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Venous Malformations Have Collagen IV Deposition Defects Associated With Increased Endothelial Cell Death
Andrew I. Zeiler, High School Student, Michael J. Schonning, MS, Carrie J. Shawber, PhD, June K. Wu, MD
Columbia University Medical Center, New York, NY, USA.

Introduction:
Collagen IV (COLIV) is an essential component of the blood vessel basement membrane (BM) that functions as a scaffold to stabilize vessels and can be proteolytically cleaved to release angiogenic peptides that regulate endothelial cell fate. In murine endothelial cells (ECs), constitutive RAS/ERK signaling leads to defects in COLIV BM deposition, which in turn promotes EC death. We have previously demonstrated ERK hyperactivation in the endothelium of venous malformations (VMs). Thus, we hypothesized that COLIV deposition is altered in VMs leading to EC death.
Methods:
Clinically confirmed VM lesions (n=11) were resected and the specimens divided and either paraffin or fixed-frozen embedded. Neonatal dermis (n=5) served as control. VM and control sections were stained for the EC marker, VECADHERIN, and either phospho-ERK or COLIV. Endothelial cell apoptosis was determined by TUNEL assay and co-staining for VECADHERIN. COLIV localization was assessed and scored as normal continuous BM expression, abnormal nuclear localization, or discontinuous BM deposition. Mean endothelial phospho-ERK expression and percent TUNEL+ ECs was quantified using ImageJ. Studentís T-Test was used for statistical analysis by Graphpad, and a p value <0.05 was considered statistically significant.
Results:
Activated phospho-ERK (pERK) expression was significantly increased in VM ECs (n=8), compared to control vessel ECs (p=0.0189; Figure 1A). COLIV defects were noted in a majority of VM tissues (10/11, 82%) which was not observed in control vessels (Figure 1B). COLIV nuclear localization (arrowheads) was observed in 27% of VM ECs, discontinuous BM deposition in 9% of VM vessels, and both abnormal COLIV deposition phenotypes were observed in 55% of VMs. VMs (n=9) had a significant increase in TUNEL+ ECs (arrowheads), when compared to control neonatal skin (Figure 2). Control vessels had 0.12% TUNEL+ ECs (0-0.6%), while VMs had an average of 31.17% (11.77%-66.43%) TUNEL+ ECs (p=0.002).
Conclusions:
We observed that VM vessels had increased ERK activation associated with COLIV mislocalization and BM deposition defects. Consistent with murine studies, the loss of COLIV in the BM correlated with a significant increase in EC apoptosis in VM tissues. These results suggest that pathological ERK hyperactivation, leads to a loss of COLIV deposition in the BM leading to endothelial cell death in VMs.


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