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. 2018 Jul;60(7):759-768.
doi: 10.1007/s00234-018-2033-1. Epub 2018 May 14.

Transcriptomic analysis of the harvested endothelial cells in a swine model of mechanical thrombectomy

Nasren Jaff  1 , Rikard Grankvist  2 , Lars Muhl  3 , Arvin Chireh  2 , Mikael Sandell  2 , Stefan Jonsson  2   4 , Fabian Arnberg  5 , Ulf Eriksson  3 , Staffan Holmin  6   7
Affiliations

Transcriptomic analysis of the harvested endothelial cells in a swine model of mechanical thrombectomy

Nasren Jaff et al. Neuroradiology. 2018 Jul.

Abstract

Purpose: In mechanical thrombectomy (MT) for ischemic stroke, endothelial cells (ECs) from intracranial blood vessels adhere to the stent retriever device and can be harvested. However, understanding the molecular biology and the role of the endothelium in different pathological conditions remains insufficient. The purpose of the study was to characterize and analyze the molecular aspect of harvested ECs using cell culture and transcriptomic techniques in an MT swine model relevant to clinical ischemic stroke.

Methods: In swine, preformed thrombi were injected into the external carotid and subclavian arteries to occlude their branches. MT was performed according to clinical routine. The stent retriever device and thrombus were treated with cell dissociation buffer. The resulting cell suspension was analyzed by immunohistochemistry and was cultured. Cultured cells were analyzed using single-cell RNA sequencing (scRNA-seq) after fluorescence-activated cell sorting (FACS).

Results: A total number of 37 samples were obtained containing CD31-positive cells. Cell culture was successful in 90% of samples, and the cells expressed multiple typical EC protein markers. Eighty-nine percent of the sorted cells yielded high-quality transcriptomes, and single-cell transcriptomes from cultured cells showed that they expressed typical endothelial gene patterns. Gene expression analysis of ECs from an occluded artery did not show distinctive clustering into subtypes.

Conclusion: ECs harvested during MT can be cultured and analyzed using single-cell transcriptomic techniques. This analysis can be implemented in clinical practice to study the EC gene expression of comorbidities, such as hypertension, diabetes mellitus, and metabolic syndrome, in patients suffering from acute ischemic stroke.

Keywords: Cell culture; Endothelial cells; Thrombectomy; Transcriptomic analysis.

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Conflict of interest statement

Funding

This study was funded by the Söderberg Foundations, Familjen Erling Perssons stiftelse, Karolinska Institutet and Karolinska University Hospital.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed in accordance with the Karolinska Institute guidelines for experiments on large animals and were approved by the ethics committee (Norra Stockholms Djurförsöksetiska nämnd, dnr N87/15 and N162/16).

Informed consent

This article does not contain any studies with human participants performed by any of the authors.

Figures

Fig. 1
Fig. 1
Anteroposterior angiographies of mechanical thrombectomy procedure. a Catheter tip in the right external carotid artery (ECA) before thrombus injection, showing patent circulation. b After a thrombus injection, a large and a smaller segments are occluded (arrows). c After two passes of mechanical thrombectomy of the occluded segments using a stent retriever device, the smaller segment and the base of the larger segment are again patent. More distal occlusions to the large segment remain
Fig. 2
Fig. 2
Immunohistochemistry staining of endothelial cells from cell culture. Endothelial cells revealed by positive CD31 membrane staining (a), vWF cytosol staining (b), VE-cadherin staining (c), and positive CD31 membrane staining and vWF cytosol staining (d). Scale bars = 100 μm
Fig. 3
Fig. 3
a FACS final gates for EC cell sorting. CD31+ and CD146+ ECs were sorted for single-cell RNA sequencing. b Histograms of counts and number of expressed genes over all cells. c Expression of gene characteristics of endothelial cells in thrombus and nonthrombus samples as well as genes that used as a negative controls (CD-11b and CD-45). Differential expression assessed by DESeq2, showing no significant difference between selected genes with a cutoff of absolute log2 fold change ≥ 1 (p > 0.05)
Fig. 4
Fig. 4
a Principal component analyses (PCA) plot, showing the first 3 PCA components
Fig. 5
Fig. 5
Heatmap for the 50 high variable genes (HVG). Rows are genes; columns are cells. Clustering is done based on Euclidean distance. No clear clustering of cells within or between mechanical thrombectomy (MT) and control is seen
See this image and copyright information in PMC

References

    1. Sumpio BE, Riley JT, Dardik A. Cells in focus: endothelial cell. Int J Biochem Cell Biol. 2002;34(12):1508–1512. doi: 10.1016/S1357-2725(02)00075-4. - DOI - PubMed
    1. Corretti MC, Anderson TJ, Benjamin EJ, Celermajer D, Charbonneau F, Creager MA, Deanfield J, Drexler H, Gerhard-Herman M, Herrington D, Vallance P, Vita J, Vogel R, International Brachial Artery Reactivity Task F Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force. J Am Coll Cardiol. 2002;39(2):257–265. doi: 10.1016/S0735-1097(01)01746-6. - DOI - PubMed
    1. Mullen MJ, Clarkson P, Donald AE, Thomson H, Thorne SA, Powe AJ, Furuno T, Bull T, Deanfield JE. Effect of enalapril on endothelial function in young insulin-dependent diabetic patients: a randomized, double-blind study. J Am Coll Cardiol. 1998;31(6):1330–1335. doi: 10.1016/S0735-1097(98)00099-0. - DOI - PubMed
    1. Thorne S, Mullen MJ, Clarkson P, Donald AE, Deanfield JE. Early endothelial dysfunction in adults at risk from atherosclerosis: different responses to L-arginine. J Am Coll Cardiol. 1998;32(1):110–116. doi: 10.1016/S0735-1097(98)00211-3. - DOI - PubMed
    1. Ludmer PL, Selwyn AP, Shook TL, Wayne RR, Mudge GH, Alexander RW, Ganz P. Paradoxical vasoconstriction induced by acetylcholine in atherosclerotic coronary arteries. N Engl J Med. 1986;315(17):1046–1051. doi: 10.1056/NEJM198610233151702. - DOI - PubMed

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