Modulation of Endothelial Bone Morphogenetic Protein Receptor Type 2 Activity by Vascular Endothelial Growth Factor Receptor 3 in Pulmonary Arterial Hypertension

Abstract

Background: Bone morphogenetic protein (BMP) signaling has multiple roles in the development and function of the blood vessels. In humans, mutations in BMP receptor type 2 (BMPR2), a key component of BMP signaling, have been identified in the majority of patients with familial pulmonary arterial hypertension (PAH). However, only a small subset of individuals with BMPR2 mutation develops PAH, suggesting that additional modifiers of BMPR2 function play an important role in the onset and progression of PAH.

Methods: We used a combination of studies in zebrafish embryos and genetically engineered mice lacking endothelial expression of Vegfr3 to determine the interaction between vascular endothelial growth factor receptor 3 (VEGFR3) and BMPR2. Additional in vitro studies were performed by using human endothelial cells, including primary lung endothelial cells from subjects with PAH.

Results: Attenuation of Vegfr3 in zebrafish embryos abrogated Bmp2b-induced ectopic angiogenesis. Endothelial cells with disrupted VEGFR3 expression failed to respond to exogenous BMP stimulation. Mechanistically, VEGFR3 is physically associated with BMPR2 and facilitates ligand-induced endocytosis of BMPR2 to promote phosphorylation of SMADs and transcription of ID genes. Conditional, endothelial-specific deletion of Vegfr3 in mice resulted in impaired BMP signaling responses, and significantly worsened hypoxia-induced pulmonary hypertension. Consistent with these data, we found significant decrease in VEGFR3 expression in pulmonary arterial endothelial cells from human PAH subjects, and reconstitution of VEGFR3 expression in PAH pulmonary arterial endothelial cells restored BMP signaling responses.

Conclusions: Our findings identify VEGFR3 as a key regulator of endothelial BMPR2 signaling and a potential determinant of PAH penetrance in humans.

Keywords: FLT4 protein, human; bone morphogenetic protein receptors, type II; endothelial cells; hypertension, pulmonary.

Fig. 1. VEGFR3 is essential for BMP signaling and closely associated with BMPR2

(A–C) Depth-encoded confocal images taken from the trunk region of 32 hours post fertilization (hpf) control (A), vegfc (B), or vegfr3 (C) morpholino (MO)-injected Tg(hsp70:bmp2b)^fr13;Tg(kdrl:eGFP)^s843 embryos upon heat shock. Overexpression of Bmp2b led to ectopic angiogenesis which was abrogated by inactivation of vegfr3. (D) Quantification on the number of somites containing ectopic angiogenic sprouts in control, vegfc, or vegfr3 MO-injected Tg(hsp70:bmp2b)^fr13;Tg(kdrl:eGFP)^s843 embryos upon heat shock. (E) BMP6-induced phosphorylation of SMADs is abrogated in VEGFR3 siRNA-treated HUVECs. (F) ID1 and ID2 are decreased in VEGFR3 siRNA-treated HUVECs. (G) Co-immunoprecipitation using BMPR2 antibody can pull down VEGFR3 in HUVECs and PAECs. (H) Co-immunoprecipitation using VEGFR3 antibody can pull down BMPR2 in HUVECs and PAECs. IP: immunoprecipitant; WCL: whole cell lysate. * P < 0.05, ** P < 0.01, *** P < 0.001.

Fig. 2. VEGFR3 and BMPR2 co-internalize upon ligand stimulation

(A) Biotinylation assay showing internalization of surface BMPR2 in response to BMP6 stimulation in a time dependent manner. (B) Inhibition of Dynamin-dependent endocytosis decreases phosphorylation of SMAD 1/5. (C) Stimulation with BMP6 induces internalization of VEGFR3 and BMPR2 in both HUVECs and PAECs in a time dependent manner. (D) Lack of VEGFR3 in HUVECs abrogated ligand-dependent internalization of BMPR2 in HUVECs.

Fig. 3. Lack of VEGFR3 in endothelial cells attenuates BMP signaling in mice

(A) Localization of YFP expression driven by the Vegfr3 promoter to the endothelial layer of bismuth contrast filled pulmonary arteriole. Scale bar = 50 μm. (B) Reduced pSMAD 1/5 levels in lung homogenates from Vegfr3 ECKO mice compared to lung homogenates from control littermates. (C) pSMAD 1/5 to total SMAD 5 ratio in control littermates and Vegfr3 ECKO mice. (D) Quantification of Id2 expression to GAPDH ratio in control and Vegf3 ECKO mice. *P < 0.05, **P < 0.01.

Fig. 4. Deletion of Vegfr3 in endothelial cells exacerbates pulmonary hypertension

(A) Right ventricle systolic pressure (RVSP) measured in control and Vegfr3 ECKO mice. (B) Weight ratio of right ventricle to left ventricle plus septum calculated in control and Vegfr3 ECKO mice. (C) Immunofluorescent staining of lung sections from control and Vegfr3 ECKO mice for endothelial cells (CD31; green), vascular smooth muscle cells (SMA; red), and nuclei (DAPI; blue). Scale bar = 50 μm. (D) Percentage of muscularized arterioles within the lung tissue obtained from control and Vegfr3 ECKO mice. (E) Thickness of SMA positive medial layer in the muscularized arteries in control and Vegfr3 ECKO mice. (F) H&E stained lung section taken from control (top) and Vegfr3 ECKO mice (bottom). Arrows point to representative images of intact (control) and obliterated (Vegfr3 ECKO) lumens of the pulmonary arterioles. Scale bar = 50 μm. (G) Percentage of pulmonary arterioles with obliterated lumen in control and Vegfr3 ECKO mice. *P < 0.05, **P < 0.01. HPF⁻¹: high power field.

Fig. 5. Attenuation of VEGFR3 contributes to the pathogenesis of pulmonary arterial hypertension in humans

(A) Immunofluorescence staining of a normal lung section (left) and a PAH lung section (right) for endothelial cells (CD31; green), VEGFR3 (red), and nuclei (DAPI; blue) showing decreased VEGFR3 expression in PAH endothelium. Scale bar = 50 μm. (B) Relative expression level of VEGFR3 mRNA in normal and PAH PAECs. The significance was calculated using Wilcoxon rank-sum test. **P < 0.01 (C) Overall decreased but heterogeneous VEGFR3 protein expression among PAH patient-derived PAECs is not dependent on mutations in BMPR2. The mean of all the expression values in the control group were used for normalization. (D–E) Expression level of VEGFR3 influences the response of PAH PAECs to BMP6 (50 ng/mL) stimulation. (D) Upon BMP6 treatment, phosphorylation of SMAD1/5 was only increased in PAH PAECs with a relatively high level of VEGFR3. (E) Phosphorylation of SMAD 1/5 did not change in PAH PAECs with low VEGFR3 expression. (F) Schematic of BMPR2 genomic loci depicting the specific gene mutation/deletion in human PAECs tested. The up-arrow depicts location of the missense mutation, and the red lines depict the locations of gene deletion. (G–I) Effect of lentiviral-mediated over-expression of VEGFR3 on BMP-ligand mediated phosphorylation of SMAD 1/5. In PAH PAEC without BMPR2 mutations with high level of VEGFR3 expression, over-expression of VEGFR3 did not increased the phosphorylation of SMAD 1/5 upon BMP6 stimulation (G), whereas in PAH PAECs with low VEGFR3 expression, over-expression of VEGFR3 restored the phosphorylation of SMAD 1/5 upon BMP6 stimulation (H). (I) In PAH PAECs from subjects with BMPR2 mutations, efficacy of rescue with VEGFR3 overexpression in enhancing BMP6 response was variable depending on the underlying BMPR2 mutation.