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Updated: Aug 7, 2026
In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing
Published on: May 11, 2021
A model of anti-angiogenesis: differential transcriptosome profiling of microvascular endothelial cells from diffuse
Betti Giusti1, Gabriella Fibbi, Francesca Margheri
1Department of Medical and Surgical Critical Care - DENOTHE, University of Florence, Florence, Italy. b.giusti@DAC.UNIFI.IT
Abstract:
The objective of this work was to identify genes involved in impaired angiogenesis by comparing the transcriptosomes of microvascular endothelial cells from normal subjects and patients affected by systemic sclerosis (SSc), as a unique human model disease characterized by insufficient angiogenesis. Total RNAs, prepared from skin endothelial cells of clinically healthy subjects and SSc patients affected by the diffuse form of the disease, were pooled, labeled with fluorochromes, and hybridized to 14,000 70 mer oligonucleotide microarrays. Genes were analyzed based on gene expression levels and categorized into different functional groups based on the description of the Gene Ontology (GO) consortium to identify statistically significant terms. Quantitative PCR was used to validate the array results. After data processing and application of the filtering criteria, the analyzable features numbered 6,724. About 3% of analyzable transcripts (199) were differentially expressed, 141 more abundantly and 58 less abundantly in SSc endothelial cells. Surprisingly, SSc endothelial cells over-express pro-angiogenic transcripts, but also show up-regulation of genes exerting a powerful negative control, and down-regulation of genes critical to cell migration and extracellular matrix-cytoskeleton coupling, all alterations that provide an impediment to correct angiogenesis. We also identified transcripts controlling haemostasis, inflammation, stimulus transduction, transcription, protein synthesis, and genome organization. An up-regulation of transcripts related to protein degradation and ubiquitination was observed in SSc endothelial cells. We have validated data on the main anti-angiogenesis-related genes by RT-PCR, western blotting, in vitro angiogenesis and immunohistochemistry. These observations indicate that microvascular endothelial cells of patients with SSc show abnormalities in a variety of genes that are able to account for defective angiogenesis.
Insights
Systemic sclerosis (SSc) impairs angiogenesis by altering endothelial cell gene expression. SSc cells show complex changes, including up-regulation of pro-angiogenic and inhibitory genes, leading to defective blood vessel formation.
Area of Science:
- Molecular Biology
- Vascular Biology
- Human Genetics
Background:
- Systemic sclerosis (SSc) is a human disease characterized by insufficient angiogenesis.
- Microvascular endothelial cells play a critical role in angiogenesis.
Purpose of the Study:
- To identify genes involved in impaired angiogenesis in SSc.
- To compare transcriptomes of endothelial cells from normal subjects and SSc patients.
Main Methods:
- Gene expression profiling using oligonucleotide microarrays (14,000 transcripts).
- Analysis of differential gene expression and functional categorization using Gene Ontology.
- Validation of key gene expression changes using quantitative PCR, Western blotting, in vitro angiogenesis assays, and immunohistochemistry.
Main Results:
- Approximately 3% of analyzed transcripts (199) were differentially expressed in SSc endothelial cells.
- SSc cells showed overexpression of pro-angiogenic transcripts alongside upregulation of negative regulators and downregulation of cell migration/cytoskeleton genes.
- Upregulation of transcripts related to protein degradation and ubiquitination was observed in SSc cells.
Conclusions:
- Microvascular endothelial cells in SSc patients exhibit widespread gene expression abnormalities.
- These genetic alterations collectively contribute to the defective angiogenesis observed in systemic sclerosis.
