Endothelial Depletion of Acvrl1 in Mice Leads to Arteriovenous Malformations Associated with Reduced Endoglin Expression

<div><p>Rare inherited cardiovascular diseases are frequently caused by mutations in genes that are essential for the formation and/or function of the cardiovasculature. Hereditary Haemorrhagic Telangiectasia is a familial disease of this type. The majority of patients carry mutations in either Endoglin (<i>ENG</i>) or <i>ACVRL1</i> (also known as <i>ALK1</i>) genes, and the disease is characterized by arteriovenous malformations and persistent haemorrhage. <i>ENG</i> and <i>ACVRL1</i> encode receptors for the TGFβ superfamily of ligands, that are essential for angiogenesis in early development but their roles are not fully understood. Our goal was to examine the role of Acvrl1 in vascular endothelial cells during vascular development and to determine whether loss of endothelial Acvrl1 leads to arteriovenous malformations. Acvrl1 was depleted in endothelial cells either in early postnatal life or in adult mice. Using the neonatal retinal plexus to examine angiogenesis, we observed that loss of endothelial Acvrl1 led to venous enlargement, vascular hyperbranching and arteriovenous malformations. These phenotypes were associated with loss of arterial Jag1 expression, decreased pSmad1/5/8 activity and increased endothelial cell proliferation. We found that Endoglin was markedly down-regulated in Acvrl1-depleted ECs showing endoglin expression to be downstream of Acvrl1 signalling <i>in vivo</i>. Endothelial-specific depletion of Acvrl1 in pups also led to pulmonary haemorrhage, but in adult mice resulted in caecal haemorrhage and fatal anaemia. We conclude that during development, endothelial Acvrl1 plays an essential role to regulate endothelial cell proliferation and arterial identity during angiogenesis, whilst in adult life endothelial Acvrl1 is required to maintain vascular integrity.</p></div

Diagrammatic Summary of Normal and Disrupted Signalling following Acvrl1 Depletion in Endothelial Cells.

<p>In normal endothelial cells endoglin promotes BMP9/10 signalling through the ACVRL1/BMPR2 receptor complex (as well as the ACVRL1/TGFBR2 complex, not shown). ACVRL1 phosphorylates SMAD1/5/8 which is then able to move to the nucleus (in combination with SMAD4) to regulate downstream expression of many genes. BMP9 signalling leads to increased endoglin expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098646#pone.0098646-Morikawa1" target="_blank">[22]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098646#pone.0098646-Upton1" target="_blank">[24]</a> which in turn promotes ACVRL1 signalling in a positive feedback loop. In the absence of ACVRL1, Smad1/5/8 signalling is reduced and endoglin is no longer expressed. On the other hand, when endoglin is depleted from endothelial cells, residual signalling through Acvrl1 is able to proceed, but at a lower efficiency.</p

Summary of key differences in the neonatal retinal vascular plexus when either Acvrl1 or Eng is depleted from endothelial cells.

<p>NB All relative terms (eg ‘increased’ and ‘reduced’) are used with respect to these features in normal (control) retina. *See reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098646#pone.0098646-Mahmoud1" target="_blank">[12]</a> for further details of the Eng-iKO^e phenotype.</p

Loss of endothelial Acvrl1 expression leads to abnormalities in the neonatal retinal vascular plexus.

<p>Acvrl1 expression in control retinas at P6 is seen in veins, arteries and capillaries (A,B) and is efficiently knocked down 40 hours after tamoxifen injection (C,D). Loss of Acvrl1 protein in endothelial cells in the Acvrl1-iKO^e mouse leads to AVMs (F, arrow), enlarged veins (compare veins in F and E as well as H and G) and hyperbranching (F, asterisks). Arteries are muscularised in both Acvrl1-iKO^e and control retinas, as indicated by staining for alpha smooth muscle actin (aSMA) positive smooth muscle cells (E,F). An AVM at higher magnification (H) contrasts with the normal capillary network seen in control retinas (G). Abbreviations a, artery; v, vein. Scale bar = 400 µm A–D; 500 µm E and F; 25 µm G and H.</p

Changes in transcript levels in ECs following knockdown of Acvrl1 expression.

<p>Quantitative PCR using a custom array of 85 genes involved in angiogenesis revealed changes in transcript levels of the 14 listed genes.</p

Reduced pericyte coverage of capillaries in neonatal Acvrl1-iKO^e retinas.

<p>Desmin staining revealed that pericyte coverage of capillaries in Acvrl1-iKO^e retinas (D–F) was reduced compared with controls (A–C). Quantitation of the ratio of desmin to CD31 staining confirmed that the increased endothelial cell density in Acvrl1-iKO^e retinas was not accompanied by an equivalent increase in pericyte density (G). *<i>p</i><0.05; ***<i>p</i><0.001. Scale bar = 20 µm A,B; 25 µm D-I.</p

Hypervascularity of the Acvrl1-iKO^e retinal plexus.

<p>Neonatal Acvrl1-iKO^e retinas show increased vascular branching compared with controls (A,B). Vessel density (C), vessel branch points (D), and density of filopodia (E) are all significantly increased in Acvrl1-iKO^e retinas (P6) compared with controls. *<i>p</i><0.05. Scale bar = 25 µm.</p

Enhanced Responses to Angiogenic Cues Underlie the Pathogenesis of Hereditary Hemorrhagic Telangiectasia 2

<div><p>Hereditary Hemorrhagic Telangiectasia (HHT) is a genetic vascular disease in which arteriovenous malformations (AVMs) manifest in skin and multiple visceral organs. HHT is caused by heterozygous mutations in endoglin (<i>ENG</i>), activin receptor-like kinase 1 (<i>ALK1</i>), or <i>SMAD4</i>. ALK1 regulates angiogenesis, but the precise function of ALK1 in endothelial cells (ECs) remains elusive. Since most blood vessels of HHT patients do not produce pathological vascular lesions, <i>ALK1</i> heterozygous ECs may be normal unless additional genetic or environmental stresses are imposed. To investigate the cellular and biochemical phenotypes of <i>Alk1</i>-null versus <i>Alk1</i>-heterozygous ECs, we have generated pulmonary EC lines in which a genotype switch from the <i>Alk1</i>-conditional allele (<i>Alk1</i>^2f) to the <i>Alk1</i>-null allele (<i>Alk1</i>^1f) can be induced by tamoxifen treatment. <i>Alk1</i>-null (1 f/1 f) ECs displayed increased migratory properties <i>in vitro</i> in response to bFGF compared with <i>Alk1</i>-het (2 f/1 f) ECs. The 1 f/1 f-ECs formed a denser and more persistent tubular network as compared with their parental 2 f/1 f-ECs. Interestingly, the response to BMP-9 on SMAD1/5 phosphorylation was impaired in both 2 f/1 f- and 1 f/1 f-ECs at a comparable manner, suggesting that other factors in addition to SMADs may play a crucial role for enhanced angiogenic activity in 1 f/1 f-ECs. We also demonstrated <i>in vivo</i> that <i>Alk1</i>-deficient ECs exhibited high migratory and invasive properties. Taken together, these data suggest that enhanced responses to angiogenic cues in ALK1-deficient ECs underlie the pathogenesis of HHT2.</p></div

Reduced pSmad1/5/8 activity and loss of endoglin expression in endothelial cells of neonatal Acvrl1-iKO^e retinas.

<p>Retinal sections stained for pSmad1/5/8 (green) reveal Smad1/5/8 activation in vascular cells and neural cells in control retinas (A). Confocal analysis of podocalyxin staining (red) was used to identify the apical surface of endothelial cells in retinal blood vessels. Reduced pSmad1/5/8 staining can be seen in endothelial cells in Acvrl1-iKO^e retinas (B), and was quantified using confocal software. Statistical analysis of pSmad1/5/8 staining intensity shows a significant reduction in endothelial cells of Acvrl1-iKO^e mutants compared with controls (C). Expression of pan-endothelial markers by rtPCR was used to confirm retinal endothelial cell (EC) purification by antibody conjugated magnetic beads. Pecam1 and Cdh5 were detected in the cDNA prepared from EC fractions compared to the non-EC (N-EC) fractions prepared from Acvrl1-iKOe and control retinas. Expression of β-actin was used as a positive control.</p

Endothelial cells from Acvrl1-iKO^e mice show loss of endoglin expression.

<p>Endoglin expression was reduced in Acvrl1-iKOe retinas (E) compared with controls (B) and this was particularly marked in the capillaries. Representative capillary regions indicated in B and E are shown in digital zoom in C and F, respectively. *p<0.05. Scale bar = 20 µm A–B; 100 µm D,E,F,H. Purified lung endothelial cells from control (Acvrl1^fl/fl) and Acvrl1-iKO^e neonatal mice were immunostained for the pan endothelial marker CD31 to confirm endothelial cell purity (G,J). Cells from the Acvrl1-iKO^e mice showed not only loss of Acvrl1 protein (K), but also reduced endoglin expression (L) compared with controls (I). Dapi was used to stain cell nuclei and inset in G shows no primary antibody control. Scale bar = 50 µm.</p