Rapamycin improves TIE2-mutated venous malformation in murine model and human subjects

Abstract

Venous malformations (VMs) are composed of ectatic veins with scarce smooth muscle cell coverage. Activating mutations in the endothelial cell tyrosine kinase receptor TIE2 are a common cause of these lesions. VMs cause deformity, pain, and local intravascular coagulopathy, and they expand with time. Targeted pharmacological therapies are not available for this condition. Here, we generated a model of VMs by injecting HUVECs expressing the most frequent VM-causing TIE2 mutation, TIE2-L914F, into immune-deficient mice. TIE2-L914F-expressing HUVECs formed VMs with ectatic blood-filled channels that enlarged over time. We tested both rapamycin and a TIE2 tyrosine kinase inhibitor (TIE2-TKI) for their effects on murine VM expansion and for their ability to inhibit mutant TIE2 signaling. Rapamycin prevented VM growth, while TIE2-TKI had no effect. In cultured TIE2-L914F-expressing HUVECs, rapamycin effectively reduced mutant TIE2-induced AKT signaling and, though TIE2-TKI did target the WT receptor, it only weakly suppressed mutant-induced AKT signaling. In a prospective clinical pilot study, we analyzed the effects of rapamycin in 6 patients with difficult-to-treat venous anomalies. Rapamycin reduced pain, bleeding, lesion size, functional and esthetic impairment, and intravascular coagulopathy. This study provides a VM model that allows evaluation of potential therapeutic strategies and demonstrates that rapamycin provides clinical improvement in patients with venous malformation.

Figure 8. Evolution of quantitative parameters from start to 12 months after rapamycin treatment.

Analysis of D-dimer levels, pain, QoL, and MRI measurements in 5 patients at start and after 12 months of rapamycin treatment.

Figure 7. Clinical photographs and MRI images of patients in the pilot study.

Clinical photographs (left panels) and MRI scans (right panels) of lesions in (A) patient 1; (B) patient 2; (C) patient 3, (D) patient 4; (E) patient 5; and (F) patient 6. L, left; R, right. Arrows point to lesions.

Figure 6. Growth of established VMs was prevented by rapamycin treatment.

(A) Treatment schematic. (B) Images of mice at day 19 after cell injection (day 7 after start of treatment). (C) Representative murine VM lesion explants after 7 days of i.p. treatment. Left panel: vehicle; right panel: rapamycin (1 mg/kg). Images taken from fixed distance. (D) HUVEC-TIE2-L914F lesional area measured by caliper every 2 days from day 13 to day 19. Data expressed as mean ± SEM, t test (n = 5 mice with 2 lesions/group). (E) Vascular volume of each lesion before (day 12) and after (day 19) treatment measured by analysis of color Doppler 3D image stacks. Data expressed as single values for each lesion and medians plotted as horizontal bars, 2-way repeated measures ANOVA. (F) HUVEC-TIE2-L914F lesional area measured with calipers every other day from day 15 after cell injection (start of treatment, vehicle or rapamycin 2 mg/kg/d) to day 35 (endpoint). Data are expressed as mean + SEM, t test (n = 10 mice with 2 lesions/group). From day 19 to day 35, values in the 2 groups are statistically different. *P < 0.01, t test. (G) Representative murine VM lesion explant at day 35 after 20 days of vehicle or rapamycin treatment. Images taken from fixed distance. Scale bars: 1 cm.

Figure 5. TIE2-TKI and rapamycin effects on signaling pathways downstream of TIE2 in HUVEC-TIE2-WT and HUVEC-TIE2-L914F.

(A) Immunoblot analysis of HUVEC-TIE2-WT and TIE2-L914F treated for 48 hours with TIE2-TKI (5 μM), DMSO, or rapamycin (15 nM). Two protein samples loaded from 2 different cell treatment sets. Tubulin served as loading control. (B) Densitometric analysis of p-TIE2, p-AKT473, p-AKT308, p-STAT1, p-ERK, p-mTOR, and p–4E-BP1 Western blot bands relative to total protein, TIE2, AKT, STAT1, ERK, mTOR, and 4E-BP1, respectively. Data expressed as mean ± SD, t test (n = 4, 2 independent experiments). Data are normalized to DMOS-treated HUVEC-TIE2-WT (dashed lines).

Figure 4. TIE2-TKI effects on TIE2 and AKT phosphorylation in TIE2-WT, TIE2-L914F, and TIE2-R1099X mutant HUVECs.

(A) Western blot analysis of HUVEC-TIE2-WT, HUVEC-TIE2-L914F, and HUVEC-TIE2-R1099X after 48 hours treatment with TIE2-TKI (0.5–10 μM). β-Actin served as loading control. (B) Densitometric analysis of p-TIE2, p-AKT473, and p-AKT308 Western blot bands relative to total TIE2 and AKT, respectively. Data are normalized to untreated HUVEC-TIE2-WT. (C) Western blot analysis of HUVEC-TIE2-WT, HUVEC-TIE2-L914F, and HUVEC-TIE2-R1099X pretreated for 2 hours with TIE2-TKI (0.5–10 μM), subsequently stimulated for 15 minutes with 1 μg/ml ANGPT1. β-Actin served as loading control. (D) Densitometric analysis of p-TIE2, p-AKT473, and p-AKT308 Western blot bands relative to total TIE2 and AKT, respectively. Data are normalized to untreated, 1 μg/ml ANGPT1 conditions.

Figure 3. TIE2-TKI and rapamycin effects on murine VMs.

(A) Pretreatment plus i.p. injection schematic. (B) Representative murine VM lesion explants at day 16. Top row: vehicle; middle row: TIE2-TKI; bottom row: rapamycin. Images taken from fixed distance. (C) HUVEC-TIE2-L914F lesional area measured by caliper every 2 days for 16 days. Data expressed as mean ± SEM, t test (n = 5 mice with 2 lesions/group). (D) Vascular volume at day 15 measured by analysis of color Doppler 3D image stacks. Data expressed as single values for each lesion (n = 5 mice with 2 lesions/group); medians shown by horizontal bars, Mann-Whitney U test. (E) Pretreatment schematic. (F) Representative murine VM lesion explants at day 16. Top row, DMSO; middle row, TIE2-TKI; bottom row, rapamycin. Images taken from fixed distance. (G) HUVEC-TIE2-L914F lesional area measured by caliper every 2 days for 16 days. Data expressed as mean ± SEM, t test (n = 5 mice with 2 lesions/group; shown is a representative experiment from 2 independent experiments). (H) Vascular volume at day 16 measured by analysis of color Doppler 3D image stacks. Data expressed as single values for each lesion; medians shown by horizontal bars, Mann-Whitney U test (n = 5 mice with 2 lesions/group; shown is a representative experiment from 2 independent experiments). (I) Representative images of VM sections immunostained for UEA-I (red), αSMA (green), and DAPI (blue). Arrows point to αSMA⁺ cells. Scale bar: 100 μM. (J) Quantification of luminal area occupied by UEA-I–stained blood vessels, expressed as percentage of total sectional area. Data expressed as mean ± SEM, t test (n = 5 fields/section, 7 mice analyzed/group). NS, P > 0.05. (K) Quantification of αSMA⁺ cells surrounding UEA-I–stained blood vessels. Data expressed as mean ± SEM (n = 5 fields/section, 7 mice analyzed/group). NS, P > 0.05.

Figure 2. HUVEC-TIE2-L914F assembled in enlarged channels filled with blood.

(A) HUVEC-TIE2-L914F (red) and HUVEC-TIE2-WT (gray) lesional area measured by caliper every 3 to 5 days for 50 days. HUVEC-TIE2-WT lesions were undetectable after day 23. Mouse weight monitored for 50 days. Data expressed as mean ± SEM, t test (n = 10 mice with 2 lesions each/group). (B) Lesions explanted 14 days after injection (n = 7 mice with 2 lesions/group). Top row: HUVEC-TIE2-L914F; bottom row: HUVEC-TIE2-WT; below, left: representative images of lesions; below, right: H&E-stained sections from middle of the explant. Scale bars: 1 cm (top panel); 500 μm (bottom panels). (C) Representative color Doppler 3D image stacks of murine VM lesion analyzed at days 7, 14, and 21 (n = 3 mice were analyzed with 3D color Doppler at days 7, 14, and 21).

Figure 1. HUVEC-TIE2-L914F formed VM lesions in immune-deficient mice.

(A) Western blot of p-TIE2 in HUVECs transfected with TIE2-WT or with mutant TIE2 (L914F). Tubulin served as loading control. (B) Representative nude mouse 7 days after injection of HUVEC-TIE2-WT (left flank) or HUVEC-TIE2-L914F (right flank) and Matrigel explants from each injected mouse (n = 8) (bottom panel). (C) Matrigel explants with HUVEC-TIE2-WT (left) or HUVEC-TIE2-L914F (middle) sectioned and stained with specific anti-human CD31 (hu-CD31), representative images (n = 4). Murine lung tissue (right) is shown as negative control for anti-human CD31 staining. Scale bar: 100 μm. (D) HUVEC-TIE2-WT and HUVEC-TIE2-L914F tissue sections stained for H&E (top panel) and UEA-I and αSMA (bottom panel). Patient-derived VM and infantile hemangioma are shown for comparison. Representative images (n = 4 WT, L914F; n = 2 patients with VM; n = 3 patients with infantile hemangioma). UEA-I (red) and αSMA (green); arrows point to areas with perivascular αSMA⁺ cell coverage in VM vessels. Scale bars: 100 μm. (E) Quantification of human CD31–stained blood vessels (vessels/mm²) and luminal area occupied by human CD31–stained blood vessels (μm²) in HUVEC-TIE2-WT or HUVEC-TIE2-L914F mid-explant sections. Data expressed as mean ± SEM, t test (n = 8).