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Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro
Published on: February 15, 2022
The Functional Interaction of EGFR with AT1R or TP in Primary Vascular Smooth Muscle Cells Triggers a Synergistic
Virginie Dubourg1, Barbara Schreier1, Gerald Schwerdt1
1Julius-Bernstein-Institute of Physiology, Martin Luther University Halle-Wittenberg, 06112 Halle, Germany.
Abstract:
In vivo, cells are simultaneously exposed to multiple stimuli whose effects are difficult to distinguish. Therefore, they are often investigated in experimental cell culture conditions where stimuli are applied separately. However, it cannot be presumed that their individual effects simply add up. As a proof-of-principle to address the relevance of transcriptional signaling synergy, we investigated the interplay of the Epidermal Growth Factor Receptor (EGFR) with the Angiotensin-II (AT1R) or the Thromboxane-A2 (TP) receptors in murine primary aortic vascular smooth muscle cells. Transcriptome analysis revealed that EGFR-AT1R or EGFR-TP simultaneous activations led to different patterns of regulated genes compared to individual receptor activations (qualitative synergy). Combined EGFR-TP activation also caused a variation of amplitude regulation for a defined set of genes (quantitative synergy), including vascular injury-relevant ones (Klf15 and Spp1). Moreover, Gene Ontology enrichment suggested that EGFR and TP-induced gene expression changes altered processes critical for vascular integrity, such as cell cycle and senescence. These bioinformatics predictions regarding the functional relevance of signaling synergy were experimentally confirmed. Therefore, by showing that the activation of more than one receptor can trigger a synergistic regulation of gene expression, our results epitomize the necessity to perform comprehensive network investigations, as the study of individual receptors may not be sufficient to understand their physiological or pathological impact.
Insights
Cell receptor interactions, like Epidermal Growth Factor Receptor (EGFR) with Angiotensin-II (AT1R) or Thromboxane-A2 (TP) receptors, show synergistic effects. Studying single receptors misses crucial biological signaling pathways impacting vascular health.
Area of Science:
- Cellular biology
- Molecular signaling
- Vascular biology
Background:
- Cells encounter multiple stimuli in vivo, making isolated stimulus effects hard to discern.
- Investigating single stimuli in vitro may not reflect complex cellular responses.
- Receptor interactions can lead to synergistic effects not predictable from individual actions.
Purpose of the Study:
- To investigate the transcriptional signaling synergy between Epidermal Growth Factor Receptor (EGFR) and Angiotensin-II Receptor Type 1 (AT1R) or Thromboxane Receptor (TP).
- To determine if combined receptor activation yields different gene expression patterns compared to individual activation.
- To experimentally validate bioinformatics predictions on the functional relevance of signaling synergy in vascular smooth muscle cells.
Main Methods:
- Murine primary aortic vascular smooth muscle cells were used.
- Transcriptome analysis (RNA sequencing) was performed to assess gene expression changes.
- Bioinformatic tools, including Gene Ontology enrichment, were employed.
- Experimental validation of predicted functional outcomes was conducted.
Main Results:
- Simultaneous activation of EGFR with AT1R or TP receptors induced distinct gene expression patterns (qualitative synergy).
- Combined EGFR-TP activation demonstrated quantitative synergy, altering gene expression amplitudes for key genes like Klf15 and Spp1.
- EGFR and TP signaling synergy impacted cell cycle and senescence, processes vital for vascular integrity.
- Bioinformatic predictions of functional relevance were experimentally confirmed.
Conclusions:
- Receptor activation can trigger synergistic gene expression regulation, highlighting the limitations of studying individual receptors.
- Comprehensive network investigations are essential for understanding the physiological and pathological roles of cellular signaling.
- Signaling synergy plays a critical role in vascular integrity and response to injury.
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