P194. Long Non-coding RNAs in Thoracic Aortic Disease – Pathophysiological Analysis and Clinical Implications.
Presented During: POD Abstracts (Available for viewing in the exhibit hall for the duration of the meeting)
LMU KLINIKUM MUNICH
Munich, NA
Germany - Contact Me
Resident and Clinician Scientist in Cardiac Surgery at the LMU Munich Hospital since January 2019. Research focus: long non-coding RNAs in thoracic aortic disease.
Thursday, April 25, 2024: 5:38 PM - 7:00 PM
Sheraton Times Square
Room: Central Park
Description
Objective: Besides for the well known connective tissue disorderes the mechanism of aortic aneurysm formation is poorly understood. The presence and quantity of non-coding RNAs, in particular long non-coding RNA (lncRNA) may offer new opportunities in understanding aortic pathology. Here, we describe promising candidates from patients with different thoracic aortic disease and by comparing their presence in different areas with more or less wall shear stress and consecutive dilation.
Methods: Single-cell RNA sequencing was performed on samples of the wall of the outer (dilated) and inner (non-dilated) curvature of thoracic ascending aortic aneurysms (TAA) (n=4). To cluster the cells, a UMAP (Uniform Manifold Approximation and Projection) analysis was performed. In a second step, the 10 most deregulated lncRNAs in vascular smooth muscle cell (VSMC) clusters were identified. Two promising representatives with almost unique expression in VSMCs were further analysed by qPCR and life-cell-imaging.
Quantitativ expression was measured by qPCR in 110 samples of TAA and acute aortic dissection (AADA), comparing inner and outer curvature. In addition, analyses were performed according to aortic valve morphology (bicuspid - BAV, tricuspid - TAV) and valve pathology (stenosis - AS, regurgitation - AI). As a control, expression was validated in 7 aortic samples from healthy heart transplant donors. CT values of more than 40 were excluded. The results were expressed as multiples of the expression at the inner curvature, which was set to 1.
Functional analyses were performed using live-cell imaging. Migration, proliferation and apoptosis were evaluated after siRNA-mediated temporary knockdown of both lncRNAs in primary VSMCs extracted from thoracic aortic tissue. All cells and tissues were provided by the Aortic Biobank of the Department of Cardiac Surgery at the LMU University Hospital, Munich, Germany.
Results: UMAP analysis identified 17 different cell clusters, including 3 VSMC cluster. Examination in the VSMC clusters for the 10 most deregulated lncRNA revealed 25 representatives with differential expression along the inner and outer curvature, notably TMEM72-AS1 and LINC00632 with almost exclusive expression in VSMC. Quantitative expression analysis showed significantly higher expression of TMEM72-AS1 and LINC00632 in dilated areas (1.96, p<0.01; 2.1, p<0.001). LINC00632 showed increased expression particularly in tissues from BAV and AI (1.99, p<0.01; 2.16, p<0.01). However, there was no significant difference in samples from AADA tissue. Conversely, TMEM72-AS1 was highly expressed in AADA tissue (1.54, p<0.01), but showed no significant difference in AI tissue samples. No differences in expression was detected for either lncRNA in healthy aortic tissue.
SiRNA-mediated silencing of TMEM72-AS1 and LINC00632 in primary VSMCs showed reduced migration and proliferation at 48 and 72 hours compared to non-transfected VSMCs (99.37% vs. 62.54%, p<0.0001 and 88.39%, p=0.025, respectively). Conversely, apoptosis was activated with a peak at 120 hours (0.5% vs. 11.25%, p<0.0001 and 14.17% p<0.0001, respectively).
Conclusion: ScRNA sequencing supports the role of lncRNAs in the pathophysiological process of thoracic aortic aneurysm formation. Functional and quantitative expression analyses of TMEM72-AS1 and LINC00632 identify these lncRNAs as targets for potential diagnostic and therapeutic approaches.
Authors
Joscha Buech (1), Jessica Pauli (2), Caroline Radner (1), Zhaolong Li (2), Linda Grefen (1), Christian Hagl (1), Maximilian Pichlmaier (1), Lars Maegdefessel (2), Sven Peterss (1)
Institutions
(1) LMU University Hospital, Munich, Germany, (2) Institute for Molecular Vascular Medicine, Technical University Munich, Munich, Germany
Methods: Single-cell RNA sequencing was performed on samples of the wall of the outer (dilated) and inner (non-dilated) curvature of thoracic ascending aortic aneurysms (TAA) (n=4). To cluster the cells, a UMAP (Uniform Manifold Approximation and Projection) analysis was performed. In a second step, the 10 most deregulated lncRNAs in vascular smooth muscle cell (VSMC) clusters were identified. Two promising representatives with almost unique expression in VSMCs were further analysed by qPCR and life-cell-imaging.
Quantitativ expression was measured by qPCR in 110 samples of TAA and acute aortic dissection (AADA), comparing inner and outer curvature. In addition, analyses were performed according to aortic valve morphology (bicuspid - BAV, tricuspid - TAV) and valve pathology (stenosis - AS, regurgitation - AI). As a control, expression was validated in 7 aortic samples from healthy heart transplant donors. CT values of more than 40 were excluded. The results were expressed as multiples of the expression at the inner curvature, which was set to 1.
Functional analyses were performed using live-cell imaging. Migration, proliferation and apoptosis were evaluated after siRNA-mediated temporary knockdown of both lncRNAs in primary VSMCs extracted from thoracic aortic tissue. All cells and tissues were provided by the Aortic Biobank of the Department of Cardiac Surgery at the LMU University Hospital, Munich, Germany.
Results: UMAP analysis identified 17 different cell clusters, including 3 VSMC cluster. Examination in the VSMC clusters for the 10 most deregulated lncRNA revealed 25 representatives with differential expression along the inner and outer curvature, notably TMEM72-AS1 and LINC00632 with almost exclusive expression in VSMC. Quantitative expression analysis showed significantly higher expression of TMEM72-AS1 and LINC00632 in dilated areas (1.96, p<0.01; 2.1, p<0.001). LINC00632 showed increased expression particularly in tissues from BAV and AI (1.99, p<0.01; 2.16, p<0.01). However, there was no significant difference in samples from AADA tissue. Conversely, TMEM72-AS1 was highly expressed in AADA tissue (1.54, p<0.01), but showed no significant difference in AI tissue samples. No differences in expression was detected for either lncRNA in healthy aortic tissue.
SiRNA-mediated silencing of TMEM72-AS1 and LINC00632 in primary VSMCs showed reduced migration and proliferation at 48 and 72 hours compared to non-transfected VSMCs (99.37% vs. 62.54%, p<0.0001 and 88.39%, p=0.025, respectively). Conversely, apoptosis was activated with a peak at 120 hours (0.5% vs. 11.25%, p<0.0001 and 14.17% p<0.0001, respectively).
Conclusion: ScRNA sequencing supports the role of lncRNAs in the pathophysiological process of thoracic aortic aneurysm formation. Functional and quantitative expression analyses of TMEM72-AS1 and LINC00632 identify these lncRNAs as targets for potential diagnostic and therapeutic approaches.
Authors
Joscha Buech (1), Jessica Pauli (2), Caroline Radner (1), Zhaolong Li (2), Linda Grefen (1), Christian Hagl (1), Maximilian Pichlmaier (1), Lars Maegdefessel (2), Sven Peterss (1)
Institutions
(1) LMU University Hospital, Munich, Germany, (2) Institute for Molecular Vascular Medicine, Technical University Munich, Munich, Germany
Presentation Duration
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