Angiopoietin-2 Inhibition Rescues Arteriovenous Malformation in a Smad4 Hereditary Hemorrhagic Telangiectasia Mouse Model

Abstract

Background: Hereditary hemorrhagic telangiectasia is an autosomal dominant vascular disorder caused by heterozygous, loss-of-function mutations in 4 transforming growth factor beta (TGFβ) pathway members, including the central transcriptional mediator of the TGFβ pathway, Smad4. Loss of Smad4 causes the formation of inappropriate, fragile connections between arteries and veins called arteriovenous malformations (AVMs), which can hemorrhage leading to stroke, aneurysm, or death. Unfortunately, the molecular mechanisms underlying AVM pathogenesis remain poorly understood, and the TGFβ downstream effectors responsible for hereditary hemorrhagic telangiectasia-associated AVM formation are currently unknown.

Methods: To identify potential biological targets of the TGFβ pathway involved in AVM formation, we performed RNA- and chromatin immunoprecipitation-sequencing experiments on BMP9 (bone morphogenetic protein 9)-stimulated endothelial cells (ECs) and isolated ECs from a Smad4-inducible, EC-specific knockout ( Smad4-iECKO) mouse model that develops retinal AVMs. These sequencing studies identified the angiopoietin-Tek signaling pathway as a downstream target of SMAD4. We used monoclonal blocking antibodies to target a specific component in this pathway and assess its effects on AVM development.

Results: Sequencing studies uncovered 212 potential biological targets involved in AVM formation, including the EC surface receptor, TEK (TEK receptor tyrosine kinase) and its antagonistic ligand, ANGPT2 (angiopoietin-2). In Smad4-iECKO mice, Angpt2 expression is robustly increased, whereas Tek levels are decreased, resulting in an overall reduction in angiopoietin-Tek signaling. We provide evidence that SMAD4 directly represses Angpt2 transcription in ECs. Inhibition of ANGPT2 function in Smad4-deficient mice, either before or after AVMs form, prevents and alleviates AVM formation and normalizes vessel diameters. These rescue effects are attributed to a reversion in EC morphological changes, such as cell size and shape that are altered in the absence of Smad4.

Conclusions: Our studies provide a novel mechanism whereby the loss of Smad4 causes increased Angpt2 transcription in ECs leading to AVM formation, increased blood vessel calibers, and changes in EC morphology in the retina. Blockade of ANGPT2 function in an in vivo Smad4 model of hereditary hemorrhagic telangiectasia alleviated these vascular phenotypes, further implicating ANGPT2 as an important TGFβ downstream mediator of AVM formation. Therefore, alternative approaches that target ANGPT2 function may have therapeutic value for the alleviation of hereditary hemorrhagic telangiectasia symptoms, such as AVMs.

Keywords: receptor, TIE-2; telangiectasia, hereditary hemorrhagic; transforming growth factor beta.

Fig. 1.. Genomic studies reveal endothelial SMAD4 targets related to HHT and the TGFβ pathway.

(a) Graphical representation of Cre-LoxP system components and Smad4 depletion timeline. (b) Representative Isolectin-IB4 staining of retinal blood vessels in Smad4^f/f control and Smad4-iECKO mutant animals depicting presence of AVMs (arrows). White dotted circle represents outgrowth of Smad4^f/f control mouse. A, arteries; V, veins. Scale bar represents 500 µm. (c) Heatmap depicting differential expression of genes in isolated retinal ECs (iREC) of Smad4^f/f and Smad4-iECKO animals. (d) Graphical plot of 1,095 upregulated and 810 downregulated genes in Smad4-iECKO compared to Smad4^f/f iREC. (e) Venn Diagram showing overlap between SMAD4 binding sites in unstimulated and 24 hour BMP9/10 stimulated Ms1 ECs. (f) Doughnut diagram of SMAD4 bound gene locations in unstimulated versus BMP9/10 stimulated ECs. (g) Top SMAD4 binding motifs in unstimulated (red) and BMP9/10 stimulated (green) ECs arranged by family. (h) Representative view of four SMAD4 binding motifs. Percentages below motifs are the percentage of binding sites where motifs were found in unstimulated and BMP9/10 stimulated ECs, respectively. (i,j) Highlighted SMAD4 binding peaks (red box) within the Eng and Acvrl1 mouse genes in unstimulated (grey) and BMP9/10 (blue) stimulated ECs. Evolutionary conserved regions between chimpanzee, human and mouse Eng and Acvrl1 genes show Smad4 binding peaks in Eng are located within non-coding evolutionary conserved regions (ECRs). Notice the lack of a SMAD4 binding site in the Acvrl1 gene.

Fig. 2.. SMAD4 deficiency leads to changes in Angpt2 and Tek expression in the endothelium.

(a) Venn Diagram depicting overlap between the isolated retinal endothelial cell (iREC) RNA-Seq and BMP9/10 stimulated Ms1 cell SMAD4 ChIP-Seq (BMP9/10 Stim ChIP) datasets revealed 212 direct, downstream targets of SMAD4. (b-d) Verification of increased Angpt2 and decreased Tek transcript levels in iRECs (b-c) and isolated lung endothelial cells (iLECs; d) of Smad4-iECKO versus Smad4^f/f animals. All values are normalized to Pecam mRNA levels. Quantifications were performed in triplicate for each n. iREC: Pecam, n = 4; Tek, n = 4; Smad4, n = 3; Angpt2, n = 4. iLECs: Pecam, n = 3; Tek, n = 3; Smad4, n = 4; Angpt2, n = 3. (e,f) In situ hybridization analysis of Angpt2 and Tek transcripts in Smad4^f/f and Smad4-iECKO P7 retinas, followed by Isolectin-IB4 (IB4) immunofluorescent staining. (e) Angpt2 transcripts are barely detectable in Smad4^f/f retinas, but levels are increased dramatically at the growing vascular front of Smad4-iECKO mice. Note Angpt2 absence in AVM (asterisk). (f) In Smad4^f/f retinas, Tek is expressed in all vessels excluding ECs at the vascular edge, whereas upon loss of Smad4, Tek levels are decreased in the vascular front and are present within the AVM (asterisks). (g) Western blot analysis verifies increased ANGPT2 and decreased TEK and SMAD4 protein levels in Smad4-iECKO versus Smad4^f/f iLECs. (h) Smad4-iECKO mice exhibit increased serum levels of ANGPT2 compared to Smad4^f/f mice. (i) ChIP-Seq analysis on unstimulated and BMP9/10 stimulated mouse Ms1 ECs revealed three SMAD4 binding peaks upstream of the Angpt2 gene. All three sites are within non-coding evolutionarily conserved regions (ECRs) between chimpanzees and humans. (j) Closeup view of SMAD4 binding sites upstream of Angpt2 (Peaks 1–3; red boxes). (k) ChIP-qPCR using SMAD4 and IgG antibodies on unstimulated and BMP9/10 stimulated Ms1 ECs verified SMAD4 binding enrichment on all three peaks upstream of Angpt2 (n = 2). Control DNA primers outside of the SMAD4 binding regions show no enrichment. Primer regions and control (C) are depicted by red lines in (j). (l) Quantification of luciferase activity normalized to renilla expression in stable Ms1 cell lines expressing nonsilencing-shRNA (NS-shRNA) or Smad4-shRNA (n = 3 for all experiments; 3 readouts were analyzed per n). Sample size (n) indicates biological replicates. Error bars represent mean ± standard error. Statistics were generated using the Mann Whitney U test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig. 3.. ANGPT2 inhibition prevents AVM formation in Smad4-iECKO mice by impeding changes in endothelial cell size and shape.

(a) Timeline depicting experimental procedures including tamoxifen (Tx) injections, drug injections (LC10 or IgG), approximate time AVMs form and retinal collection. (b-e) Smad4^f/f and Smad4-iECKO P7 retinas injected with 30 μg of IgG (b,c) or LC10 (d,e), and stained for Isolectin-IB4 (magenta) and αSMA (blue). White dotted circles represent outgrowth of Smad4^f/f injected with IgG (b). (c) Smad4-iECKO P7 retinas injected with IgG revealed increased vessel diameters and AVMs (arrows). (d) Smad4^f/f P7 retinas injected with LC10 exhibited a reduction in vascular outgrowth. (e) Smad4-iECKO P7 retinas injected with LC10 did not exhibit AVMs or increased vessel diameters. A, arteries; V, veins. Scale bars represent 500 μm. (f-i) Quantification of AVM number (f), retinal vascular outgrowth (g), and vein (h) and artery (i) diameters from experimental retinas in b-e. Notice that administration of LC10 prevents vessel enlargement associated with SMAD4 loss (Smad4-iECKO + IgG versus Smad4-iECKO + LC10). (j-m) Close up images of retinal veins stained with PECAM to mark EC junctions/boundaries revealed changes in size and morphology of ECs in Smad4^f/f and Smad4-iECKO P7 retinas injected with IgG versus LC10. Several cells are uniformly enlarged below in black to highlight cell morphological changes. Scale bars represent 50 μm. (n,p) Quantification of venous and arterial EC areas. Administration of LC10 prevents endothelial cell enlargement. (o,q). Shape factor quantification of venous and arterial ECs. A cell is given a numerical value of 1 to 0 based on its shape: circle = 1; elongated line = 0. (o) Loss of Smad4 causes venous ECs to acquire an elongated, arterial shape, whereas administration with LC10 prevents this phenotype. Sample size (n) indicates biological replicates which were used for the corresponding quantifications. Mice were taken from three separate litters. Error bars represent mean ± standard error. Statistics were generated using ordinary one-way ANOVA with Tukey’s multiple comparison test applied. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig. 4.. ANGPT2 inhibition rescues AVM formation and EC morphology in Smad4-iECKO mice.

(a) Timeline depicting experimental procedures including tamoxifen (Tx) injections, approximate time AVMs form, drug injections (30 μg of LC10 or IgG) and retinal collection. (b-e) Smad4^f/f and Smad4-iECKO P8 retinas injected with either IgG or LC10 and stained with Isolectin-IB4 (magenta) and αSMA (blue). Scale bars represent 500 μm. White dotted circles represent outgrowth of Smad4^f/f injected with IgG. Compared to Smad4-iECKO mice treated with IgG (c), administration of LC10 to Smad4-iECKO mice results in a substantial reduction in the number of retinal AVMs and normalization of artery and vein calibers (e). (f-i) Quantification of retinal AVM number (f), vascular outgrowth (g), and vein (h) and artery (i) diameters from experimental retinas in b-e. Notice that the increases in vessel caliber in Smad4-iECKO mice treated with IgG revert to control sizes (Smad4^f/f + IgG) when LC10 is administered to Smad4-iECKO mice. (j-m) Close up images of retinal veins stained with PECAM to mark EC junctions/boundaries revealed changes in size and morphology of ECs in Smad4^f/f and Smad4-iECKO P8 retinas injected with IgG versus LC10. Several cells are uniformly enlarged below in black to highlight cell morphological changes. Scale bars represent 50 μm. (n,p) Quantification of venous and arterial EC areas. The increased cell areas observed in Smad4-iECKO mice injected with IgG revert to control sizes when LC10 is given to Smad4-iECKO mice (o,q). Shape factor quantification of venous and arterial ECs. A cell is given a numerical value of 1 to 0 based on its shape: circle = 1; elongated line = 0. (o) Loss of Smad4 causes venous ECs to acquire an elongated, arterial shape, whereas administration with LC10 significantly reverses this phenotype and cells return to a more cuboidal shape. Sample size (n) indicates biological replicates which were used for the corresponding quantifications. Mice were taken from three separate litters. Error bars represent mean ± standard error. Statistics were generated using ordinary one-way ANOVA with Tukey’s multiple comparison test applied. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig 5.. Model: SMAD4 directly represses Angpt2 transcription in ECs to maintain a normal vascular network and prevent HHT associated phenotypes.

SMAD4 is a direct repressor of Angpt2 transcription in ECs. Loss of SMAD4 causes increased Angpt2 expression and leads to changes in EC size and shape that results in blood vessel dilation and AVM formation. Inhibition of ANGPT2 function can prevent and alleviate AVM formation and vessel caliber increases by regulating EC size and shape.