6 General discussion
6.2 Angiogenesis in von Willebrand disease in the porcine female
In the first part of our study, the porcine model of VWD type 1 and 3 was characterized particularly regarding the influence of VWD on the non-pregnant
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female reproductive tract. For this purpose, uteri, oviducts, and ovaries of six mature sows of the three different genotypes were analyzed for histologic morphology and gene and protein expression of several angiogenic factors probably connected to VWF.
While storage of VWF in WPB was shown for the wildtype and VWD type 1 animals, this was not seen in VWD type 3 animals, concordant with in vitro results on BOEC (Selvam et al. 2017). The general impact of this loss of WPB on expression and release of Ang-2 -as one of the many proteins stored as cargo of WPB- seems unclear so far. In our study, a trend for changed genetic expression of ANG2 was observed only in the VWD type 3 ovaries, as it was decreased compared to wildtype and VWD type 1 animals. Regarding Ang-2 protein, varying distribution among the animals and genotypes was observed. These findings confirm the observed heterogeneity of Ang-2 expression and distribution in different in vitro models, in BOEC of patients with different disease-causing mutations and in vivo. Heart tissue of VWF-knockout mice showed increased gene expression accompanied by vascular defects, while liver and kidney tissue did not display expression changes (Yuan et al. 2016). Increased gene expression was also seen in HUVEC and some BOEC, which also displayed increased protein release. BOEC of other patients did not show expression changes and decreased protein release. Furthermore, Ang-2 protein levels were decreased in VWD patients’ plasma (Starke et al. 2011;
Selvam et al. 2017; Groeneveld et al. 2018). Thus, loss of VWF and consequential alterations or absence of WPB does not always seem to result in increased Ang-2 expression but can affect Ang-2 levels rather in both ways. The subsequent influence on angiogenesis is mediated via Ang/Tie signaling and hence needs further observation of the ratios of the respective components. Regarding ANG2/ANG1 ratio, the ovary of one VWD type 3 animal displayed pronounced overexpression of ANG2 while the ovary of the other VWD type 3 animal showed overexpression of ANG1. As Ang-2 acts as an antagonist on Ang-1/Tie-2 signaling and thus, has destabilizing and angiogenic effects on blood vessels, a high ratio would be expected in pro-angiogenic tissue. Apart from that, ANG2 overexpression is found in angiodysplastic lesions of the small bowel of humans (Holleran et al. 2015). The observed relatively high expression of ANG2 might therefore lead to vessel alterations, which could influence the process of ovulation probably by pronounced bleeding. This may subsequently lead to hemorrhages seen by ultrasound probably at the time of
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ovulation resulting in a fibrotic cap surrounding the ovary and rendering the VWD type 3 sows sterile (personal communication Claire Bal dit Sollier, Hôpital Lariboisière, Paris, France).
When investigating expression of the Tie receptors, increased TIE2 expression was found in the ovaries of VWD type 3 animals and the oviducts of VWD type 1 animals, which was also described in venous malformations (Vikkula et al. 1996). Additionally, relatively higher ANG1 expression over TIE2 expression was seen in ovaries of VWD type 3 animals only. In contrast to balanced Ang-1/Tie-2 signaling, ANG1 overexpression was described to result in vascular remodeling with widening of the vessel diameter (Redondo et al. 2007). This indicates that the ANG/TIE system of these animals might be influenced by high TIE2 expression levels as well as relatively high ANG1 expression levels, which can both result in impaired vascular development. Additionally, imbalanced Ang-1/Ang-2 ratios in connection with VWD were proposed to alter vascularization previously (Randi et al. 2018).
Internalization of integrin αVβ3, which was shown in HUVEC, was observed in uterine epithelial cells of all VWD animals. In HUVEC, this was accompanied by decreased ITGB3 expression (Starke et al. 2011), which was also shown for all but one tissue sample of the VWD type 3 animals. The impact of these findings on angiogenesis might be determined by the respective microenvironment, as integrin αVβ3 can have both pro- and anti-angiogenic effects. Considering the increased angiogenic properties of the VWF-knockdown HUVEC and the angiodysplastic blood vessels in the VWD type 3 uteri, the internalization and reduced expression might mimic the situation of an integrin β3-knockout, which would subsequently lead to increased VEGFR-2 recycling and VEGF sensitivity (Reynolds et al. 2009). This is also supported by the increased VEGF expression in the uteri of VWD type 3 animals. In the HUVEC model, connection of pro-angiogenic properties, decreased integrin αVβ3
and VEGF/VEGFR-2 signaling was confirmed. However, angiogenic properties were not increased in all BOEC and in this study, angiodysplasia was observed in the uteri only. This seems to be in accordance with the described bimodal effect of integrin αVβ3 on angiogenesis and the fact that it is expressed in angiogenic or pathological tissues only (Brooks et al. 1995).
Angiogenesis in the non-pregnant human endometrium occurs in correlation to the menstrual cycle during menstruation, the proliferative phase and during secretory phase (Gargett and Rogers 2001). Hence, the environment is often in a
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angiogenic state. In the ovary, angiogenesis mainly occurs during late maturation of the follicles and during establishment of the corpus luteum directly at the side of the ovulated follicle (Plendl 2000). In contrast, vascular supply of the porcine non-pregnant oviduct seems to be stably established before puberty (Gutierrez et al.
2015) and the human oviduct even seems to be in an anti-angiogenic state during passage of the oocyte (Hess et al. 2013). Therefore, the most pronounced influence of integrin αVβ3 on angiogenesis would be expected in the uterus and a less pronounced influence in the oviduct, which might explain the fact that angiodysplasia was seen in both VWD type 3 uteri only. Furthermore, VWF expression was shown to vary between tissues and even between single EC of the same tissue. According to that, control of VWF over Ang-2 was already proposed to be tissue specific (Randi et al. 2018), which might be true for other probably connected angiogenic mediators as well. Combining these findings with results of our study, the idea of tissue specific influence of VWF on angiogenesis is supported and this influence might be pronounced particularly in the uterus.
Overall, this part of the study confirms influence of VWD on the VEGF/VEGFR-2 system, integrin αVβ3 and the ANG/TIE system in the porcine non-pregnant female reproductive tract in vivo, which may result in enhanced or impaired angiogenesis.
This influence seems to be heterogenic depending on the disease-causing mutation and on the tissue. Angiodysplasia in the non-pregnant VWD type 3 uteri was demonstrated, which is the first description of angiodysplasia in a VWD animal model.