Fig 1.
CD31, VECADHERIN, and VEGFR2 expression was altered in the VM endothelium.
(A) Representative images of VM and control neonatal dermis co-stained for CD31 and VECADHERIN. White arrowheads mark discontinuous CD31 and VECADHERIN expression observed in VM vessels. Yellow arrowheads mark CD31-/VECADHERIN+ ECs. (B) Representative images of VMs and control neonatal dermis co-stained for CD31 and VEGFR2. White arrowheads mark CD31+/VEGFR2+ ECs. (A, B) Boxed areas are enlarged to the right. Scale bars—50μm. A-artery, V-vein, VC-VM channel. (C) Quantification of percent VECADHERIN+ ECs in VMs (n = 16) and controls (n = 5). Bar represents median value, *p<0.005. D) Quantification of percent CD31+ ECs in VMs (n = 16) and controls (n = 5). Bar represents median value, *p<0.001. (E) Mean VEGFR2 expression determined as signal intensity normalized by vessel length in VMs (n = 6) and controls (n = 3) tissues. Bar represents median value, *p<0.05.
Fig 2.
Venous endothelial proteins were altered in VM endothelium.
(A) Representative images of VMs and control neonatal dermis co-stained for VECADHERIN (VECAD) and COUP-TFII. White arrowheads mark VECADHERIN+/COUP-TFII+ ECs. (B) Representative sections of VMs and control neonatal dermis co-stained for VECADHERIN and EPHB4. White arrowheads mark CD31+/EPHB4+ cells. (A, B) Boxed areas are enlarged to the right. V-vein, VC-VM channel. Scale bars—50μm. V-vein, VC-VM channel. (C) Percent COUP-TFII+ ECs in VMs (n = 10) and controls (n = 3). (D) Mean EPHB4 expression normalized by vessel length in VMs (n = 10) and controls (n = 3). (C, D) Bars represent median values, *p<0.05.
Fig 3.
VM endothelium misexpressed arterial EC proteins.
(A) Representative images of VM and control neonatal dermis co-stained for CD31 and DLL4. White arrowheads mark CD31+/DLL4+ ECs. Yellow arrowheads mark CD31+/DLL4- ECs. (B) Representative images of VM and control neonatal dermis co-stained for CD31 and EPHRINB2. White arrowheads mark CD31+/EPHRINB2+ cells. (A, B) Boxed areas are enlarged to the right. A-artery, V-vein, VC-VM channel. Scale bars– 50μm. (C). Mean DLL4 expression normalized by vessel length in VMs (n = 5) and controls (n = 3). Bar represents median value; ANOVA, p<0.0005; T-Test *p< 0.01, **p<0.0001, ns, non-significant. (D) Mean EPHRINB2 expression normalized by vessel length in VMs (n = 5) and controls (n = 3). Bar represents median value. V-vein, Art-artery, VM-venous malformation.
Fig 4.
VMs had increased and disorganized αSMA expressing mural cells.
(A) Representative images of VM and control neonatal dermis co-stained for CD31 and αSMA. White arrowheads mark CD31-/αSMA+ mural cells. (B) Representative sections of VMs and control neonatal dermis co-stained for VECADHERIN and NOTCH3. White arrowheads mark VECADHERIN-/NOTCH3+ mural cells. (A, B) Boxed areas are enlarged to the right. A-artery, V-vein, VC-VM channel. Scale bars– 50μm. (C) Percentage of mural cell phenotype (arterial-like, continuous multi layer; vein-like, single layer/no layer, and discontinuous layers) in arteries and veins of controls (n = 3), and VMs (n = 11).
Fig 5.
NOTCH3 and PDGFRβ were expressed in the endothelium of VMs.
(A) Representative images of VMs and control neonatal dermis co-stained for VECADHERIN and NOTCH3. Yellow arrowheads mark VECADHERIN-/NOTCH3+ mural cells, and white arrowheads mark VECADHERIN+/NOTCH3+ ECs. Boxed areas are enlarged to the right. A-artery, V-vein, VC-VM channel. Scale bars—50μm. (B) Quantification of percent NOTCH3+ ECs in VMs (n = 11) and controls (n = 5). Bar represents median value, *p<0.0001. (C) Quantification of percent PDGFRβ+ ECs in VMs (n = 7) and controls (n = 3). Bar represents median value, *p<0.005.
Fig 6.
VM endothelium expressed progenitor markers.
(A) Representative images of VM and control neonatal dermis co-stained for VECADHERIN and CD133. White arrowheads mark VECADHERIN+/CD133+ ECs. (B). Representative images of VM and control neonatal dermis co-stained for VECADHERIN and CKIT. White arrowheads mark VECADHERIN+/CKIT+ ECs. (A, B) Boxed areas are enlarged to the right. A-artery, V-vein, VC-VM channel. Scale bars—50μm. (C) Quantification of percent CD133+ ECs in VMs (n = 10) and controls (n = 3). Bar represents median value, *p<0.02. (D) Quantification of percent CKIT+ ECs in VMs (n = 8) and controls (n = 3). Bar represents median value, *p = 0.05. (E) Mean CD146 expression normalized by vessel length in VMs (n = 5) and controls (n = 2). Bar represents median value.
Fig 7.
Increased in proliferative ECs in VM vessels.
(A) Representative images of VM and control neonatal dermis co-stained for VECADHERIN and KI67. White arrowheads mark VECADHERIN+/KI67+ ECs. Yellow arrowhead marks a KI67+ non-EC in the control. Scale bars—50μm. (B) Quantification of percent KI67+ ECs in VMs (n = 10) and controls (n = 5). Bar represents median value, *p<0.0002. (C) Quantification of EC length in VMs (n = 10) and controls (n = 5). Bar represents median value.