SMAD4 Deficiency Leads to Development of Arteriovenous Malformations in Neonatal and Adult Mice

Abstract

Background Hereditary hemorrhagic telangiectasia ( HHT ) is a rare genetic vascular disorder caused by mutations in endoglin ( ENG ), activin receptor-like kinase 1 ( ACVRL 1; ALK 1), or SMAD 4. Major clinical symptoms of HHT are arteriovenous malformations ( AVM s) found in the brain, lungs, visceral organs, and mucosal surface. Animal models harboring mutations in Eng or Alk1 recapitulate all of these HHT clinical symptoms and have been useful resources for studying mechanisms and testing potential drugs. However, animal models representing SMAD 4 mutations have been lacking. The goal of this study is to evaluate Smad4-inducible knockout ( iKO ) mice as an animal model of HHT and compare the phenotypes with other established HHT animal models. Methods and Results Global Smad4 deletion was induced at neonatal and adult stages, and hemoglobin levels, gastrointestinal hemorrhage, and presence of aberrant arteriovenous connections were examined. Neonatal Smad4- iKO mice exhibited signs of gastrointestinal bleeding and AVM s in the brain, intestine, nose, and retina. The radial expansion was decreased, and AVM s were detected on both distal and proximal retinal vasculature of Smad4- iKO s. Aberrant smooth muscle actin staining was observed in the initial stage AVM s and their connecting veins, indicating abnormal arterial flow to veins. In adult mice, Smad4 deficiency caused gastrointestinal bleeding and AVM s along the gastrointestinal tract and wounded skin. HHT -related phenotypes of Smad4- iKO s appeared to be comparable with those found in Alk1- iKO and Eng- iKO mice. Conclusions These data further confirm that SMAD signaling is crucial for normal arteriovenous network formation, and Smad4- iKO will be an alternative animal model of AVM research associated with HHT .

Keywords: animal model of human disease remodeling; arteriovenous fistula; arteriovenous malformation; hereditary hemorrhagic telangiectasia; transgenic model.

Figure 1

Smad4 deletion results in gastrointestinal hemorrhages and arteriovenous malformations in the intestines, head, and brain. A, Schematic overview of the experimental setup. 100 μg of tamoxifen (TM) was intragastrically injected for 3 consecutive days. B, Decreased body weight of Smad4‐inducible knockout (iKO) pups at postnatal day 7 (P7). All data represent mean±SD. ***P<0.001. C, Morphology of stomachs (upper panels) and intestines (lower panels) of control and Smad4‐iKO pups at P7. Arrows indicate the areas showing hemorrhagic signs. Scale bars: 2 mm. D, Mesenteric and intestinal vessels of controls and Smad4‐iKO visualized by latex dye perfusion via the left ventricle. Red and yellow dots indicate arteries and veins, respectively. White arrows indicate arteriovenous shunting vessels. Scale bars: 1 mm. E, Lateral (upper panels) and bottom‐up (lower panels) views of vasculature in snout areas of control and Smad4‐iKO mice. Apparent arteriovenous shunts were detected in Smad4 mutants. Scale bars: 1 mm. F, Vasculature in the area of the hippocampus of control and Smad4‐iKO visualized by latex dye. Numerous arteriovenous shunts and latex‐filled superior sagittal sinuses were detected in Smad4‐iKOs. Red and yellow dots indicate arteries and veins, respectively. White arrows mark arteriovenous shunts. Scale bars: 1 mm. a indicates artery; DNV, dorsal nasal vein; MPA, major palatine artery; P0, P1, P2, and P3, postnatal day 0, 1, 2, and 3, respectively; SS, straight sinus; TS, transverse sinus; UI, upper incisor; v, vein.

Figure 2

Smad4 deficiency induces spontaneous arteriovenous malformations (AVMs) in developing retina. A and B, Immunofluorescence staining of CD31 (red) in postnatal day 5 retinas of control (A) and Alk1‐inducible knockout (iKO) (B) mice, in which 50 μg of tamoxifen was intragastrically administrated to postnatal day 3 pups. C through N, CD31 (green) and smooth muscle α actin (red) staining of postnatal day 7 retinas from control (C through F) and Smad4‐iKO (G through N). 100 μg of tamoxifen was injected for 3 consecutive days from postnatal day 1. I and J, Magnified images of immature AVMs in (G and H). M and N, Magnified images of developed AVMs of Smad4‐iKO retina in the boxes of (K and L). In (C, G, and K), arteries and veins are indicated by red and yellow dots, respectively. Arrows and arrowheads indicate developed and immature AVMs, respectively. Scale bars in (A through D, G, H, K, and L) indicate 500 μm; (E, F, I, J, M, and N), 100 μm. O, Quantification of retinal AVMs in Smad4‐iKO pups. P, Percentage of retinas containing only immature or both immature and developed AVMs in Smad4‐iKO retinas. Q, Decreased progression of vascular plexus towards the edge of the retina. All data represent mean±SD. ***P<0.001. a indicates artery; v, vein.

Figure 3

Immature arteriovenous malformation areas shown in a postnatal day 7 (P7) retina of Smad4‐inducible knockout (iKO). Immunofluorescence staining of CD31 (green; A, D, and G), smooth muscle α actin (SMA [red]; B, E, and H), and merged images (C, F, and I) of P7 retina of Smad4‐iKO. Aberrant SMA‐positive veins and connecting vessels to neighboring arteries indicate initial stages of arteriovenous shunting. Scale bars indicate 100 μm. a indicates artery; v, vein.

Figure 4

Global deletion of Smad4 in adult mice leads to gastrointestinal hemorrhages and arteriovenous malformations (AVMs). A and B, Reduction of body weight (b.w.; A) and hemoglobin levels (B) in Alk1‐, Eng‐, and Smad4‐inducible knockout (iKO) mice 6 days after tamoxifen treatment. *P<0.05, ***P<0.001. C, Hemorrhagic signs shown in gastrointestinal tracks of Smad4‐iKO mice. Scale bars indicate 1 mm. D, Visualized gastric and intestinal AVMs with latex dye perfusion. While the perfused dye was detectable only in arteries of control mice, it was observed in both dilated and tortuous arteries and veins in the stomachs, intestines, and Peyer's patches of Smad4‐, Alk1‐, and Eng‐iKO mice. Scale bars indicate 1 mm. E, Percentages of AVMs formed in intestine (upper panel) or stomach (lower panel) in Alk1‐, Eng‐, and Smad4‐iKO mice. D0 indicates day 0; D6, day 6.

Figure 5

Smad4 deficiency causes skin arteriovenous malformations (AVMs) in response to wounding in adults. A and B, Western blot (A) of wounded skin and immunostaining of wounded ears with anti‐SMAD4 antibodies demonstrate that the SMAD4 protein is diminished in Smad4‐inducible knockout (iKO) mice. ACTIN was used as a loading control. Scale bars in B=100 μm. C through H’, Latex dye–perfused vasculature surrounding the wound in the dorsal skin of control (C), Alk1‐iKO (D), Eng‐iKO (E), and Smad4‐iKO (F through H’) mice 6 days after tamoxifen treatment. While most of the Alk1‐iKO and Eng‐iKO mice displayed well‐developed AVMs, Smad4‐iKO mice exhibited a range of severity of skin AVMs: no AVM (F), immature AVMs (G and G’), and developed AVMs (H and H’). Arrows indicate fully developed AVMs and arrowhead marks show immature AVMs. The wound sites are indicated by asterisks. The scale bars indicate 2 mm. I, Percentage of categorized wound‐induced AVMs in hereditary hemorrhagic telangiectasia model animals. J, Blood vessels containing the latex dye are processed for quantification. Representative converted images to quantify the vascular area containing latex. The wound sites are indicated by asterisks. The scale bars indicate 2 mm. K, Quantification of vascular density per a given area. All data represent mean±SD. *P<0.05, **P<0.01, and ***P<0.001. Cont indicates control.

Figure 6

SMAD4 depletion causes loss of canonical ALK1/endoglin (ENG) signaling. A through D, Western blot analyses of SMAD4, phosphorylated and total R‐SMADs, and ENG in human umbilical vein endothelial cells (HUVECs) treated with scrambled control small interfering RNA (siRNA) or SMAD4‐siRNA for 24 and 48 hours in normal culture media (A). Quantification of SMAD4 proteins (B), pSMAD1, 5, and 8 levels (C), and ENG proteins (D) at 48 hours after siRNA transfection. E through H, Protein levels of SMAD4, phosphorylated and total R‐SMADs, and ENG in control and SMAD4‐KD HUVECs treated with or without bone morphogenetic protein 9 (BMP9) in serum‐starved condition at 24 and 70 hours after siRNA transfection (E). Quantification shows a >95% decrease of SMAD4 (F), responses to BMP9 (G), and reduction of ENG proteins (H) in SMAD4‐KD HUVECs 70 hours after transfection. 10 ng/mL of human BMP9 was treated for 2 hours after serum starvation for 16 hours. I through N, Protein levels of SMAD4, phosphorylated and total R‐SMADs, and ENG in primary pulmonary endothelial cells (pECs) isolated from wild‐type (WT) and Smad4‐inducible knockout (iKO) mice (I). Treatment with 4 hydroxyl‐tamoxifen (OH‐TM) did not affect SMAD4 and R‐SMAD expressions in WT cells but abolished SMAD4 expression and elevated pSMAD1, 5, and 8 levels in Smad4‐iKO cells (I and K). In serum‐free condition, SMAD1, 5, and 8 are activated by BMP9 treatment in both SMAD4‐depleted and normal pECs (J and L). ENG protein level is decreased in Smad4‐iKO pECs (I, J, M, and N). O, Quantitative reverse transcription polymerase chain reaction demonstrates that responses to BMP9 in upregulating BMP9‐ALK1 downstream genes ID1,ID3,JAG1, and HEY2 are markedly attenuated in SMAD4‐depleted HUVECs. ACTIN was used for normalization. All data represent mean±SD. *P<0.05, **P<0.01, ***P<0.001. Scr indicates scrambled‐siRNA; Vehi, vehicle.