Hereditäre Hämorrhagische Teleangiektasie Typ II: Eine Untersuchung zum Einfluss der ACVRL1-Expression auf die Angiogenese von HMEC-1 Endothelzellen
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Philipps-Universität Marburg
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Abstract
Hereditary hemorrhagic telangiectasia (HHT), also known as Osler's disease, is a rare
disease of a more common type. It occurs with an incidence of 1:5,000 and is inherited
in an autosomal dominant pattern. The most commonly affected genes are endoglin
(ENG, HHT 1) and activin A receptor like type 1 (ACVRL1, HHT 2). The predominant
pathology is the development of arteriovenous malformations, which occur throughout
the body and can cause life-threatening hemorrhages. The nasal mucosa is particularly
affected, leading to recurrent nosebleeds (epistaxis). The constant nosebleeds can
severely limit a person's quality of life. Other common sites of bleeding include the skin
and mucous membranes, the gastrointestinal tract, the central nervous system and the
lungs. A clinical diagnostic tool are the so-called Curaçao criteria, which include Epistaxis,
mucocutaneous telangiectases at typical predisposition sites (lips, oral cavity, fingers
and nose), visceral involvement (e.g. gastrointestinal, cerebral, pulmonary, hepatic) and
a positive family history of first-degree relatives affected. If at least three of the above
criteria are met, the diagnosis of HHT can be made. If two or less of the criteria are
positive, a molecular genetic diagnosis can be sought. The focus of this study was to gain
a better understanding of the impaired angiogenesis in ACVRL1 deficiency and
overexpression. Particular attention was paid to the ability of the vasculature to form
tubes when ACVRL1 protein expression is altered, as well as the alteration of
angiogenesis-associated genes under these conditions.
Due to the limited data available on transfection by electroporation of the endothelial
cell line HMEC-1, an establishment test was performed first. After optimization of the
transfection efficiency, editing of the ACVRL1 gene using CRISPR-Cas9 was attempted,
which did not yield an ACVRL1-deficient cell clone. Nevertheless, the goal was to better
understand the impact of ACVRL1 on angiogenesis. Overexpression of ACVRL1 after
plasmid vector transfection demonstrated a significant reduction in tube formation
capacity. There was a significant reduction in the number of junctions, crossings, total
segment length and an increase in the number of isolated segments over the entire
observation period.
On the opposite, downregulation by RNAi knockdown of the ACVRL1 gene was
performed to simulate the conditions in the cellular context of HHT patients. A
significant increase in the tube-forming ability of ACVRL1-knockdown cells was
observed. This was reflected 24 h after cell seeding, by a significant increase in the
number of crossings, junctions and an increase in the total length of the segments, as
well as a decrease in the number of isolated segments.
An in vivo experiment was performed with ACVRL1-knockdown cells which were
successfully xenotransplanted onto the chorionallantois membrane (CAM) of a chicken
embryo. Microscopically, the HHT 2 cells showed increased tumour formation compared
to the control. This was also confirmed by 18FDG-PET. On the other hand, the results of
the immunhistochemical analysis were inconsistent and could not be interpreted
unambiguously. Further experiments are therefore planned to clarify this discrepancy.
This will allow us to pursue the overall goal of drug testing with the two therapeutic
endpoints of tumour size and (anti-)angiogenesis. The influence of the downregulation
of the ACVRL1 gene contrasted with the observations of the tube formation assay. Here,
pro-angiogenic gene expression was inhibited. Overall, the work at the cellular level
allowed conclusions to be drawn about the angiogenic behaviour of HHT 2. An improved
tube formation ability was confirmed, which, however, did not correlate with the gene
expression results. Here further investigations should take place, which can address
these given differences. Furthermore, a stable ACVRL1 knockout HMEC-1 cell line should
be established and the in vivo assay should be extended to the chicken CAM to enable
drug testing in the future.
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Dates
Created: 2025Issued: 2025-05-15Updated: 2025-05-15
Faculty
Medizin
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
Keywords
ACVRL1ACVRL1angiogenesishereditary haemorrhagic telangiectasia
DFG-subjects
Hereditäre Hämorrhagische Teleangiektasie Typ IIMorbus OslerAngiogeneseHHT
DDC-Numbers
000
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Rusche, Johanna Rosa: Hereditäre Hämorrhagische Teleangiektasie Typ II: Eine Untersuchung zum Einfluss der ACVRL1-Expression auf die Angiogenese von HMEC-1 Endothelzellen. : Philipps-Universität Marburg 2025-05-15. DOI: https://doi.org/10.17192/z2025.0196.