Wnt7a deficit is associated with dysfunctional angiogenesis in pulmonary arterial hypertension

Ananya Chakraborty, Abinaya Nathan, Mark Orcholski, Stuti Agarwal, Elya A. Shamskhou, Natasha Auer, Ankita Mitra, Eleana Stephanie Guardado, Gowri Swaminathan, David F. Condon, Joyce Yu, Matthew McCarra, Nicholas H. Juul, Alden Mallory, Roberto A. Guzman-Hernandez, Ke Yuan, Vanesa Rojas, Joseph T. Crossno, Lai-Ming Yung, Paul B. Yu, Thomas Spencer, Robert A. Winn, Andrea Frump, Vijaya Karoor, Tim Lahm, Haley Hedlin, Jeffrey R. Fineman, Robert Lafyatis, Carsten N.F. Knutsen, Cristina M. Alvira, David N. Cornfield, Vinicio A. de Jesus Perez
European Respiratory Journal 2023 61: 2201625; DOI: 10.1183/13993003.01625-2022

Abstract

Introduction: Pulmonary arterial hypertension (PAH) is characterised by loss of microvessels. The Wnt pathways control pulmonary angiogenesis but their role in PAH is incompletely understood. We hypothesised that Wnt activation in pulmonary microvascular endothelial cells (PMVECs) is required for pulmonary angiogenesis, and its loss contributes to PAH.

Methods: Lung tissue and PMVECs from healthy and PAH patients were screened for Wnt production. Global and endothelial-specific Wnt7a−/– mice were generated and exposed to chronic hypoxia and Sugen-hypoxia (SuHx).

Results: Healthy PMVECs demonstrated >6-fold Wnt7a expression during angiogenesis that was absent in PAH PMVECs and lungs. Wnt7a expression correlated with the formation of tip cells, a migratory endothelial phenotype critical for angiogenesis. PAH PMVECs demonstrated reduced vascular endothelial growth factor (VEGF)-induced tip cell formation as evidenced by reduced filopodia formation and motility, which was partially rescued by recombinant Wnt7a. We discovered that Wnt7a promotes VEGF signalling by facilitating Y1175 tyrosine phosphorylation in vascular endothelial growth factor receptor 2 (VEGFR2) through receptor tyrosine kinase-like orphan receptor 2 (ROR2), a Wnt-specific receptor. We found that ROR2 knockdown mimics Wnt7a insufficiency and prevents recovery of tip cell formation with Wnt7a stimulation. While there was no difference between wild-type and endothelial-specific Wnt7a−/– mice under either chronic hypoxia or SuHx, global Wnt7a+/– mice in hypoxia demonstrated higher pulmonary pressures and severe right ventricular and lung vascular remodelling. Similar to PAH, Wnt7a+/– PMVECs exhibited an insufficient angiogenic response to VEGF-A that improved with Wnt7a.

Conclusions: Wnt7a promotes VEGF signalling in lung PMVECs and its loss is associated with an insufficient VEGF-A angiogenic response. We propose that Wnt7a deficiency contributes to progressive small vessel loss in PAH.

Abstract

Wnt7a deficiency in PAH reduces angiogenic response in pulmonary microvascular endothelial cells and promotes small vessel loss in PAH https://bit.ly/3xZtEf9

Introduction

Pulmonary arterial hypertension (PAH) is a rare disorder associated with endothelial dysfunction, progressive loss and remodelling of pulmonary microvessels [1, 2]. Endothelial cells (ECs) regenerate lost or damaged vessels by activating a repair programme known as angiogenesis, a process predominantly driven by the vascular endothelial growth factor (VEGF) signalling pathway [3]. Following injury, VEGF-A accumulates and instructs ECs in local vessels to initiate angiogenic sprouting via VEGF receptor 2 (VEGFR2), a tyrosine kinase receptor that activates angiogenic pathways. Despite increased VEGF-A and VEGFR2 expression in PAH vascular lesions, pulmonary microvascular ECs (PMVECs) from PAH patients demonstrate reduced angiogenic responses to VEGF-A [4]. While mutations in VEGFR2 have recently been associated with hereditary PAH [5], the low penetrance suggests that additional genetic modifiers are involved in the development of PAH.

We previously reported that endothelial secretion of Wnt5a, a ligand of the Wnt signalling pathways, promotes lung vessel maturation through pericyte recruitment by activating the planar cell polarity (PCP) pathway, which requires that Wnts interact with a surface receptor complex featuring receptor tyrosine kinase-like orphan receptor 2 (ROR2), a tyrosine kinase membrane receptor [6]. Our group has shown that Wnt signalling can drive expression of VEGF-A in ECs from the large pulmonary artery and that crosstalk with the bone morphogenetic protein pathway promotes angiogenesis in response to injury [7]. Given that Wnt pathways crosstalk with VEGF during embryonic development and in various tissues [8, 9], we hypothesised that crosstalk between Wnt and VEGF signalling is required for appropriate pulmonary angiogenesis (figure 1a).

FIGURE 1
FIGURE 1
FIGURE 1

Wnt7a expression is reduced in pulmonary microvascular endothelial cells (PMVECs) and vascular lesions of pulmonary arterial hypertension (PAH) patients. a) Summary of sprouting angiogenesis and hypothetical role for Wnts. VEGF-A: vascular endothelial growth factor A. b) SYBR Green quantitative PCR analysis for Wnt ligands in the healthy donor (HD) (n=6) and PAH (n=6) PMVECs cultured in semi-confluent versus confluent conditions. The expression of each gene is shown relative to that of confluent cells. **: p<0.005, Mann–Whitney test. c) Western blot of Wnt7a in HD (n=3) and PAH PMVEC (n=5) lysates. Densitometry analysis shows the relative expression of Wnt7a versus tubulin. *: p<0.05, Mann–Whitney test. d) Confocal images of HD (n=3, top) and PAH (n=3, bottom) lung sections stained for endothelial cells (lectin, green) and Wnt7a (red). Arrows point to Wnt7a in the endothelium. Scale bar: 50 µm.

Materials and methods

Detailed methods can be found in the supplementary material.

Animals

Global Wnt7a+/– and Wnt7a−/– were obtained by crossing C57BL/6J Wnt7aflox/flox mice and Wnt7a+/flox with Rosa26 Cre-ERT2 mice. Mice were treated with 75 mg·kg−1 of tamoxifen (20 mg·mL−1 dissolved in corn oil) for 5 consecutive days to induce the Wnt7a knockout (KO) in all transgenic strains. After tamoxifen treatments, mice were allowed to rest for 7–10 days before experimentation.

Cell culture

Healthy control and PAH PMVECs were obtained from the Pulmonary Hypertension Breakthrough Initiative (PHBI), with additional healthy donor cells obtained from PromoCell (PromoCell GmbH, Heidelberg, Germany). All PMVECs were cultured in 5% fetal bovine serum endothelial cell media with 1× endothelial cell growth supplement. Donor clinical information can be found in supplementary table S1.

Statistical analysis

All statistical analyses were performed using Prism 9 software (GraphPad). A p-value <0.05 was considered significant and reported in the graphs. Statistical comparisons between two groups for in vitro studies were performed using an unpaired t-test or Mann–Whitney for nonparametric data. Comparison among three or more groups was performed using one-way ANOVA followed by a Dunnet or Bonferroni post hoc analysis if the data followed a normal distribution. Otherwise, a nonparametric Kruskal–Wallis test with Dunn post hoc analysis was used.

Results

Wnt7a expression is reduced in PAH endothelium

First, we compared the Wnt ligand gene expression profile of healthy and PAH PMVECs cultured in semi-confluent versus confluent conditions [10]. Despite an ∼2-fold increase in several Wnt ligands (Wnt3a, Wnt4, Wnt5a, Wnt7b, Wnt11), Wnt7a mRNA was >6-fold greater in semi-confluent versus confluent healthy PMVECs; in contrast, only an ∼2-fold Wnt7a increase was seen in PAH PMVECs (figure 1b). Wnt7a is a master regulator of central nervous system angiogenesis and lung branching morphogenesis [11, 12]. A study by Liu et al. [13] showed that excess Wnt7a expression can lead to pulmonary smooth muscle cell hyperplasia; however, the effect of Wnt7a in lung ECs remains unknown. Compared to donors, PAH PMVECs demonstrated significantly lower Wnt7a protein expression (figure 1c). Confocal imaging demonstrated higher Wnt7a expression in endothelium versus other vascular layers (figure 1d, upper panels); however, PAH lesions demonstrated reduced Wnt7a expression (figure 1d, lower panels). In two independent single-cell RNA-sequencing (scRNA-seq) databases of healthy and PAH lungs [14, 15], Wnt7a expression was very low across endothelial cell subtypes, with higher abundance in Aerocytes (supplementary figure S1a). We found that alveolar type 1 (AT1) cells displayed the highest level of Wnt7a expression among all lung cell populations (supplementary figure S1b). Similar findings were documented in scRNA-seq analysis of PAH lungs [14] (supplementary figure S2a). Confocal imaging of AT1 cells demonstrated that Wnt7a was almost absent in PAH versus donors (supplementary figure 2b). Finally, we assessed Wnt7a expression in lungs of monocrotaline (MCT) and Sugen-hypoxia (SuHx) rats [15]. As with PAH samples, we found that Wnt7a expression was significantly lower in MCT and SuHx rats versus controls (supplementary figure S3).

FIGURE 2
FIGURE 2
FIGURE 2

Loss of Wnt7a is associated with impaired endothelial response to vascular endothelial growth factor A (VEGF-A). a) Cell count proliferation assay of control (n=5) and Wnt7a-specific small interfering RNA (siWnt7a) (n=5) pulmonary microvascular endothelial cells (PMVECs) stimulated with VEGF-A (10 ng·mL−1), Wnt7a (100 ng·mL−1) or both for 24 h. **: p=0.008, multiple Mann­–Whitney test. ##: p=0.01, control baseline versus cells stimulated with VEGF-A+Wnt7a; ###: p=0.0005, control baseline versus control VEGF-A; Kruskal–Wallis test with Dunn's comparison. b) Boyden chamber motility assay of control (n=5) versus siWnt7a (n=5) PMVECs stimulated with VEGF-A (10 ng·mL−1) or VEGF-A+Wnt7a (100 ng·mL−1) for 6 h. The migration index is calculated as the ratio of migrated cells relative to the baseline. *: p=0.008, Mann–Whitney test. #: p=0.0148, control baseline versus control VEGF-A; ##: p=0.009, control baseline versus cells stimulated with VEGF-A+Wnt7a; Kruskal–Wallis test with Dunn's comparison. c, d) Live imaging cell migration assay of control (n=6) and siWnt7a (n=4) PMVECs stimulated with VEGF-A (10 ng·mL−1) or VEGF-A+Wnt7a (100 ng·mL−1) for 24 h. The total distance was calculated using the Image J plugin (NIH). *: p<0.05; **: p=0.009, Kruskal­–Wallis test with Dunn's comparison. e, f) Representative images of Matrigel assay for control and siWnt7a PMVECs stimulated with VEGF-A (10 ng·mL−1) or VEGF-A+Wnt7a (100 ng·mL−1) for 6 h (n=5 per condition). Tube length was measured using Image J. Scale bar: 200 µm. ***: p<0.001, Kruskal–Wallis test with Dunn's comparison. g) Cell count proliferation assay of healthy donor (HD) and pulmonary arterial hypertension (PAH) PMVECs stimulated with VEGF-A (10 ng·mL−1), Wnt7a (100 ng·mL−1) or both for 24 h (n=5 per condition). **: p=0.008, Mann–Whitney test. #: p=0.0243, HD baseline versus control VEGF-A; ##: p=0.0053, HD baseline versus cells stimulated with VEGF-A+Wnt7a; Kruskal–Wallis test with Dunn's comparison. h) Boyden chamber motility assay of HD and PAH PMVECs stimulated with VEGF-A (10 ng·mL−1), Wnt7a (100 ng·mL−1) or both for 6 h (n=5 per condition). The migration index is calculated as the ratio of migrated cells relative to unstimulated. *: p=0.039; **: p=0.007; Mann–Whitney test. #: p=0.0127, HD baseline versus control VEGF-A; ###: p<0.0001, HD baseline versus cells stimulated with VEGF-A+Wnt7a; Kruskal–Wallis test with Dunn's comparison. i, j) Live imaging cell migration assay of HD and PAH PMVECs stimulated with VEGF-A (10 ng·mL−1) or VEGF-A+Wnt7a (100 ng·mL−1) for 24 h (n=5 per condition). The total distance was calculated using the Image J plugin (NIH). **: p<0.01, multiple Mann–Whitney tests. k, l) Representative images of Matrigel assay for HD and PAH PMVECs stimulated with VEGF-A (10 ng·mL−1) or VEGF-A+Wnt7a (100 ng·mL−1) for 6 h (n=5 per condition). Tube length was measured using Image J. Scale bar: 200 µm. **: p<0.01; ***: p<0.001, Kruskal–Wallis test with Dunn's comparison.

Wnt7a insufficiency is associated with an impaired PMVEC response to VEGF-A

To determine the impact of Wnt7a insufficiency on angiogenesis, we transfected healthy PMVECs with Wnt7a-specific small interfering RNA (siWnt7a) (supplementary figure S4) and measured proliferation, motility and tube formation in response to VEGF-A (10 ng·mL−1). Compared to controls, siWnt7a PMVECs demonstrated significantly reduced proliferation (figure 2a) and migration (figure 2b) in response to VEGF-A. Live imaging cell tracking demonstrated that VEGF-A-stimulated siWnt7a cells remained near their point of origin (figure 2c) and moved shorter distances (figure 2d) than controls. In Matrigel, siWnt7a PMVEC formed smaller networks (figure 2e) and shorter tubes than controls (figure 2f). While recombinant Wnt7a (100 ng·mL−1) alone did not influence proliferation or motility (figure 2a, b), it increased tube length of VEGF-A-stimulated siWnt7a PMVECs (figure 2f). Importantly, co-stimulation with VEGF-A and Wnt7a increased proliferation, motility, translocation and tube formation responses in siWnt7a PMVECs compared to controls (figure 2a–f). Similar to siWnt7a cells, PAH PMVECs demonstrated significantly lower proliferation (figure 2g) and motility (figure 2h) responses to VEGF-A or Wnt7a alone, which improved when both proteins were added to the media. Cell tracking also showed that co-stimulation with Wnt7a+VEGF-A increased PAH PMVEC translocation (figure 2i) and distance travelled (figure 2j). Finally, Wnt7a+VEGF-A improved the network size (figure 2k) and tube length (figure 2l) of PAH PMVECs in Matrigel.

Wnt7a facilitates tip cell formation

Tip cells are specialised filopodia-rich ECs induced by VEGFR2 activation [3, 16] that steer vascular sprouting in response to VEGF-A (figure 1a). To determine whether Wnt7a insufficiency prevents tip cell formation, we looked at filopodia formation in control and siWnt7a PMVECs using actin staining. Compared to control (figure 3a, right upper panel), siWnt7a PMVECs demonstrated minimal filopodia formation in response to VEGF-A (figure 3a, right lower panel). In Matrigel, filopodia-rich control PMVECs could be seen during early tube formation (figure 3b, left upper panel); by contrast, fewer filopodia-enriched siWnt7a PMVECs were found within cell clusters (figure 3b, left lower panel), a finding that was confirmed by scanning electron microscopy (figure 3b, right panels).

FIGURE 3
FIGURE 3
FIGURE 3

Reduced Wnt7a expression is associated with reduced filopodia formation in response to vascular endothelial growth factor A (VEGF-A). a) Filopodia formation in control (n=20) and Wnt7a-specific small interfering RNA (siWnt7a) (n=20) pulmonary microvascular endothelial cells (PMVECs) at baseline and after 1 h of VEGF-A (10 ng·mL−1) stimulation. Arrows point at the filopodia. Actin filaments were visualised using a phalloidin stain (green). Average filopodia length and numbers per cell were measured using Image J. Scale bar: 10 µm. ***: p<0.001, Mann–Whitney test. b) Matrigel filopodia formation assay by control (n=5) and siWnt7a (n=5) PMVECs after 1 h of VEGF-A (10 ng·mL−1) stimulation. Representative scanning electron microscope images of tip cells in Matrigel (right panels) showing densely clustered filopodia in control versus siWnt7a PMVECs. Scale bar: 500 µm. ***: p<0.001, Mann–Whitney test. c) Three-dimensional Cytodex bead assay showing control and siWnt7a PMVECs stimulated with VEGF-A alone or in combination with Wnt7a (n=15–20 per condition). Scale bar: 10 µm. ***: p<0.001, Mann–Whitney test. d) Three-dimensional collagen invasion assay of control versus siWnt7a on gels enriched with VEGF-A or VEGF-A+Wnt7a (n=5 per condition). Broad arrows point at filopodia in tip cells, and thin arrows point at the lumen. Scale bar: 20 µm. **: p=0.0082; ***: p<0.0001; Kruskal–Wallis with Dunn's comparison. e) Matrigel filopodia formation assay by healthy donor (HD) and pulmonary arterial hypertension (PAH) PMVECs after 1 h of VEGF-A (10 ng·mL−1) stimulation (n=5 per condition). Scale bar: 50 µm. *: p<0.05; **: p<0.01; Mann–Whitney test. f) Three-dimensional collagen invasion assay of HD and PAH PMVECs on gels enriched with VEGF-A or VEGF-A+Wnt7a (n=5 per condition). Filopodia length and number were measured using Image J. Scale bar: 40 µm. *: p=0.269; **: p=0.0078; ***: p<0.0001; Kruskal–Wallis with Dunn's comparison.

To overcome the limitations of two-dimensional angiogenesis assays, we developed two complementary three-dimensional models of sprouting angiogenesis that captured tip cell behaviour during sprouting angiogenesis. Control PMVECs seeded on collagen-suspended Cytodex beads (supplementary figure S5a) differentiated into tip cells (figure 3c, upper panel), whereas siWnt7a PMVECs exhibited a triangular (tipi-like) shape with fewer and shorter filopodia (figure 3c, middle panel), which improved with VEGF-A+Wnt7a co-stimulation (figure 3c, lower panel). Collagen invasion assays in gels containing VEGF-A alone or combined with Wnt7a (supplementary figure S5b) showed angiogenic sprout formation by control PMVECs, featuring distinct tip cells (figure 3d, left upper panel). Interestingly, the number and invasion distance of angiogenic sprouts arising from VEGF-stimulated siWnt7a PMVECs were significantly lower (figure 3d, left lower panel). We also found that VEGF-A+Wnt7a increased filopodia formation, number and invasion distance by siWnt7a PMVECs (figure 3d, right lower panel) to levels comparable to those seen in control cells (figure 3d, right upper panel).

We used the same assays to assess tip cell formation and sprouting angiogenesis by donor and PAH PMVECs. In Matrigel, healthy PMVECs formed multiple tip-like cells within clusters, which gave rise to tube-like structures (figure 3e, left panel). In contrast, PAH PMVEC formed fewer tip-like cells with fewer filopodia (figure 3e, right panel). In collagen invasion studies, VEGF-A-stimulated PAH PMVECs formed fewer angiogenic sprouts (figure 3f, left lower panel), although invasion distance was comparable to healthy donors (figure 3f, left upper panel). With VEGF-A+Wnt7a, the number of PAH angiogenic sprouts and filopodia increased without change in the invasion distance (figure 3f, right lower panel). Interestingly, VEGF-A+Wnt7a also induced healthy donor sprouts to bifurcate and grow thicker in diameter (figure 3f, right upper panel).

Sprouting angiogenesis by PAH and siWnt7a organoids is significantly reduced

To assess sprouting angiogenesis in real-time, we imaged endothelial organoids in collagen I gel droplets containing VEGF-A alone or with Wnt7a over 10 h (figure 4a). In the presence of VEGF-A, control organoids started growing sprouts at 3 h, which then increased in number and length (figure 4b and supplementary video S1). In contrast, siWnt7a organoids grew significantly fewer sprouts that receded and remained stagnant over the 10 h (figure 4b and supplementary video S2). With VEGF-A+Wnt7a, the sprout number and stability increased in both control (figure 4b, d and supplementary video S3) and siWnt7a organoids (figure 4b, d and supplementary video S4). Similarly, compared to donor organoids (figure 4c and supplementary video S5), VEGF-stimulated PAH organoids demonstrated short sprouts confined to discrete areas (figure 4c and supplementary video S6). Similar to siRNA-treated cells, both donor (figure 4c and supplementary video S7) and PAH (figure 4c and supplemental video S8) PMVECs exhibited a significant increase in the number and length of sprouts with VEGF-A+Wnt7a (figure 4e).

FIGURE 4
FIGURE 4
FIGURE 4

Organoid assay reveals differences in sprouting angiogenesis in Wnt7a-specific small interfering RNA (siWnt7a) and pulmonary arterial hypertension (PAH) of pulmonary microvascular endothelial cells (PMVECs). a) Schematic figure of the three-dimensional organoid platform. b, c) Still images of control and siWnt7a (b) and healthy donor (HD) and PAH PMVEC (c) organoids filmed over 10 h. Gels were enriched with vascular endothelial growth factor A (VEGF-A) and VEGF+Wnt7a. Scale bar: 1000 µm. d, e) Maximum sprout numbers for control and siWnt7a (d) and HD and PAH PMVEC (e) organoids. Measurements were carried out using Image J. Experiments were repeated three times per condition. Shown are mean±sem. ***: p<0.001; ****: p<0.0001; one-way ANOVA with Dunnett post hoc test.

Wnt7a regulates VEGFR2 activity through the ROR2 receptor

VEGF binding to VEGFR2 results in the phosphorylation of several intracellular tyrosine residues such as Y1175, which activate multiple downstream mitogen activated protein (MAP) kinases, including p38, involved in proliferation, survival and angiogenesis [17, 18]. Western immunoblotting demonstrated no differences in pY1175 VEGFR2 in control versus siWnt7a PMVECs at baseline; however, after 5 min of VEGF-A, there was a significant increase in pY1175 VEGFR2 in both control (figure 5a, left panels) and siWnt7a (figure 5a, right panels) PMVECs that gradually decreased over 1 h. Interestingly, pY1175 VEGFR2 was significantly greater in siWnt7a at 5 min of VEGF-A stimulation. To assess whether downstream signalling activity changed in response to pY1175 VEGFR2, we measured phospho-p38, a MAP kinase required for VEGF-induced tip cell induction [18]. In alignment with pY1175 VEGFR2 status, phospho-p38 increased in control PMVECs, with the highest levels seen after 15 min of VEGF-A stimulation (figure 5a, left panels). In contrast, phospho-p38 was significantly greater in siWnt7a cells after 5 min of VEGF-A stimulation, followed by reduction at 15 min (figure 5a, right panels). We also probed donor and PAH PMVEC lysates for pY1175 VEGFR2 and phospho-p38 at baseline and after 5 min of VEGF-A. While there were no differences between donor and PAH, there was a trend towards higher pY1175 VEGFR2 in PAH at 5 min (figure 5b, right panels), while phospho-p38 levels showed a higher trend at baseline (figure 5b, left panel). We speculate that the discrepancies in pY1175 VEGFR2 between siWnt7a and PAH could indicate differences in VEGFR2 dynamics between the cells, such as variable internalisation and recycling, which are known to regulate VEGFR2 signalling activity and duration [19]. We also analysed VEGF-A-induced changes in the expression of established tip cell markers (DLL4, EphB2, Sox17) [19, 20]. We found lower EphB2 and Sox17 in siWnt7a, which supports a requirement for Wnt7a in VEGF-mediated tip cell formation (supplementary figure S6). To elucidate how Wnt7a regulates VEGFR2 phosphorylation, we studied the signalling mechanism by which Wnt7a triggers filopodia formation in PMVECs. Filopodia formation depends on localised formation and extension of actin microfilaments, a process dependent on coordinated activation of Rac1 and cdc42 [21, 22]. Because these GTPases are downstream targets of Wnt/PCP (figure 5c) [7, 23], we tested whether Wnt7a activates Rac1 and cdc42 in healthy PMVECs; after 1 h of Wnt7a, donor PMVECs demonstrated a significant increase in both Rac1 and cdc42 (figure 5d).

FIGURE 5
FIGURE 5
FIGURE 5

Vascular endothelial growth factor receptor 2 (VEGFR2) phosphorylation is altered in Wnt7a-deficient and pulmonary arterial hypertension (PAH) pulmonary microvascular endothelial cells (PMVECs) and correlates with reduced receptor tyrosine kinase-like orphan receptor 2 (ROR2) expression. a) Western blot showing expression of total VEGFR2, pY1175-VEGFR2, phospho-p38 (P-p38) and total p38 in control small interfering RNA (siControl) (n=3) versus Wnt7a-specific small interfering RNA (siWnt7a) (n=3) stimulated with vascular endothelial growth factor A (VEGF-A) (10 ng·mL−1) over 1 h. Densitometry analysis was carried for phospho- versus total VEGFR2 and P-p38 versus total p38. **: p<0.01, multiple Mann–Whitney test. b) Western blot showing expression of total and pY1175 VEGFR2 and P-p38 and total p38 in healthy donor (HD) (n=3) and PAH (n=3) PMVECs stimulated with VEGF-A (10 ng·mL−1) over 5 min. **: p<0.01, multiple Mann–Whitney test. c) Diagram of Wnt/planar cell polarity pathway. d) Western blot of active Rac1 and cdc42 in HD PMVECs (n=3) stimulated with Wnt7a (100 ng·mL−1). Densitometry analysis was done against tubulin. **: p<0.01. e) SYBR Green quantitative PCR analysis for Wnt receptors in HD (n=5) and PAH (n=5) PMVECs cultured in semi-confluent versus confluent conditions. The expression of each gene is shown relative to that of confluent cells. The data presented are the result of three independent studies. ***: p=0.002, Mann–Whitney test. f) Western blot of Wnt7a and ROR2 in HD (n=3) and PAH (n=5) PMVEC lysates. Densitometry analysis shows the relative expression of Wnt7a and ROR2 versus tubulin. ***: p=0.004, unpaired t-test. g) Proximity ligation assay of HD PMVECs (n=5 per condition) stimulated with VEGF-A, Wnt7a or both for 1 h. Dots per cell were quantified using Image J. ***: p<0.001 versus VEGF; ###: p<0.001 Wnt7a versus VEGF+Wnt7a; Kruskal–Wallis with Dunn's comparison. h) Representative images of Matrigel assay for control (n=5) and ROR2-specific small interfering RNA (siROR2) (n=5) PMVECs stimulated with VEGF-A (10 ng·mL−1) or VEGF-A+Wnt7a (100 ng·mL−1) for 4 h. Tube length was measured using Image J. Scale bars: 200 µm. **: p<0.01, multiple Mann–Whitney tests. i) Filopodia formation assay in Matrigel by siControl (n=5) and siROR2 (n=5) PMVECs after 1 h of VEGF-A (10 ng·mL−1) stimulation. Filopodia length and number were measured using Image J. Scale bar: 100 µm. ***: p<0.001, Mann–Whitney test. OD: optical density.

Given that Wnt/PCP activation requires Wnt interaction with the tyrosine kinase receptor ROR2 (figure 5c), we decided to measure ROR2 expression in PAH PMVECs. Quantitative PCR demonstrated an ∼6-fold increase in ROR2 in semi- versus confluent healthy PMVECs, which was significantly lower in PAH PMVECs (figure 5e). Furthermore, protein levels of ROR2 were reduced in PAH PMVECs and correlated with the lower levels of Wnt7a expression (figure 5f). Finally, confocal analysis of PAH lesions demonstrated a significant reduction in ROR2 signal within the endothelium (supplementary figure S7). Because previous studies have shown that ROR2 can regulate neighbouring receptors through physical association [24, 25], we sought to determine whether ROR2 is capable of interacting with VEGFR2 by using co-immunoprecipitation and a proximity ligation assay (PLA) [26]. With PLA, we found a significant increase in ROR2–VEGFR2 complex formation with VEGF-A+Wnt7a compared to either agent alone (figure 5g).

We assessed whether ROR2 knockdown would mimic the effects of siWnt7a on PMVECs. Cell tracking demonstrated significantly reduced siROR2 PMVEC translocation and distance covered that did not improve with Wnt7a (supplementary figure S8). Furthermore, siROR2 PMVECs in Matrigel formed significantly shorter tube-like structures and smaller networks (figure 5h), while tip cell formation was also negatively affected (figure 5i).

Wild-type and endothelial Wnt7a KO mice develop similar pulmonary hypertension and vascular remodelling with hypoxia and SuHx

Wild-type (WT) mice in hypoxia demonstrated an increase in Wnt7a lung protein expression that peaked between days 1 and 7 and returned to baseline by day 14 (supplementary figure S9). To test the effect of endothelial-specific Wnt7a deletion in mice, we generated tamoxifen-inducible vascular endothelial (VE)-cadherin (PAC)-CreERT2/Wnt7aflox/flox (Wnt7a ECKO) mice and exposed them to chronic hypoxia and SuHx (figure 6a). We found no significant differences in haemodynamic profiles (figure 6b), right ventricular remodelling (figure 6c), vessel number (figure 6d) or muscularisation (figure 6e) between WT and Wnt7a ECKO mice under either condition. We did not identify any major differences in ROR2 or VEGFR2 expression in Wnt7a ECKO versus WT lungs (supplementary figure S10).

FIGURE 6
FIGURE 6
FIGURE 6

Endothelial-specific loss of Wnt7a is not associated with pulmonary hypertension in mice. a) Vascular endothelial-cadherin (PAC)-CreERT2/Wnt7aflox/flox (Wnt7a ECKO) model generation and experimental design for hypoxia and Sugen-hypoxia (SuHx) studies. RV: right ventricular. b–e) Right ventricular systolic pressure (RVSP), Fulton index (weight ratio of the right ventricle to the sum of the left ventricle and septum (RV/(LV+S)), vessel number per 100 alveoli and per cent muscularisation of microvessels in wild-type (WT) versus Wnt7a ECKO mice under normoxia, hypoxia and SuHx (hatched) (n=5–8 per condition). **: p<0.01; ***: p<0.001; one-way ANOVA with Dunnett post hoc test. f, g) Confocal images of WT and Wnt7a ECKO mice in normoxia (f) and hypoxia (g). Arrows indicate perivascular cells expressing Wnt7a. Endothelium (lectin, green), RAGE (AT1 marker, white), Wnt7a (red) and DAPI (blue). Scale bars: 30 µm.

One possible explanation for the lack of a distinct Wnt7a ECKO phenotype is compensatory Wnt7a production by other pulmonary cells. Similar to human lungs (supplementary figure S1), Wnt7a expression is low across all EC subtypes but highest in AT1 cells (supplementary figure S11). Confocal analysis of Wnt7a ECKO lungs confirmed that Wnt7a expression was preserved in AT1 cells under normoxia (figure 6f) and hypoxia (figure 6g). As our next step, we decided to repeat our studies in mice with global Wnt7a KO.

Wnt7a+/– mice in chronic hypoxia develop severe pulmonary hypertension and vascular remodelling

We generated conditional homozygous and heterozygous Wnt7a global KO mice by crossing Wnt7aflox/flox mice with ROSA26-CreERT2 mice and exposed them to chronic hypoxia (figure 7a). Wnt7a+/– mice exhibited >50% reduction in Wnt7a protein levels following tamoxifen induction (figure 7b). Compared to normoxia, Wnt7a+/– mice demonstrated significantly greater right ventricular systolic pressure (figure 7c), right ventricular remodelling (figure 7d) and reduction in vessel number (figure 7e) and increased muscularisation (figure 7f) versus WT. Wnt7a+/– mice had more muscularised microvessels (figure 7g, upper panels) and exhibited greater medial thickening versus WT (figure 7g, lower panels). To perform Matrigel studies, we isolated murine PMVECs from the lungs of WT and Wnt7a+/– mice and found that, similar to PAH, Wnt7a+/– PMVECs formed shorter tubes and smaller networks, which improved with Wnt7a (figure 7h). Of note, Wnt7a−/– mice demonstrated failure to thrive 5 days after tamoxifen (supplementary figure S12a). Necropsy revealed cardiomegaly, lung nodules and interstitial thickening from the expansion of endothelial and alveolar epithelial cells (supplementary figure S12b–f).

FIGURE 7
FIGURE 7
FIGURE 7

Wnt7a+/– mice develop more severe pulmonary hypertension and vascular remodelling in chronic hypoxia. a) Wnt7a+/– model generation and experimental design for hypoxia studies. RV: right ventricular. b) Western blot of Wnt7a in wild-type (WT) (n=3) and Wnt7a+/– (n=3) lung lysates. Densitometry analysis shows the relative expression of Wnt7a versus total p38. OD: optical density. **: p<0.001, unpaired t-test. c–f) Right ventricular systolic pressure (RVSP), Fulton index (weight ratio of the right ventricle to the sum of the left ventricle and septum (RV/(LV+S)), vessel number per 100 alveoli and per cent muscularisation of microvessels in WT versus Wnt7a+/– mice under normoxia and hypoxia (n=5–7 per condition). *: p<0.05; **: p<0.01; ***: p<0.001; one-way ANOVA with Bonferroni's post hoc test. g) Representative low (upper panels) and high (lower panels) magnification confocal images of lungs of hypoxic WT and Wnt7a+/– mice. Endothelium was labelled for vascular endothelial-cadherin (green), smooth muscle actin (red) and DAPI (blue). Scale bar: 50 µm. h) Representative images of Matrigel assay for WT (n=3) and Wnt7a+/– (n=3) pulmonary microvascular endothelial cells stimulated with vascular endothelial growth factor A (VEGF-A) (10 ng·mL−1) or VEGF-A+Wnt7a (100 ng·mL−1) for 4 h. Tube length was measured using Image J. Scale bars: 100 µm. *: p<0.05; ***: p<0.001; one-way ANOVA with Bonferroni's post hoc test.

Angiogenesis is a key driver of alveolarisation postnatally starting at birth [27], and exposure to hyperoxia is associated with reduced alveolarisation and impaired angiogenesis [28, 29]. In this context, we hypothesised that ECs from postnatal lungs of WT and hyperoxia-exposed mice would display increased expression of Wnt7a and other tip cell genes. Two independent neonatal mouse lung scRNA-seq datasets [30, 31] also identify AT1 cells as the main lung cell type expressing Wnt7a mRNA. Notably, confocal analysis of hyperoxia lungs demonstrated immunoreactivity for Wnt7a throughout the distal lung (supplementary figure S13) [32].

To complement the murine studies, we used a lamb model of congenital heart disease and compensatory lung angiogenesis [33]. Despite a mild increase in Wnt7a mRNA (supplementary figure S14), we found Wnt7a protein reduction in shunt lung lysates (supplementary figure S14b) and pulmonary alveolar epithelial cells (supplementary figure S14), reminiscent of PAH PMVECs (figure 1d).

Discussion

For many years, there has been an active debate regarding the role of VEGF signalling in the pathogenesis of PAH [34]. We show for the first time that Wnt7a is a critical modifier of VEGF signalling required for tip cell formation and sprouting angiogenesis (figure 8). As a master regulator of angiogenesis, VEGFR2 controls a comprehensive portfolio of downstream signalling pathways responsible for many aspects of EC behaviour in angiogenesis [17]. Besides confirming that Wnt7a is required for pulmonary vascular homeostasis, our murine studies paint a complex picture that opens exciting opportunities to delineate further how Wnt7a acts in the lung. The lack of a phenotype in Wnt7a ECKO may result from AT1 cells and other intraparenchymal cells serving as sources of Wnt7a in the lung [35], thereby compensating for the lack of Wnt7a production by ECs. These results differ from our prior study, where we generated Wnt5a ECKO mice and demonstrated a pulmonary vascular phenotype associated with chronic hypoxia [6]. Our studies also demonstrate a gene dosage effect with Wnt7a that could play a role in the extent of endothelial dysfunction seen in PAH. The fulminant course seen in Wnt7a−/– mice is difficult to reconcile with the relatively circumscribed lesions, and greater insight into the pathophysiology will require a more detailed assessment of the mice over time. Wnt7a is a tumour-suppressive agent associated with lung carcinogenesis and displays a high level of expression in AT1 and AT2 pneumocytes [36]. We are presently conducting AT1-selective Wnt7a KO studies combined with fate mapping to further delineate the role of the alveolar epithelium in the vascular phenotype seen in these animals.

FIGURE 8
FIGURE 8

Proposed model. In healthy conditions, Wnt7a primes vascular endothelial growth factor receptor 2 (VEGFR2) phosphorylation via receptor tyrosine kinase-like orphan receptor 2 (ROR2) and activates Rac1/cdc42 to trigger cytoskeletal changes and actin filament formation. Sprouting angiogenesis occurs after tip cell formation, and vascular repair are completed. Reduced expression of Wnt7a lowers VEGFR2 activity, resulting in reduced tip cell formation and inability to initiate sprouting angiogenesis. PAH: pulmonary arterial hypertension.

In conclusion, our study is the first to uncover the role of Wnt7a in angiogenesis in pulmonary ECs and how its loss can influence the angiogenic potential of the ECs. Understanding how Wnt7a and its receptors crosstalk and participate in forming new vessels will provide a more in-depth understanding of vascular remodelling in PAH.

Supplementary material

Supplementary Material

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Supplementary figures and tables ERJ-01625-2022.Supplement

Supplementary video 1 ERJ-01625-2022.Video_S1

Supplementary video 2 ERJ-01625-2022.Video_S2

Supplementary video 3 ERJ-01625-2022.Video_S3

Supplementary video 4 ERJ-01625-2022.Video_S4

Supplementary video 5 ERJ-01625-2022.Video_S5

Supplementary video 6 ERJ-01625-2022.Video_S6

Supplementary video 7 ERJ-01625-2022.Video_S7

Supplementary video 8 ERJ-01625-2022.Video_S8

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Acknowledgements

Lung tissues from PAH and control patients were provided by the Pulmonary Hypertension Breakthrough Initiative, funded by the NIH and managed at Stanford University by Marlene Rabinovitch and Roham T. Zamanian. The tissues were procured at the Transplant Procurement Centers at Stanford University, Cleveland Clinic and Allegheny General Hospital and de-identified patient data were obtained via the Data Coordinating Center at the University of Michigan. The authors thank all patients and their proxies who participated in this study. The authors are also grateful to Patricia Angeles del Rosario and Matthew Bill (Stanford University Medical Center) for helping with the collection and processing of blood samples, and Andrew Hsi (Stanford University Medical Center) for helping organise the patient database.

Footnotes

  • This article has an editorial commentary: https://doi.org/10.1183/13993003.00504-2023

  • Author contribution: A. Chakraborty, A. Nathan and V.A. de Jesus Perez were responsible for overseeing study performance, data analysis and drafting of the manuscript. All authors contributed to the design, performance and analysis of the studies included in the manuscript. All authors were involved in reviewing and approving the final manuscript.

  • Conflict of interest: V.A. de Jesus Perez reports support for the present manuscript from the National Institutes of Health National Heart, Lung, and Blood Institute; and outside the submitted work, holds a leadership position as AHA Chair of Diversity subcommittee. All other authors have nothing to disclose.

  • Support statement: This work was supported by the National Institutes of Health (R01 HL134776, R01 HL134776-02 and R01 HL159886-01), American Heart Association Beginning Grant in Aid, Stanford Cardiovascular Institute and Translational Research and Applied Medicine to V.A. de Jesus Perez. A. Chakraborty was supported by a Stanford Maternal Child Health Research Institute Postdoctoral Fellowship Grant. K. Yuan was supported by the American Heart Association Scientist Development Grant (15SDG25710448), the Parker B. Francis Fellowship and the Pulmonary Hypertension Association Proof of Concept Award. Funding information for this article has been deposited with the Crossref Funder Registry.

  • Received August 18, 2022.
  • Accepted February 21, 2023.
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Wnt7a deficit is associated with dysfunctional angiogenesis in pulmonary arterial hypertension
Ananya Chakraborty, Abinaya Nathan, Mark Orcholski, Stuti Agarwal, Elya A. Shamskhou, Natasha Auer, Ankita Mitra, Eleana Stephanie Guardado, Gowri Swaminathan, David F. Condon, Joyce Yu, Matthew McCarra, Nicholas H. Juul, Alden Mallory, Roberto A. Guzman-Hernandez, Ke Yuan, Vanesa Rojas, Joseph T. Crossno, Lai-Ming Yung, Paul B. Yu, Thomas Spencer, Robert A. Winn, Andrea Frump, Vijaya Karoor, Tim Lahm, Haley Hedlin, Jeffrey R. Fineman, Robert Lafyatis, Carsten N.F. Knutsen, Cristina M. Alvira, David N. Cornfield, Vinicio A. de Jesus Perez
European Respiratory Journal Jun 2023, 61 (6) 2201625; DOI: 10.1183/13993003.01625-2022
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