Site-Specific Genetic and Functional Signatures of Aortic Endothelial Cells at Aneurysm Predilection Sites in Healthy and AngII ApoE−/− Mice

Academic Background

Aortic aneurysm (AA) is characterized by pathological dilation at specific predilection sites of the vessel, potentially leading to life-threatening vascular rupture. Aortic aneurysms typically develop at specific sites, such as the aortic arch and abdominal aorta. While hemodynamic factors play a significant role in aneurysm formation, it remains unclear whether intrinsic differences in the vascular wall also contribute. Recent evidence suggests that endothelial cells (ECs) play a crucial role in the pathogenesis of aortic aneurysms. Endothelial cells exhibit significant heterogeneity across different organs and various segments of the vascular tree, which may be related to the susceptibility of aneurysm formation.

To further investigate the role of aortic endothelial cells in aneurysm formation, researchers developed a modified “Häutchen method” to isolate endothelial cells from specific segments of the mouse aorta and analyzed their spatial heterogeneity through RNA sequencing (RNA-seq). The study aimed to reveal the genetic and functional signatures of aortic endothelial cells at aneurysm predilection sites in healthy mice and explore their potential role in disease development.

Source of the Paper

The study was conducted by Alexander Brückner, Adrian Brandtner, Sarah Rieck, and others, with the research team from the University of Bonn, the University of Lübeck, and Ruhr University Bochum, among others. The paper was published online on July 4, 2024, in the journal Angiogenesis, titled Site-specific genetic and functional signatures of aortic endothelial cells at aneurysm predilection sites in healthy and AngII ApoE−/− mice.

Research Process

1. Isolation and Processing of Endothelial Cells

The researchers first used a modified “Häutchen method” to isolate endothelial cells from different segments of the aorta in healthy C57BL/6 mice and AngII ApoE−/− mice. The specific steps are as follows:

• Aorta Isolation: The thoracic and abdominal cavities of the mice were opened, and the aorta was dissected free of connective tissue and perfused with heparin (250 IU/mL).
• Aorta Segmentation: The aorta was divided into four segments representing the ascending and descending parts of the aortic arch, as well as the thoracic and abdominal aorta.
• Endothelial Cell Isolation: The modified “Häutchen method” was used to separate endothelial cells and medial/adventitial cells from the aortic segments. Specifically, the aortic segments were cut open and placed endothelial-side down on a glass coverslip. A pre-cooled glass coverslip was then placed on the adventitial side, and mechanical force was applied to transfer the superficial cell layers to the glass.
• RNA Extraction: RNA was extracted from the isolated endothelial cells using the RNeasy Plus Micro Kit (Qiagen) and subjected to RNA-seq analysis.

2. Establishment of the AngII ApoE−/− Mouse Model

To study aneurysm formation, the researchers used the AngII ApoE−/− mouse model. The specific steps are as follows:

• Mouse Treatment: ApoE−/− mice aged 10-18 weeks were randomly assigned to the AngII group or the control group. The AngII group received continuous infusion of AngII (1000 ng/kg/min) via implanted Alzet osmotic pumps for 14 or 28 days.
• Disease Monitoring: Aneurysm formation was monitored using the Vevo 3100 ultrasound machine.

3. RNA-seq Analysis

The researchers performed RNA-seq analysis on the isolated endothelial cells to compare gene expression differences across different aortic segments. The specific steps are as follows:

• Library Preparation: RNA-seq libraries were prepared using the Trio RNA-seq Library Preparation Kit (Tecan) and sequenced on the NextSeq 500 sequencer.
• Data Analysis: Bioinformatics analysis of RNA-seq data was performed using the Galaxy platform, including differential gene expression analysis, Gene Ontology (GO) analysis, and heatmap generation.

4. Measurement of Endothelial Cell Mechanical Properties

The researchers used atomic force microscopy (AFM) to measure the cortical stiffness of endothelial cells, assessing changes in mechanical properties at aneurysm predilection sites.

Main Results

1. Isolation and Identification of Endothelial Cells

Using the modified “Häutchen method,” the researchers successfully isolated highly purified endothelial cells from different segments of the aorta in healthy mice. RNA-seq analysis revealed significant differences in gene expression profiles between endothelial cells and medial/adventitial cells, further validating the effectiveness of the method.

2. Heterogeneity in Endothelial Cell Gene Expression

The researchers found significant heterogeneity in gene expression among endothelial cells from different segments of the aorta in healthy mice. Specifically, endothelial cells at aneurysm predilection sites (e.g., the ascending aortic arch and abdominal aorta) exhibited upregulation of genes related to extracellular matrix remodeling, angiogenesis, and inflammation.

3. Gene Expression Changes in the AngII ApoE−/− Mouse Model

In the AngII ApoE−/− mouse model, the gene expression patterns of endothelial cells at aneurysm formation sites resembled those at predilection sites in healthy mice. This suggests that the heterogeneity of aortic endothelial cells in healthy mice may determine the location and pathological changes of aneurysm formation.

4. Changes in Endothelial Cell Mechanical Properties

Using AFM, the researchers found that the cortical stiffness of endothelial cells at aneurysm predilection sites was significantly increased, which may be an early sign of endothelial dysfunction.

Conclusion

The study revealed the genetic and functional heterogeneity of aortic endothelial cells at aneurysm predilection sites in healthy mice and suggested that this heterogeneity may determine the location and pathological changes of aneurysm formation. The findings provide new insights into the pathogenesis of aortic aneurysms and offer potential targets for future therapeutic strategies.

Research Highlights

• Modified “Häutchen Method”: The study developed a modified “Häutchen method” that efficiently isolates endothelial cells from specific segments of the aorta, providing an important tool for studying endothelial cell heterogeneity.
• Heterogeneity in Endothelial Cell Gene Expression: The study found significant heterogeneity in gene expression among endothelial cells from different segments of the aorta in healthy mice, particularly at aneurysm predilection sites, where endothelial cells exhibited upregulation of genes related to extracellular matrix remodeling, angiogenesis, and inflammation.
• Validation in the AngII ApoE−/− Mouse Model: In the AngII ApoE−/− mouse model, the gene expression patterns of endothelial cells at aneurysm formation sites resembled those at predilection sites in healthy mice, further validating the importance of endothelial cell heterogeneity in aneurysm formation.
• Changes in Endothelial Cell Mechanical Properties: The study found that the cortical stiffness of endothelial cells at aneurysm predilection sites was significantly increased, which may be an early sign of endothelial dysfunction, providing a potential basis for early diagnosis of aneurysms.

Research Significance

The study not only revealed the important role of aortic endothelial cells in aneurysm formation but also provided new insights for future therapeutic strategies. Targeting endothelial cell heterogeneity may help prevent or treat aortic aneurysms. Additionally, the modified “Häutchen method” offers an important tool for studying endothelial cell heterogeneity in other vascular diseases.