ALK1 controls hepatic vessel formation, angiodiversity, and angiocrine functions in hereditary hemorrhagic telangiectasia of the liver

Abstract

Background and aims: In hereditary hemorrhagic telangiectasia (HHT), severe liver vascular malformations are associated with mutations in the Activin A Receptor-Like Type 1 ( ACVRL1 ) gene encoding ALK1, the receptor for bone morphogenetic protein (BMP) 9/BMP10, which regulates blood vessel development. Here, we established an HHT mouse model with exclusive liver involvement and adequate life expectancy to investigate ALK1 signaling in liver vessel formation and metabolic function.

Approach and results: Liver sinusoidal endothelial cell (LSEC)-selective Cre deleter line, Stab2-iCreF3 , was crossed with Acvrl1 -floxed mice to generate LSEC-specific Acvrl1 -deficient mice ( Alk1HEC-KO ). Alk1HEC-KO mice revealed hepatic vascular malformations and increased posthepatic flow, causing right ventricular volume overload. Transcriptomic analyses demonstrated induction of proangiogenic/tip cell gene sets and arterialization of hepatic vessels at the expense of LSEC and central venous identities. Loss of LSEC angiokines Wnt2 , Wnt9b , and R-spondin-3 ( Rspo3 ) led to disruption of metabolic liver zonation in Alk1HEC-KO mice and in liver specimens of patients with HHT. Furthermore, prion-like protein doppel ( Prnd ) and placental growth factor ( Pgf ) were upregulated in Alk1HEC-KO hepatic endothelial cells, representing candidates driving the organ-specific pathogenesis of HHT. In LSEC in vitro , stimulation or inhibition of ALK1 signaling counter-regulated Inhibitors of DNA binding (ID)1-3, known Alk1 transcriptional targets. Stimulation of ALK1 signaling and inhibition of ID1-3 function confirmed regulation of Wnt2 and Rspo3 by the BMP9/ALK1/ID axis.

Conclusions: Hepatic endothelial ALK1 signaling protects from development of vascular malformations preserving organ-specific endothelial differentiation and angiocrine signaling. The long-term surviving Alk1HEC-KO HHT model offers opportunities to develop targeted therapies for this severe disease.

Graphical abstract

FIGURE 1

Alk1 ^HEC‐KO mice exhibit a vascular parenchymal enhancement and dilated hepatic veins. (A) Computed tomography sections in sagittal, axial, and coronal planes (n = 5). (B) Quantification of intrahepatic large vessel density in (A) (n = 5). (C) Diameters of hepatic vein branches (n ≥ 4). Error bars: Mean with standard deviation. (B,C) Welch's t test. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 2

Posthepatic volume overload of the vena cava in Alk1 ^HEC‐KO mice. (A) Diameters of vena cava segments (n = 5). (B) Doppler sonography of vena cava before and after confluence of hepatic veins (n ≥ 5). (C–E) Blood flow velocity in vena cava before and after confluence of hepatic veins (n ≥ 5). HV, hepatic vein; VC, vena cava. Error bars: Mean with standard deviation. (A,C–E) Welch's t test. *p < 0.05.

FIGURE 3

Alk1 deficiency causes disturbed hepatic vessel architecture. (A) Macroscopic images of livers (n ≥ 7). (B) Hematoxylin and eosin (H&E) and Sirius Red staining of livers (n ≥ 7). (C) Immunofluorescence staining of livers for Endomucin (EMCN) and Endoglin, EMCN and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE‐1), and CD31 and alpha‐SMA (ACTA2) (n = 5). (A) Scale bar: 5 mm; (B,C) scale bars: 100 μm.

FIGURE 4

Arterialization of hepatic vessels and loss of pericentral genes in Alk1 ^HEC‐KO mice. (A) Heatmap of significantly regulated genes in RNA‐sequencing (RNA‐seq) data of hepatic endothelial cells with annotation for gene sets from Kalucka et al. and Halpern et al. (n = 5). (B,C) Gene set enrichment analysis (GSEA) of RNA‐seq data using (B) the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (n = 5) and (C) Molecular Signatures Database (MSigDB) hallmark gene sets (n = 5). (D) Overrepresentation analysis (ORA) for significantly regulated genes using the Gene Ontology biological processes gene sets (n = 5). (E) Heatmap for stalk‐ and tip cells marker genes with annotation of significance in RNA‐seq data of hepatic endothelial cells from Alk1 ^HEC‐KO mice (n = 5).

FIGURE 5

Induction of proangiogenic factors including prion‐like protein doppel (Prnd) and placental growth factor (Pgf) in Alk1‐deficient mice. (A) Heatmap of top 20 upregulated and downregulated genes ordered by fold change in isolated hepatic endothelial RNA (n = 5). (B) Quantitative polymerase chain reaction (qPCR) for identified proangiogenic factors Prnd and Pgf using isolated hepatic endothelial RNA (n = 5). (C) In situ hybridization for Prnd and Pgf in mouse liver (n = 6). (D) Quantification of in situ hybridizations in (C) (n = 6). (E) Fluorescent in situ hybridization for Prnd or Pgf costained with Cdh5. (F) Enzyme‐linked immunosorbent assay for placental growth factor (PlGF)‐2 of blood serum (n = 8). Scale bars: 100 μm. Error bars: mean with standard deviation. (B,D) Mann–Whitney U test; (F) Welch's t test. **p < 0.01; ***p < 0.001.

FIGURE 6

Loss of endothelial Wnt factors correlates with disrupted metabolic zonation in Alk1 ^HEC‐KO mice. (A) Heatmap of significantly dysregulated angiocrine factors in isolated hepatic endothelial RNA (n = 5). (B) Quantitative polymerase chain reaction (qPCR) for Wnt2, Wnt9b, and R‐spondin‐3 (Rspo3) using isolated hepatic endothelial RNA (n = 5). (C) In situ hybridization for Wnt2, Wnt9b, and Rspo3 in livers (n ≥ 4). (D) Immunofluorescent staining of glutamine synthetase (GLUL) and arginase (ARG) in livers (n = 5). (E) Heatmap of significantly dysregulated genes in whole liver RNA‐sequencing (RNA‐seq) data (n = 5). Genes are also annotated for the zone in which the genes are expressed in wild‐type livers using published single cell RNA‐seq data. (F) Enrichment for pericentral (p < 0.001; normalized enrichment score [NES] = −2.97) and periportal (p < 0.001; NES = 2.57) genes in whole liver RNA‐seq data. Scale bars: 100 μm. Error bars: Mean with standard deviation. (B) Mann–Whitney U test. Wnt, portmanteau of the names Wingless and Int‐1. **p < 0.01.

FIGURE 7

ALK1 signaling enhances expression of Wnt factors and suppresses transcripts of prion‐like protein doppel (Prnd) and placental growth factor (Pgf). (A) Wild‐type (Wt) liver sinusoidal endothelial cells (LSEC) were stimulated in vitro with bone morphogenetic protein (BMP)‐9, BMP‐10, and ALK1‐FC followed by quantitative polymerase chain reactions (qPCRs) for known ALK1 targets Id1, Id2, and Id3; Wnt factors Wnt2, Wnt9b, and R‐spondin‐3 (Rspo3); and proangiogenic factors Prnd and Pgf (n ≥ 3). (B) Wt LSEC were stimulated in vitro with BMP‐9, BMP‐9, Pan‐ID antagonist AGX51, and transforming growth factor (TGF)‐β1 followed by qPCRs for Wnt2 and Rspo3 (n = 4). (A) One‐way ANOVA followed by Dunnett's multiple comparisons test. (B) One‐way ANOVA followed by Tukey's multiple comparisons test. Wnt, portmanteau of the names Wingless and Int‐1. *p < 0.05; **p < 0.01; ***p < 0.001; **** p < 0.0001.

FIGURE 8

Loss of metabolic zonation in human hereditary hemorrhagic telangiectasia (HHT) samples of the liver. (A) Immunofluorescence staining for CD34, CD32b, and glutamine synthetase (GLUL) in livers from patients with HHT and healthy individuals (n = 4). Scale bars: 100 μm. (B) Graphical summary of results in this study. Created with BioRender.com.