scholarly journals Characterization of a Porcine Model for Von Willebrand Disease Type 1 and 3 Regarding Expression of Angiogenic Mediators in the Nonpregnant Female Reproductive Tract

2019 ◽  
Vol 69 (5) ◽  
pp. 401-412 ◽  
Author(s):  
Hanna Allerkamp ◽  
Stefanie Lehner ◽  
Mahnaz Ekhlasi-Hundrieser ◽  
Carsten Detering ◽  
Christiane Pfarrer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Animal Model ◽  
Growth Factor ◽  
Reproductive Tract ◽  
Porcine Model ◽  
Angiogenic Factors ◽  
Von Willebrand Disease ◽  
Female Reproductive Tract ◽  
Type 3 ◽  
Von Willebrand

Von Willebrand disease (VWD), a blood coagulation disorder, is also known to cause angiodysplasia. Hitherto, no animal model has been found with angiodysplasia that can be studied in vivo. In addition, VWD patients tend to have a higher incidence of miscarriages for reasons unknown. Thus, we aimed to examine the influence of von Willebrand factor (VWF) on the female reproductive tract histology and the expression and distribution of angiogenic factors in a porcine model for VWD types 1 and 3. The disease-causing tandem duplication within the VWF gene occurred naturally in these pigs, making them a rare and valuable model. Reproductive organs of 6 animals (2 of each mutant genotype and 2 wildtype (WT) animals) were harvested. Genotype plus phenotype were confirmed. Several angiogenic factors were chosen for possible connections to VWF and analyzed alongside VWF by immunohistochemistry and quantitative gene expression studies. VWD type 3 animals showed angiodysplasia in the uterus and shifting of integrin αVβ3 from the apical membrane of uterine epithelium to the cytoplasm accompanied by increased vascular endothelial growth factor (VEGF) expression. Varying staining patterns for angiopoietin (Ang)-2 were observed among the genotypes. As compared with WT, the ovaries of the VWD type 3 animals showed decreased gene expression of ANG2 and increased gene expression of TIE (tyrosine kinase with immunoglobulin and epidermal growth factor homology domains) 2, with some differences in the ANG/TIE-system among the mutant genotypes. In conclusion, severely reduced VWF seems to evoke angiodysplasia in the porcine uterus. Varying distribution and expression of angiogenic factors suggest that this large animal model is promising for investigation of influence of VWF on angiogenesis in larger groups.

scholarly journals Expression Profile of VEGF and VEGFR2 in the Female Reproductive Tract at the Time of Placentation in a Porcine Model of Von Willebrand Disease Type 1

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3776-3776
Author(s):  
Mario von Depka ◽  
Mahnaz Ekhlasi-Hundrieser ◽  
Carsten Detering ◽  
Stefanie Lehner ◽  
Christiane Pfarrer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Early Pregnancy ◽  
Reproductive Tract ◽  
Porcine Model ◽  
Conflicts Of Interest ◽  
Von Willebrand Disease ◽  
Female Reproductive Tract ◽  
Von Willebrand

Abstract Objectives: Clinical studies showed that women affected by von Willebrand disease (VWD) trend towards higher rates of miscarriages, but the underlying pathomechanisms remain unclear. Several in vitro studies demonstrated an influence of von Willebrand factor (VWF) on angiogenesis, which seems to be partly mediated via VEGF. Since angiogenesis in the reproductive organs is essential to establish and maintain pregnancy, we aimed to investigate gene expression of VWF, VEGF, and its receptor VEGFR2 in non-pregnant and pregnant individuals, using a porcine model of VWD type 1. Methods: Tissue samples of uterus, oviduct and ovary were harvested from eight female pigs of which four were pregnant on day 30 (time of placentation) and four were non-pregnant and in estrus. Of each group, two were affected by VWD type 1 and two were wildtype (WT) individuals. The gene expression of VWF, VEGF, and VEGFR2 was measured by qRT-PCR and relatively quantified against the endothelial specific housekeeping genes PROCR and CD31 using the ΔΔCT method and calculating the respective x-fold changes. The gene expression differences were compared between both genotypes as well as between both reproductive states Mean differences were taken into account, if the divergences were consistent in both individuals of each group and not within the range of the group they were compared to. Results: Regarding the non-pregnant sows, VWF expression was lower in uterus and ovary of the VWD type 1 animals. This difference was not seen comparing the pregnant animals of each genotype, but the VWD type 1 animals showed higher VEGF expression in oviduct and higher VEGF and VEGFR2 expression in the ovary. The expression of VEGFR2 was reduced in the uterus. Comparing the non-pregnant with the pregnant animals within each genotype, the following results were found: the pregnant WT pigs showed increased expression of VEGFR2 in uterus and ovary, but decreased expression of VEGF in the uterus. For the pregnant VWD type 1 animals increased expression was found for VWF and VEGFR2 in the ovary, and decreased expression for VEGF in uterus and oviduct and VEGFR2 in the uterus. Discussion and Conclusion: Comparison of the different groups revealed differences of gene expression in the female reproductive tract during early pregnancy. The expectedly lower expression of VWF in the VWD type 1 animals was not found for the pregnant animals. Apparently, there is an increase of VWF levels during pregnancy as seen in women. While VEGFR2 expression in the uterus increases during placentation in the WT animals, it decreases in the VWD type 1 animals. This suggests altered regulation of early angiogenesis, which is essential during placentation. Expression of VEGF and VEGFR2 was increased in the ovaries of the VWD type 1 animals. This points to an enhanced involvement of this pathway during conversion of the ruptured follicles to sufficient corpora lutea graviditates, which may affect this process. Enhanced angiogenesis via the VEGF/VEGFR2-pathway due to lack of VWF was already shown in vitro. Our study shows that the expression of VEGF and VEGFR2 differs during early pregnancy in VWD type 1 compared to wildtype sows. Therefore, this pathway may influence angiogenesis in the reproductive tract. Disclosures No relevant conflicts of interest to declare.

Expression of angiogenic factors in the uteroplacental unit is altered at time of placentation in a porcine model of von Willebrand disease type 1

2019 ◽  
Vol 19 (4) ◽  
pp. 412-420
Author(s):  
Hanna Allerkamp ◽  
Stefanie Lehner ◽  
Mahnaz Ekhlasi-Hundrieser ◽  
Carsten Detering ◽  
Mario von Depka Prondzinski ◽  
...  
Keyword(s):  
Porcine Model ◽  
Disease Type ◽  
Angiogenic Factors ◽  
Von Willebrand Disease ◽  
Von Willebrand

Von Willebrand Disease influences the expression of angiogenic factors and the blood vessel conformation in the uterus in a porcine model

Placenta ◽  
2017 ◽  
Vol 57 ◽  
pp. 271-272
Author(s):  
Hanna Allerkamp ◽  
Stefanie Lehner ◽  
Mahnaz Ekhlasi-Hundrieser ◽  
Carsten Detering ◽  
Mario von Depka Prondzinski ◽  
...  
Keyword(s):  
Blood Vessel ◽  
Porcine Model ◽  
Angiogenic Factors ◽  
Von Willebrand Disease ◽  
Von Willebrand

scholarly journals Immunohistochemical Analyses on Angiogenesis in a Porcine Von Willebrand Disease Model

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2564-2564
Author(s):  
Mario Von Depka ◽  
Hanna Allerkamp ◽  
Mahnaz Ekhlasi-Hundrieser ◽  
Christiane Pfarrer ◽  
Carsten Detering ◽  
...  
Keyword(s):  
Blood Vessels ◽  
Lamina Propria ◽  
Angiogenic Factors ◽  
Von Willebrand Disease ◽  
Heterozygous Carriers ◽  
Type 3 ◽  
Von Willebrand ◽  
Arteries And Veins ◽  
Hematoxylin Eosin ◽  
Immunohistochemical Analyses

Abstract Objectives: Women with von Willebrand Disease (VWD) type 3 may develop miscarriage during pregnancy. There is evidence that besides its role in coagulation von Willebrand Factor (VWF) is also involved in angiogenesis and defects of this pathway could play a role in miscarriage. For this reason, VWF as well as proteins with known functions in angiogenesis were analysed by immunohistochemistry of several tissues in a pig model comprising all VWD type 3 genotypes (VWD, heterozygous carriers, wildtype). Methods: Uterus tissue samples including the endometrium were collected from a total of 6 pigs. Of these, two were affected by VWD type 3, two were heterozygous carriers, and the remaining two were wildtype individuals, which served as controls. Hematoxylin-eosin-staining was implemented for morphological evaluation of the tissues, especially with regard to possible differences in structure and pattern of blood vessels. In a second step immunohistochemical analyses comparing the expression of angiogenic factors including VWF were performed in VWD-animals, genetic carriers and wildtype pigs. Results: Hematoxylin-eosin-staining of histological sections of the porcine uteri comprising the different genotypes revealed differences in the vascular architecture within the lamina propria. Compared to non pregnant wild type pigs where blood vessels in the lamina propria of the endometrium usually are of smaller caliber (mostly categorized as arterioles, venules and capillaries), the lamina propria of VWD type 3 animals contained multiple large arteries and veins (of the type usually found in the uterine wall). The immunohistochemical analysis for VWF showed almost no VWF expression in the pigs affected by VWD. In wildtype and heterozygous pigs, expression was obvious in the endothelium. A narrow band of VWF expression apically on the epithel cells as well as partial expression in glands was seen only in wildtype pigs, but not in heterozygous pigs. For some, but not all further angiogenic factors analysed, expression differences were present among the porcine VWD genotypes. Conclusion: The results of our study confirm effects of VWF on the uterine vascular architecture and on the expression of specific angiogenic factors. Pigs affected by VWD showed almost no expression of VWF and the blood vessels within the uterine lamina propria had a surprisingly high percentage of large arteries and veins indicating a crucial role of vWF for uterine angiogenesis. Expression differences among the genotypes were obvious for specific angiogenic factors. Disclosures Von Depka: Octapharma: Other: Study Investigator, Speakers Bureau.

scholarly journals Altered Angiogenic Gene Expression Profiles in Porcine Von Willebrand Disease

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4917-4917
Author(s):  
Mario von Depka ◽  
Katharina Kaiser ◽  
Carsten Detering ◽  
Mahnaz Ekhlasi-Hundrieser ◽  
Christiane Pfarrer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Tissue Growth ◽  
Von Willebrand Disease ◽  
Endothelial Cell Proliferation ◽  
Altered Expression ◽  
Physical Interactions ◽  
Von Willebrand

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 ΔΔCT 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. Disclosures No relevant conflicts of interest to declare.

A Novel Mutation Glyl672→Arg in Type 2A and a Homozygous Mutation in Type 2B von Willebrand Disease

1996 ◽  
Vol 76 (02) ◽  
pp. 253-257 ◽  
Author(s):  
Takeshi Hagiwara ◽  
Hiroshi Inaba ◽  
Shinichi Yoshida ◽  
Keiko Nagaizumi ◽  
Morio Arai ◽  
...  
Keyword(s):  
Missense Mutation ◽  
Novel Mutation ◽  
Point Mutations ◽  
Japanese Patients ◽  
Von Willebrand Disease ◽  
Homozygous Mutation ◽  
Missense Mutations ◽  
Reaction Products ◽  
Type 3 ◽  
Von Willebrand

SummaryGenetic materials from 16 unrelated Japanese patients with von Willebrand disease (vWD) were analyzed for mutations. Exon 28 of the von Willebrand factor (vWF) gene, where point mutations have been found most frequent, was screened by various restriction-enzyme analyses. Six patients were observed to have abnormal restriction patterns. By sequence analyses of the polymerase chain-reaction products, we identified a homozygous R1308C missense mutation in a patient with type 2B vWD; R1597W, R1597Q, G1609R and G1672R missense mutations in five patients with type 2A; and a G1659ter nonsense mutation in a patient with type 3 vWD. The G1672R was a novel missense mutation of the carboxyl-terminal end of the A2 domain. In addition, we detected an A/C polymorphism at nucleotide 4915 with HaeIII. There was no particular linkage disequilibrium of the A/C polymorphism, either with the G/A polymorphism at nucleotide 4391 detected with Hphl or with the C/T at 4891 detected with BstEll.

Evaluation of the Abnormal Platelet Function in von Willebrand Disease by the Blood Filtration Test

1996 ◽  
Vol 76 (03) ◽  
pp. 460-468 ◽  
Author(s):  
Francesco I Pareti ◽  
Marco Cattaneo ◽  
Luca Carpinelli ◽  
Maddalena L Zighetti ◽  
Caterina Bressi ◽  
...  
Keyword(s):  
Platelet Function ◽  
Relevant Information ◽  
Von Willebrand Disease ◽  
Type I ◽  
Cumulative Number ◽  
Primary Hemostasis ◽  
Normal Platelet ◽  
Filter Test ◽  
Type 3 ◽  
Von Willebrand

SummaryWe have evaluated platelet function in different subtypes of von Willebrand disease (vWD) by pushing blood through the capillarysized channels of a glass filter. Patients, including those with type IIB vWD, showed lower than normal platelet retention and increased cumulative number of blood drops passing through the filter as a function of time. In contrast, shear-induced platelet aggregation, measured in the cone-and-plate viscometer, was paradoxically increased in type IIB patients. Treatment with l-desamino-8-D-arginine vasopressin (DDAVP) tended to normalize the filter test in patients with type I-platelet normal and type I-platelet low vWD, but infusion of a factor VUI/von Willebrand factor (vWF) concentrate lacking the largest vWF multimers was without effect in type 3 patients. Experiments with specific monoclonal antibodies demonstrated that the A1 and A3 domains of vWF, as well as the glycoproteins Ibα and Ilb-IIIa on platelets, are required for platelet retention in the filter. Thus, the test may reflect vWF function with regard to both platelet adhesion and aggregation under high shear stress, and provide relevant information on mechanisms involved in primary hemostasis.

Toward Personalized Treatment for Patients with Low von Willebrand Factor and Quantitative von Willebrand Disease

2021 ◽  
Vol 47 (02) ◽  
pp. 192-200
Author(s):  
James S. O'Donnell
Keyword(s):  
Von Willebrand Factor ◽  
Bleeding Risk ◽  
Von Willebrand Disease ◽  
Personalized Treatment ◽  
Treatment Plans ◽  
Type 3 ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractThe biological mechanisms involved in the pathogenesis of type 2 and type 3 von Willebrand disease (VWD) have been studied extensively. In contrast, although accounting for the majority of VWD cases, the pathobiology underlying partial quantitative VWD has remained somewhat elusive. However, important insights have been attained following several recent cohort studies that have investigated mechanisms in patients with type 1 VWD and low von Willebrand factor (VWF), respectively. These studies have demonstrated that reduced plasma VWF levels may result from either (1) decreased VWF biosynthesis and/or secretion in endothelial cells and (2) pathological increased VWF clearance. In addition, it has become clear that some patients with only mild to moderate reductions in plasma VWF levels in the 30 to 50 IU/dL range may have significant bleeding phenotypes. Importantly in these low VWF patients, bleeding risk fails to correlate with plasma VWF levels and inheritance is typically independent of the VWF gene. Although plasma VWF levels may increase to > 50 IU/dL with progressive aging or pregnancy in these subjects, emerging data suggest that this apparent normalization in VWF levels does not necessarily equate to a complete correction in bleeding phenotype in patients with partial quantitative VWD. In this review, these recent advances in our understanding of quantitative VWD pathogenesis are discussed. Furthermore, the translational implications of these emerging findings are considered, particularly with respect to designing personalized treatment plans for VWD patients undergoing elective procedures.

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