Essential role for TMEM100 in vascular integrity but limited contributions to the pathogenesis of hereditary haemorrhagic telangiectasia

Abstract

Aims: TMEM100 was previously identified as a downstream target of activin receptor-like kinase 1 (ALK1; ACVRL1) signalling. Mutations on ALK1 cause hereditary haemorrhagic telangiectasia (HHT), a vascular disorder characterized by mucocutaneous telangiectases and visceral arteriovenous malformations (AVMs). The aims of this study are to investigate the in vivo role of TMEM100 at various developmental and adult stages and to determine the extent to which TMEM100 contributed to the development of AVMs as a key downstream effector of ALK1.

Methods and results: Blood vasculature in Tmem100-null embryos and inducible Tmem100-null neonatal and adult mice was examined. We found that TMEM100 deficiency resulted in cardiovascular defects at embryonic stage; dilated vessels, hyperbranching, and increased number of filopodia in the retinal vasculature at neonatal stage; and various vascular abnormalities, including internal haemorrhage, arteriovenous shunts, and weakening of vasculature with abnormal elastin layers at adult stage. However, arteriovenous shunts in adult mutant mice appeared to be underdeveloped without typical tortuosity of vessels associated with AVMs. We uncovered that the expression of genes encoding cell adhesion and extracellular matrix proteins was significantly affected in lungs of adult mutant mice. Especially Mfap4, which is associated with elastin fibre formation, was mostly down-regulated.

Conclusion: These results demonstrate that TMEM100 has essential functions for the maintenance of vascular integrity as well as the formation of blood vessels. Our results also indicate that down-regulation of Tmem100 is not the central mechanism of HHT pathogenesis, but it may contribute to the development of vascular pathology of HHT by weakening vascular integrity.

Keywords: ALK1 (ACVRL1); Hereditary haemorrhagic telangiectasia; MFAP4; TMEM100; Vascular integrity.

Figure 1

Expression of Tmem100 in developing organs. (A) Whole-mount X-gal-stained hearts of Tmem100^+/SIBN mice at various embryonic, postnatal, and adult stages followed by clearing. X-gal-positive coronary arteries were indicated by arrowheads. LacZ expression was first detected at E17.5 (ii) and disappeared at weaning age (vi). Note X-gal-positive coronary artery (CA) and X-gal-negative coronary vein (CV) in the heart before clearing (iv). (B) Whole-mount X-gal-stained lungs followed by clearing (i–iv) and sections of X-gal-stained lungs counterstained with haematoxylin (v) or nuclear fast red (vi–vii). Only main pulmonary arteries were X-gal positive at E15.5 (i,v); however, LacZ expression spread as development proceeded (ii–iv and vi–vii). (C) X-gal-stained eye (i), spleen (ii), thymus (iii), and skin (iv) at P2 stage. Scale bars: A, Bi, and C, 500 μm; Bii–iv, 1 mm; Bv,vii, 50 μm; Bvi, 200 μm. More than three samples of each experiment were subjected to X-gal staining.

Figure 2

Abnormal development of retinal vasculature in Tmem100-iKO newborn mice. (A) FITC-conjugated isolectin B₄ labelling showed complete formation of superficial vascular plexus in the control mice but delayed progression of the angiogenic front in the Tmem100-iKO mutant mice. The progression of the vascular plexus towards the edge of the retina was measured by the ratio of the vascular radius (blue arrow) and the retina's radius (red arrow). (B) Dilated and hyperbranched vascular plexus were observed in the mutants. Note that these defects were severer in the angiogenic front region. Arrows indicate abnormally dilated vessels resembling AV shunts. (C) High magnified image of the angiogenic front region represented increased number of filopodial protrusions (arrows) in the tip cells. (D–G), Comparison of several angiogenic parameters between the control and mutant retinas; progression of the vascular plexus towards the edge of the retina (D), number of capillary junctions within a field of view (×200) (E), vascular density (the covered area by blood vessels in the vascular plexus region) within a field of view (×25) (F), number of sprouts (filopodial protrusions in the tip cells) within a field of view (×100) (G). *P < 0.05, ***P < 0.001. Scale bars: A, 1 mm; B, 200 μm; C, 100 μm.

Figure 3

Vascular defects of Tmem100-iKO adult mice. (A–D) Reduced haemoglobin level (Control, n = 11; Mutant, n = 11, *P < 0.05) (A) and signs of internal haemorrhages in the lungs (B: left panel, mild haemorrhage; right panel, severe haemorrhage; 8/16), liver (C; 6/6), and intestine (D; 8/16) of Tmem100-iKO mutants. Two-tailed Student's t-test was performed for statistical analysis. (E–H) Blood vessels in the small intestine (E), liver (F), back skin (G), and lung (H) visualized by latex dye perfusion. Presence of the latex dye in both arteries (A) and veins (V) would indicate AV shunts (arrows). (E) The focal AV shunts in the intestine were detected close to haemorrhagic areas indicated by an asterisk. Control, n = 7; Mutant, n = 5 (four of them showed AV shunts). (F) Note direct connection (arrowhead) between arteries (A) and veins (V) in the AV shunts of the liver. Control, n = 7; Mutant, n = 8 (four of them showed AV shunts). (G) No sign of AV shunts was observed in the wounded skin area of control (n = 4) and Tmem100-iKOs (n = 4), compared with wounded skin of tamoxifen-treated Alk1^2loxP/2loxP;ROSA26^+/CreER mice (Alk1-iKO; n = 20). *, wound; red arrowheads, arteries; blue arrowheads, veins. (H) Leakage of the latex dye was detected in the pulmonary vasculature. Scale bars: B–H, 1 mm; enlarged images, 0.25 mm.

Figure 4

Gene Set Enrichment Analysis (GSEA) of biological processes and molecular functions for differentially expressed genes in the lungs between control and Tmem100-iKO mice. Significantly enriched biological processes (A) and molecular functions (B) are represented with their Fisher P-value (<0.01). Expression of genes encoding cell adhesion proteins (biological process) and ECM proteins (molecular function) is most significantly affected in the lungs of tamoxifen-treated Tmem100-iKO mutant mice.

Figure 5

Mfap4 is down-regulated in the lungs of Tmem100-iKO mice. (A) Immunohistochemical staining of control (n = 4) and Tmem100-iKO mutant (n = 8) lungs with anti-MFAP4 antibody. The MFAP4-positive staining was predominantly detected in the pulmonary vessels with various sizes and alveolar walls in the control mice. However, the MFAP4 expression was diminished in small vessels and alveolar walls of Tmem100-iKO mice. Arrows indicate MFAP4-positive vessels. (B) Quantitative real-time PCR showing 5.5-fold down-regulation of Mfap4 in the lungs of Tmem100-iKO mice. **P < 0.01. Control, n = 3; Mutant, n = 3. (C) Number of vessels positive for MFAP4. Pulmonary vessels were divided into three groups according to their diameters (Group I: <50 µm diameter; Group II: 50–100 µm diameter; Group III: >100 µm diameter). Note that the numbers of MFAP4-positive vessels with diameter <100 µm were markedly reduced in the Tmem100-iKO lungs. However, the numbers of MFAP4-positive vessels with diameter larger than 100 µm were not affected. Two-tailed Student's t-test was performed for statistical analysis. ***P < 0.001. Control, n = 4; Mutant, n = 8. Three microscopic fields of each lung section were analysed. (D) Hart's stain showing reduced number of small vessels possessing elastin layers in the Tmem100-iKO lung. Control, n = 4; Mutant, n = 8. (E, F) Reduced number of organized blood vessels in the lung of Tmem100-iKO mice. Smooth muscle cells (SMCs) and vascular SMC-associated endothelial cells were immunostained by anti-α-smooth muscle actin (SMA) and anti-von Willebrand factor (vWF) antibodies, respectively. Note disarrayed vascular structure in the affected region and a ruptured blood vessel (indicated by an asterisk) of the Tmem100-iKO lung. Control, n = 4; Mutant, n = 8. Scale bars: A and D, 100 μm; E and F, 200 μm; enlarged images, 50 μm.