Figure 1.
Loss of endothelial Acvrl1 expression leads to abnormalities in the neonatal retinal vascular plexus.
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-iKOe 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-iKOe 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.
Figure 2.
Hypervascularity of the Acvrl1-iKOe retinal plexus.
Neonatal Acvrl1-iKOe 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-iKOe retinas (P6) compared with controls. *p<0.05. Scale bar = 25 µm.
Figure 3.
Reduced pericyte coverage of capillaries in neonatal Acvrl1-iKOe retinas.
Desmin staining revealed that pericyte coverage of capillaries in Acvrl1-iKOe 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-iKOe retinas was not accompanied by an equivalent increase in pericyte density (G). *p<0.05; ***p<0.001. Scale bar = 20 µm A,B; 25 µm D-I.
Figure 4.
Loss of arterial identity and increased EC proliferation in neonatal Acvrl1-iKOe retinas.
Retinal veins show retention of venous marker EphB4 similar to control (A,B) and AVMs are also EphB4 positive (arrow, B). Jag1 expression in arteries of Acvrl1-iKOe retinas is reduced compared with controls (C,D). EdU labelling (E,F) reveals a significant increase in EC proliferation in mid plexus capillaries (G) and veins (H) of Acvrl1-iKOe retinas compared with controls. Increased EC proliferation is also associated with AVMs in Acvrl1-iKOe mutants (arrow, F). *p<0.05; **p<0.01. Scale bar = 50 µm A and B; 200 µm C and D; 100 µm E and F.
Figure 5.
Reduced pSmad1/5/8 activity and loss of endoglin expression in endothelial cells of neonatal Acvrl1-iKOe retinas.
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-iKOe 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-iKOe 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.
Figure 6.
Endothelial cells from Acvrl1-iKOe mice show loss of endoglin expression.
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 (Acvrl1fl/fl) and Acvrl1-iKOe neonatal mice were immunostained for the pan endothelial marker CD31 to confirm endothelial cell purity (G,J). Cells from the Acvrl1-iKOe 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.
Table 1.
Changes in transcript levels in ECs following knockdown of Acvrl1 expression.
Figure 7.
Diagrammatic Summary of Normal and Disrupted Signalling following Acvrl1 Depletion in Endothelial Cells.
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 [22]–[24] 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.
Table 2.
Summary of key differences in the neonatal retinal vascular plexus when either Acvrl1 or Eng is depleted from endothelial cells.