Genetic and Pharmacological Targeting of mTORC1 in Mouse Models of Arteriovenous Malformation Expose Non-Cell Autonomous Signalling in HHT

Hereditary Hemorrhagic Telangiectasia (HHT) is a genetic disorder caused by mutations leading to the loss of function in either Activin Receptor-Like Kinase 1 (ACVRL1) or Endoglin (ENG), which act as receptors and co-receptors for Bone Morphogenetic Proteins (BMPs) 9 and 10. The manifestation of HHT includes the formation of pathological high-flow connections between arteries and veins, known as Arteriovenous Malformations (AVMs), and recurrent bleeding often causing anemia. AVMs also lead to reduced peripheral resistance, affecting heart function, and local tissue hypoxia. Despite previous studies indicating the role of the PI3K/AKT/mTORC1 signaling pathway in HHT pathology, the precise contribution of mTORC1 in AVM formation remains unclear.

This study aims to investigate the cell-autonomous and non-cell autonomous roles of the mTORC1 signaling pathway in HHT-related AVM formation, using genetic and pharmacological interventions to elucidate the specific contributions of mTORC1 in AVM biology.

Source of the Paper

The paper was authored by Antonio Queiro-Palou, Yi Jin, and Lars Jakobsson, all affiliated with the Division of Vascular Biology at Karolinska Institutet, Sweden. It was published in 2025 in the journal Angiogenesis, titled Genetic and pharmacological targeting of mTORC1 in mouse models of arteriovenous malformation expose non-cell autonomous signalling in HHT.

Research Process and Experimental Design

1. Animal Models

The study utilized several genetically modified mouse models, including:

• Engflox/flox mice: For inducing endothelial cell (EC)-specific deletion of the Endoglin (Eng) gene.
• Tsc1flox/flox mice: For inducing EC-specific deletion of the Tsc1 gene, thereby activating the mTORC1 signaling pathway.
• Rptorflox/flox mice: For inducing EC-specific deletion of the Rptor gene, thereby inhibiting the mTORC1 signaling pathway.

These mice were crossed with Cdh5(pac)-CreERT2 mice to generate EC-specific, tamoxifen-inducible knockout models.

2. Experimental Procedures

a) Tamoxifen-Induced Gene Deletion

Tamoxifen (100 µg) was administered via intraperitoneal injection on postnatal day 1 or 3 to induce gene deletion. Experiments were conducted at different time points, including postnatal days 4, 5, and 6.

b) Rapamycin Treatment

Following gene deletion induction on postnatal day 3, rapamycin (1 mg/kg/day) or vehicle control was administered on postnatal days 4 and 5, with tissue collection on day 6.

c) BMP9/10 Antibody-Induced AVM

Following gene deletion induction on postnatal day 1, BMP9 and BMP10 antibodies (15 mg/kg) or control antibodies were administered on postnatal days 3, 4, and 5, with tissue collection on day 6.

d) Whole-Mount Retinal Vasculature Staining

Postnatal mouse eyes were collected, fixed, and retinas were dissected for immunostaining. Various antibodies were used to label endothelial cells and mTORC1 signaling-related proteins, with imaging performed using confocal microscopy.

3. Data Analysis

ImageJ software was used for image analysis, quantifying vascular area, AVM count, AVM thickness, and mTORC1 signaling activation levels (assessed via phosphorylated RPS6 immunoreactivity).

Key Findings

1. Endoglin Deletion Leads to Retinal AVM and mTORC1 Activation

The study found that EC-specific deletion of the Endoglin gene resulted in the formation of AVMs in the retina, accompanied by mTORC1 signaling activation in both ECs and non-ECs within AVM regions. This suggests that mTORC1 activation is not solely cell-autonomous but also influenced by non-ECs.

2. Rapamycin Inhibits mTORC1 Signaling and Reduces AVM Formation

Rapamycin treatment significantly reduced the number and thickness of AVMs and nearly completely inhibited mTORC1 signaling activation. This indicates that mTORC1 plays a crucial role in AVM formation and expansion.

3. mTORC1 Activation Occurs After AVM Formation

The study found that mTORC1 activation occurred after AVM formation, suggesting that mTORC1 activation is a consequence rather than a cause of AVM development.

4. EC-Specific mTORC1 Inhibition Has Limited Impact on AVM

EC-specific deletion of Rptor (inhibiting mTORC1) only showed a mild reduction in AVM severity, indicating that mTORC1 signaling within ECs plays a limited role in AVM formation.

5. EC-Specific mTORC1 Activation Reduces AVM Severity

EC-specific deletion of Tsc1 (activating mTORC1) unexpectedly reduced the severity of BMP9/10 antibody-induced AVMs, suggesting that excessive mTORC1 activation may inhibit AVM formation.

Conclusions and Significance

This study reveals the complex role of the mTORC1 signaling pathway in HHT-related AVM formation. While mTORC1 signaling within ECs plays a limited role, non-EC mTORC1 activation significantly contributes to AVM progression. Systemic inhibition of mTORC1 with rapamycin effectively reduces AVM formation, suggesting that mTORC1 targeting could be a viable therapeutic strategy for HHT.

Highlights of the Study

1. Dual Role of mTORC1 Signaling: The study demonstrates that mTORC1 activation in both ECs and non-ECs differentially impacts AVM formation and expansion.
2. Therapeutic Potential of Rapamycin: Rapamycin, by systemically inhibiting mTORC1 signaling, significantly reduces AVM formation, offering a new therapeutic avenue for HHT.
3. Importance of Non-Cell Autonomous Signaling: The study highlights the critical role of non-EC mTORC1 activation in AVM pathology, suggesting that future treatments should consider multi-cellular interactions.

Research Value

This study not only deepens the understanding of the pathogenesis of HHT-related AVMs but also provides experimental evidence for developing new therapeutic strategies. By elucidating the specific role of the mTORC1 signaling pathway in AVM formation, the research offers important theoretical support for future drug development and clinical treatments.