Altered Angiogenic Gene Expression Profiles in Porcine Von Willebrand Disease

Objectives: Von Willebrand Factor (VWF) is a main factor of coagulation, but also known for its influence on angiogenesis by modifying the expression of several genes. The mechanisms were assumed to base on shared genetic pathways and also on an altered storage capability of certain proteins within the VWF-dependent Weibel-Palade Bodies (WPBs). As women affected by Von Willebrand Disease (VWD), a disease caused by a deficiency of VWF, may develop miscarriage and other complications during pregnancy which highly relies on proper angiogenesis, we analyzed the effect of VWD on the expression of genes potentially influenced by a deficiency of VWF within uterus tissue.

Methods: Uterus tissue samples were taken from each two non-pregnant VWD pigs suffering from VWD type 1 (T1, heterozygous) and type 3 (T3, homozygous), resp., during late estrus. Two healthy pigs served as controls (WT, wild type). The genes F8 (coagulation factor VIII), FGA (fibrinogen alpha chain), FGB (fibrinogen beta chain), FGG (fibrinogen gamma chain), PLG (plasminogen), CXCL8 (C-X-C motif chemokine ligand 8), CALR (calreticulin), CTGF (connective tissue growth factor), and IGFBP7 (insulin like growth factor binding protein 7) were chosen for analysis and specific primers were designed. The expression of these genes was analyzed by qRT-PCR and relatively quantified against the endothelial specific housekeeping genes PROCR and CD31 using the ΔΔC_T method and the Wilcoxon-Mann-Whitney statistic test. The differences in gene expression were compared among the genotypes.

Results:F8, FGA, FGG, PLG, CXCL8, CTGF, and IGFBP7 showed no significantly altered expression profiles. However, genes showing differential expression with significant (p ≤ 0.05) or suggestive (p ≤ 0.1) p-values among the three genotypes in uterus tissue were CALR and FGB. CALR expression was reduced to 36.7% and 33.3% within uterus tissue of T1 and T3 pigs, respectively, compared to WT (p ≤ 0.1). When combining the VWD pigs of both types and comparing them to WT, their expression was reduced to 35.0% reaching significance (p ≤ 0.05). The expression of FGB was increased to about five-fold in T1 pigs compared to the WT (p ≤ 0.1), while it was not significantly changed in T3 individuals.

Discussion and Conclusions: CALR protein is stored in the VWF-dependent WPBs and shows physical interactions with VWF. As a reduction of VWF goes along with a reduction of WPBs, adequate CALR release might be diminished. Additionally, CALR is known to inhibit endothelial cell proliferation and to strongly affect angiogenesis. FGB shows strong physical interactions with CALR. It is part of the coagulation system and a homozygous dysfunction of this gene causes afibrinogenemia and consequently bleeding disorders. The observed higher expression of FGB in T1 uterus tissue compared to the WT might therefore reflect a higher production of fibrinogen. However, this needs to be confirmed in further studies. The observed reduced expression of CALR in uterus tissue of VWD-affected pigs might therefore contribute to the antiangiogenic alterations observed in VWD patients and contribute to the pregnancy related complications in VWD.