Hereditary Hemorrhagic Telangiectasia (HHT) Cell Models for Research

Hereditary Hemorrhagic Telangiectasia (HHT), also known as Osler-Weber-Rendu syndrome, is an autosomal dominant genetic disorder affecting approximately 1 in 5,000 to 10,000 individuals worldwide (WHO, 2023). It is characterized by the development of telangiectases on skin and mucous membranes, and arteriovenous malformations (AVMs) in internal organs such as lungs, liver, and brain. The disease has a significant clinical impact: recurrent epistaxis occurs in over 90% of patients, and AVMs can lead to life-threatening complications like hemorrhagic stroke, high-output heart failure, and severe gastrointestinal bleeding. The 5-year survival rate is generally good, but it is reduced in patients with untreated pulmonary AVMs (NCI, 2022). The disease is underdiagnosed, and many patients remain asymptomatic until a major complication occurs.

HHT is an ideal model for studying angiogenesis and vascular development because it is caused by mutations in genes involved in the TGF-beta/BMP signaling pathway, which is crucial for endothelial cell homeostasis. The disease offers a clear genotype-phenotype correlation: mutations in ENG cause HHT1, ACVRL1 cause HHT2, and SMAD4 cause a combined syndrome of juvenile polyposis and HHT. Public datasets, such as those from the Genotype-Tissue Expression (GTEx) project and the Human Protein Atlas, provide expression data for these genes, but there is a need for functional models to understand the molecular mechanisms. Open questions include the role of modifier genes, the triggers for AVM formation, and the development of targeted therapies. Gene-edited cell models are essential to dissect these pathways and test potential drugs.

HHT is not a cancer, but it involves dysregulated angiogenesis. The major pathways involved are:

• • TGF-beta/BMP Signaling Pathway: This pathway is central to HHT. Mutations in ENG (endoglin) or ACVRL1 (ALK1) impair the signaling through the BMP9/BMP10-ALK1-endoglin axis, leading to abnormal endothelial cell proliferation and migration.

1. BMP9/10 ligands bind to ALK1 and endoglin on endothelial cells.

2. This activates SMAD1/5/8 phosphorylation.

3. Phosphorylated SMADs complex with SMAD4 and translocate to the nucleus.

4. They regulate transcription of genes involved in angiogenesis, such as ID1, ID3, and NOTCH pathway components.

• • NOTCH Signaling: NOTCH signaling is also implicated in HHT. Mutations in NOTCH pathway genes can exacerbate the vascular phenotype. The NOTCH pathway interacts with TGF-beta/BMP signaling to regulate endothelial cell fate.
• • VEGF Signaling: Vascular endothelial growth factor (VEGF) is a key pro-angiogenic factor. In HHT, there is an imbalance between pro- and anti-angiogenic factors, leading to the formation of fragile vessels. VEGF levels are often elevated in HHT patients, and anti-VEGF therapies are being explored.

Data from ClinVar and COSMIC (2023).

The deregulated signaling networks in HHT include:

• • TGF-beta/BMP Network: Key nodes include ENG, ACVRL1, SMAD1/5/8, SMAD4, and inhibitory SMADs (SMAD6/7).
• • NOTCH Network: Components include NOTCH1, DLL4, JAG1, and downstream effectors like HEY1/2.
• • VEGF/VEGFR Network: Involves VEGFA, VEGFR2 (KDR), and downstream PI3K/AKT and MAPK pathways.
• • PI3K/AKT Pathway: Activated by VEGF and other growth factors, promoting endothelial cell survival and proliferation.
• • MAPK/ERK Pathway: Also activated by VEGF and involved in cell proliferation and migration.

These networks cross-talk and are critical for maintaining vascular integrity. Mutations in ENG or ACVRL1 lead to an imbalance, favoring pro-angiogenic signaling and vessel instability.

Organoids offer a more physiologically relevant 3D environment and can be used to model vascular malformations. They are particularly useful for studying the interaction between endothelial cells and pericytes.

• • Genetically Engineered Mouse Models (GEMMs): Knockout mice for Eng or Acvrl1 are embryonic lethal, so conditional knockouts are used. For example, endothelial-specific Eng knockout mice develop AVMs. These models are valuable for studying disease progression and testing therapies.
• • Induced Models: Chemical or viral induction of gene mutations in adult mice can model sporadic AVMs.
• • Patient-Derived Xenografts (PDX): Not commonly used for HHT because it is not a cancer, but xenografts of endothelial cells from patients can be used to study vascular abnormalities.
• • Zebrafish Models: Zebrafish with mutations in eng or acvrl1 have been used to study angiogenesis and are useful for high-throughput drug screening.

Gene-edited cell models are powerful tools for studying HHT. Using CRISPR/Cas9 technology, researchers can create isogenic cell lines with specific mutations in ENG, ACVRL1, or SMAD4. These models are commercially available from various sources and are sequence-verified to ensure accuracy.

Examples include:

• • ENG knockout HUVEC lines: These cells lack endoglin expression, mimicking HHT1. They can be used to study the effects of endoglin loss on endothelial cell function, such as migration and tube formation.
• • ACVRL1 knock-in lines: These cells carry a specific missense mutation (e.g., c.1231G>A, p.Gly411Arg) found in HHT2 patients. They allow for the study of mutant ALK1 function in a controlled background.
• • SMAD4 knockout lines: These are useful for studying the combined JP-HHT syndrome and the role of SMAD4 in TGF-beta/BMP signaling.

These isogenic pairs (wild-type vs. mutant) are essential for functional studies, drug screening, and target validation. They eliminate genetic background variability, making results more reliable.

Gene-edited cell lines are used to validate the function of genes implicated in HHT. For example, knocking out ENG in endothelial cells and then performing transcriptomic analysis can identify downstream targets of endoglin. Similarly, introducing a specific ACVRL1 mutation can reveal how that mutation affects signaling pathways. These models are also used in CRISPR screens to identify modifier genes that influence the severity of the disease. By knocking out candidate genes in an HHT background, researchers can determine which genes contribute to the vascular phenotype.

Isogenic pairs (wild-type and mutant) are ideal for drug screening. For instance, HHT endothelial cells with ENG knockout can be used to test drugs that modulate angiogenesis, such as anti-VEGF agents or TGF-beta pathway inhibitors. The isogenic background ensures that any observed effect is due to the mutation, not genetic variability. Additionally, these models can be used to study drug resistance. For example, if a drug is effective in wild-type cells but not in ENG knockout cells, it suggests that endoglin is required for the drug's action. This information is critical for developing targeted therapies.

CRISPR-based synthetic lethality screens can identify genes that are essential for the survival of HHT-mutant cells but not wild-type cells. These genes could serve as potential therapeutic targets. For example, if ENG knockout cells are sensitive to knockdown of a particular gene, that gene might be a target for drug development. Additionally, gene-edited cells can be used to identify biomarkers of disease progression by analyzing secreted proteins or exosomes. These biomarkers could be used for early diagnosis or monitoring of HHT.