Immunohistochemical Analyses and Quantitative RT-PCR of Angiogenic Factors in a Porcine Von Willebrand Disease Model

Objectives: Von Willebrand Factor (VWF) is a main factor in coagulation. In addition, it was shown to affect angiogenesis and the distribution of angiogenic factors. A severe deficit of this factor, as seen in patients of Von Willebrand Disease (VWD) type 3, can be expected to have impact on all these issues. As women with VWD may develop miscarriage during pregnancy, we analysed VWF as well as the angiogenic factors Angiopoietin-2 (ANG2) and Integrin αVβ3 (ITG) using immunohistochemistry (IHC) and quantitative PCR of uterus and caecum tissues in a female pig model comprising all VWD type 3 genotypes.

Methods: Tissue samples of uterus and caecum were harvested from a total of 5 sexually mature female pigs. One of these was affected by VWD type 3, two were heterozygous carriers, and two were wildtype individuals, which served as controls. The expression of VWF, ITG, and ANG2 proteins were compared semiquantitatively by IHC in a double-blind manner. In addition, qRT-PCR was performed for the genes VWF, ANGPT2 (coding for ANG2 protein), and ITGAV (coding for ITG protein subunit)and analysed by relative quantification against the endothel-specific gene PROCR using the ΔΔCT method.

Results: The expression of the angiogenic factors analysed showed remarkable differences amongst the genotypes. Using IHC, the pig affected by VWD showed almost no VWF expression within the examined tissues, while in wildtype and heterozygous pigs, expression was obvious in the endothelium. ITG showed strong granular intracellular staining in the uterine epithelium and glands of the VWD pig, but the staining was diffuse and moderate in heterozygous individuals. In wildtype pigs, the strongest staining of the epithelium was detected at the apical cell membrane. On the contrary, the apical cell membrane of the uterine epithelium in the VWD pig showed distinct ANG2 staining. qRT-PCR showed a higher expression of ITGAV in uterus and caecum (p-values: 0.1 and 0.05, respectively) and a significantly higher expression of ANG2 in caecum tissue (p-value: 0.03).

Discussion and Conclusion: We were able to show that angiogenic factors in different tissues show alternative expression in pigs affected by VWD compared with heterozygous and wildtype pigs. In pigs affected by VWD, altered expression of ITG as well as ANG2 was seen, next to an extremely reduced expression of VWF. VWF seems to possess a stabilizing effect on ITG on apical membranes. Therefore, ITG in VWD affected pigs was detected to a higher degree in the cytoplasm, probably due to increased internalization. The generally increased expression of ITGAV in VWD affected pigs might be caused by negative feedback due to the reduced level of this protein at its physiological location. The increased expression and the staining pattern of ANG2 in pigs affected by VWD might be caused by the missing of Weibel-Palade-Bodies. The production of these storage organelles depends on VWF. Therefore, no storage of ANG2 can occur in VWD affected pigs and ANG2 may be released steadily.