Overexpression of Activin Receptor-Like Kinase 1 in Endothelial Cells Suppresses Development of Arteriovenous Malformations in Mouse Models of Hereditary Hemorrhagic Telangiectasia

Abstract

Rationale: Hereditary hemorrhagic telangiectasia (HHT) is a genetic disease caused by mutations in ENG, ALK1, or SMAD4. Since proteins from all 3 HHT genes are components of signal transduction of TGF-β (transforming growth factor β) family members, it has been hypothesized that HHT is a disease caused by defects in the ENG-ALK1-SMAD4 linear signaling. However, in vivo evidence supporting this hypothesis is scarce.

Objective: We tested this hypothesis and investigated the therapeutic effects and potential risks of induced-ALK1 or -ENG overexpression (OE) for HHT.

Methods and results: We generated a novel mouse allele (ROSA26^Alk1) in which HA (human influenza hemagglutinin)-tagged ALK1 and bicistronic eGFP expression are induced by Cre activity. We examined whether ALK1-OE using the ROSA26^Alk1 allele could suppress the development of arteriovenous malformations (AVMs) in wounded adult skin and developing retinas of Alk1- and Eng-inducible knockout (iKO) mice. We also used a similar approach to investigate whether ENG-OE could rescue AVMs. Biochemical and immunofluorescence analyses confirmed the Cre-dependent OE of the ALK1-HA transgene. We could not detect any pathological signs in ALK1-OE mice up to 3 months after induction. ALK1-OE prevented the development of retinal AVMs and wound-induced skin AVMs in Eng-iKO as well as Alk1-iKO mice. ALK1-OE normalized expression of SMAD and NOTCH target genes in ENG-deficient endothelial cells (ECs) and restored the effect of BMP9 (bone morphogenetic protein 9) on suppression of phosphor-AKT levels in these endothelial cells. On the other hand, ENG-OE could not inhibit the AVM development in Alk1-iKO models.

Conclusions: These data support the notion that ENG and ALK1 form a linear signaling pathway for the formation of a proper arteriovenous network during angiogenesis. We suggest that ALK1 OE or activation can be an effective therapeutic strategy for HHT. Further research is required to study whether this therapy could be translated into treatment for humans.

Keywords: activin receptor-like kinase 1; arteriovenous malformation; endoglin; endothelial cell; hereditary hemorrhagic telangiectasia; mice; signal transduction.

Figure 1.. Endothelial-specific induction of exogenous ALK1 in L1Cre; ALK1-OE mice.

A and B, Schematic diagram of the R26^Alk1-off (A), and R26^Alk1-on (B) alleles. Mouse ALK1 carrying an HA tag at the C-terminus is bicistronically transcribed with eGFP by the CMV/chick β-actin (CAGGS) promoter from the R26^Alk1-on allele due to the excision of transcription stop sequences in the presence of Cre recombinase. LoxP sequences are indicated by arrowheads. tpA, transcription stop sequences. C-J, Fluorescence stereomicroscopic images on brains (C-F) and lungs (G and J) of control (L1Cre-negative ALK1-OE, C, G, and I) and L1Cre;ALK1-OE (D-F and H and J) mice. I and J, CD31 (red) immunostaining on control (I) and L1Cre;ALK1-OE (J) lung section. GFP is detected in CD31-positive pulmonary ECs, but not in bronchial epithelial or smooth muscle cells (J). br, bronchus. K. Quantitative RT-PCR on Alk1 transcripts in control and L1Cre;ALK1-OE mouse lungs. β-Actin was used for normalization. n=3 per each group. Unpaired t-test. L. Western blot analyses against HA and ALK1 proteins in control and L1Cre;ALK1-OE lung. M. Quantification of ALK1 levels normalized with β-ACTIN. n=4 per each group. Unpaired t-test. All data are means ± SDs. Scale bars: C-H, 1 mm; I and M, 100 μm.

Figure 2.. ALK1-OE rescues the vascular phenotypes of ENG- as well as ALK1-deficient mice.

A, Alteration of hemoglobin levels in SclCreER;Alk1-iKO (n=8) and SclCreER;Alk1-iKO;ALK1-OE (n=13) on the first day (D0) and 8 days (D8). Two-way ANOVA with Tukey’s correction. B, Gastrointestinal hemorrhage index of SclCreER;Alk1-iKO (n=8) and SclCreER;Alk1-iKO;ALK1-OE (n=13) mice. The unpaired t-test was performed for statistical analysis. Welch t-test. C, Representative images of latex dye-perfused blood vessels (upper panels) and processed images (lower panels) on the dorsal skin of control (SclCreER-negative Alk1-iKO;ALK1-OE (n=4), SclCreER;Alk1-iKO (n=6), and SclCreER;Alk1-iKO;ALK1-OE (n=7) mice on 8 days after wounding. D, Quantification of the vascular area containing latex with the processed images. One-way ANOVA with Tukey’s correction. E, Latex perfusion images in SclCreER;Eng-iKO (n=11) and SclCreER;Eng-iKO;ALK1-OE (n=10). F, Quantification of vascular density containing latex. Mann-Whitney test. All data are means ± SDs. The wound sites are marked by asterisks. Scale bars: 1 mm.

Figure 3.. ALK1-OE in retinal ECs suppresses the development of AVMs caused by ALK1- or ENG-deficiency.

A-E, IB4 staining of postnatal day 5 (PN5) retinas from control (A, n=12), SclCreER;Alk1-iKO (B and D, n=9), and SclCreER;Alk1-iKO;ALK1-OE (C and E, n=5) mice. The boxed region in B is a magnified image showing direct connections between arteries and veins (D). SclCreER-mediated GFP reporter expression in SclCreER;Alk1-iKO;ALK1-OE retinas (E). F, Quantification of AVM numbers in PN5 retinal vasculature. Kruskal–Wallis test with Dunn’s correction. G-K, IB4 staining of PN7 retinas from control (G, n=46), SclCreER;Eng-iKO (H and J, n=24), and SclCreER;Eng-iKO;ALK1-OE (I and K, n=22) mice. The boxed region in H is a magnified image showing arteriovenous shunts (J). Bicistronic GFP reporter expression in SclCreER;Eng-iKO;ALK1-OE retinas (K). L, Quantification of the number of AVMs. Yellow arrowheads indicate AVMs in the developing retinas. Kruskal–Wallis test with Dunn’s correction. All data are means ± SDs. a, artery; v, vein. Scale bars: A-C and G-I, 500 μm; D, E, J, and K, 100 μm.

Figure 4.. ALK1-OE restores aberrant SMC coverage observed in Eng-iKO retinal vasculature.

A-F, ENG (red) and IB4 (blue) fluorescence staining with retinas isolated from PN7 control (SclCreER-negative Eng-iKO, A and D), SclCreER;Eng-iKO (B and E), and SclCreER;Eng-iKO;ALK1-OE (C and F) mice. G-I. Double staining of IB4 (blue) and SMA (red) in PN7 retinas of controls (G), SclCreER;Eng-iKO (H), and SclCreER;Eng-iKO;ALK1-OE (I) mice. GFP reporter expression in IB4-positive retinal ECs in SclCreER;Eng-iKO;ALK1-OE (F and I). Yellow arrowheads indicate developed retinal AVMs. a, artery; v, vein. Scale bars: 100 μm.

Figure 5.. Disrupted arteriovenous EC identities in Eng-deficient vasculature are rescued by ALK1-OE.

A-F, Immunostaining on EMCN (red) and IB4 (blue) in PN 7 retinas of control (SclCreER-negative Eng-iKO, A and D), SclCreER;Eng-iKO (B and E), and SclCreER;Eng-iKO;ALK1-OE (C and F) mice. Merged images of EMCN, GFP, and IB4 (D-F). G-L, JAG1 (red) expression in PN7 retinas of controls (G and J), SclCreER;Eng-iKO (H and K), and SclCreER;Eng-iKO;ALK1-OE (I and L) mice. ECs are marked with IB4 (blue) staining. GFP signals in IB4-stained retinal ECs (J-L). Yellow arrowheads indicate AVMs in the retinal vasculature. a, artery; v, vein. Scale bars: 100 μm.

Figure 6.. Altered NOTCH and SMAD downstream genes in Eng-iKO ECs are restored by ALK1-OE.

Quantitative RT-PCR analysis of Eng (A) and Alk1 (B), and Id1 (C)and NOTCH downstream genes [Hey1 (D), Jag1 (E), and Jag2 (F)] in retinal ECs purified from control (SclCreER-negative Eng-iKO, n=6), SclCreER;Eng-iKO (n=6), and SclCreER;Eng-iKO;ALK1-OE (n=6) mice. The mRNA levels of the genes are normalized with actin. One-way ANOVA with Tukey’s correction. All data are means ± SDs.

Figure 7.. ALK1–OE leads to the rescue of disrupted SMAD and AKT signaling in Eng-iKO ECs.

A, Western blot analyses of phosphorylated SMAD1,5,8 (pSMAD1,5,8), ENG, and HA in pulmonary ECs from R26^CreER;Eng-iKO or R26^CreER;Eng-iKO;ALK1-OE. HA indicates transgenic ALK1 expression. One μM of 4-hydroxy-tamoxifen was treated to delete the Eng gene and to induce HA-ALK1 expression. BMP9 (0.5 ng/mL) was added to serum-starved pulmonary ECs for 45 min in the static or flow condition. B and C, Quantification of SMAD1,5,8 phosphorylation levels in static condition (B) and flow stimulation (C). pSMAD1,5,8 levels were normalized with total SMAD1 and β-ACTIN. n=5 per each group. Two-way ANOVA with Tukey’s correction. D, Western blot analyses of phosphorylated AKT (pAKT) and ENG in pulmonary ECs of R26^CreER;Eng-iKO (left panel) or R26^CreER;Eng-iKO;ALK1-OE (right panel). HA indicates ALK1 overexpression. One μM of OH-TM was used to delete the Eng gene. BMP9 (10 ng/mL) was treated for 90 min, followed by 30 min exposure to flow. E-H, Quantification of relative phosphor-AKT levels normalized with total AKT in static condition (E and F) and flow stimulation (G and H). n=5 per each group. Two-way ANOVA with Tukey’s correction. All data are means ± SDs.

Figure 8.. ENG-OE does not overcome the vascular phenotype of Alk1-iKO mice.

A-C, Latex perfusion images in control (A, n=9), SclCreER;Eng-iKO (B, n=6), and SclCreER;Eng-iKO;hENG-OE (C, n=8) on 8 days after wounding. D and E, Quantification of vascular density containing latex using the processed images. Welch t-test (D). One-way ANOVA with Tukey’s correction (E). F-I, Visualization of wound-induced skin AVMs using latex infusion in R26^CreER;Alk1-iKO (F, n=10), R26^CreER;Alk1-iKO;hENG-OE (G, n=7), SclCreER;Alk1-iKO (H, n=7) and SclCreER;Alk1-iKO;hENG-OE (I, n=7) mice. J-L, IB4 (red) staining of PN5 retinal vasculature from control (J, n=20), R26^CreER;Alk1-iKO (K, n=4), and R26^CreER;Alk1-iKO;hENG-OE (L, n=10) mice. Yellow arrowheads indicate AVMs in the retinal vasculature. M, Quantification of the number of AVMs per retina. Kruskal–Wallis test with Dunn’s correction. N, Western blot analyses in the effects of hENG-OE on the SMAD1,5,8 phosphorylation and endogenous mENG level. O and P, Quantification of pSMAD1,5,8 levels normalized to normalized with SMAD1 and β-ACTIN (O) and mouse ENG levels normalized with β-ACTIN (P). N=5 per each group. One-way ANOVA with Tukey’s correction. All data are means ± SDs. Scale bars: A-C and F-I, 1 mm; J-L, 500 μm.