-WT mice (set at 1). Scale bars represents 10 μm. Plasma levels of ET-1 in samples from End.TGFβRII-WT and End.TGFβRII-KO mice uninjured (no IVC ligation; n=10 per group) and 21 days after IVC ligation (n=6 per group). Exact p-values, as determined by One-Way ANOVA followed by Bonferroni’s multiple comparisons test (4 comparisons), are shown. Non-significant p-values are not shown. Representative images of endothelin-1 protein expression in thrombosed vein wall segments 21 days after IVC ligation of End.TGFβRII-WT and End.TGFβRII-KO mice (D). Scale bars represent 10 μm. Quantitative analysis of thrombus area at day 21 after IVC ligation in End.TGFβRII-WT and End.TGFβRII-KO controls and mice treated with bosentan (n=5 per group; 5 mg/kg BW; E). Representative ultrasound images are shown in (F). Quantitative flow cytometry analysis of CD31- and CD31/FSP1 (n=4 per group; G) or CDH5- and CDH5/SMA (n=4 per group; H) double-positive cells residing within 21 day-old thrombi of End.TGFβRII-KO control- and bosentan-treated mice. Results in mice treated with bosentan are labeled in green. Exact p-values, as determined by Two-Way ANOVA followed by Bonferroni’s multiple comparisons test (6 comparisons; E) and multiple t test (G and H), are shown. Non-significant p-values are not shown.