Ccn2 deficiency causes smooth muscle cell de-differentiation and severe atherosclerosis in hyperlipidemic mice

Larsen, J. H.; Stubbe, J.; Beck, H. C.; Overgaard, M.; Lindegaard, C. A.; Hansen, D. R.; Goldschmeding, R.; Rodriguez-Diez, R. R.; Ruiz-Ortega, M.; Pyke, C.; Wirka, R.; Lindholt, J. S.; Rasmussen, L. M.; Steffensen, L. B.

doi:10.1101/2023.01.16.524161

bioRxiv · 10.1101/2023.01.16.524161

Ccn2 deficiency causes smooth muscle cell de-differentiation and severe atherosclerosis in hyperlipidemic mice

Larsen, J. H.·Stubbe, J.·Beck, H. C.·Overgaard, M.·Lindegaard, C. A.·Hansen, D. R.·Goldschmeding, R.·Rodriguez-Diez, R. R.·Ruiz-Ortega, M.·Pyke, C.·Wirka, R.·Lindholt, J. S.·Rasmussen, L. M.·Steffensen, L. B.

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Abstract

Cellular communication network factor 2 (CCN2/CTGF) is a matricellular protein with an established role in fibrotic diseases and cancers, and therapies targeting CCN2 is currently in Phase II and III clinical trials for idiopathic pulmonary fibrosis, pancreatic cancer and Duchenne Muscular Dystrophy. Recent studies have highlighed a protective role of CCN2 in aortic aneurysm disease, but its role in atherosclerosis remains to be investigated. We identified arteries as having the highest relative expression of CCN2 across 54 human tissues. In aortas, CCN2 was among the highest expressed genes, and in situ hybridization of human internal thoracic arteries revealed vascular smooth muscle cells (SMCs) as its principal source. Hypothesizing a role for CCN2 in SMC phenotype maintenance and athero-protection, we investigated inducible Ccn2 knockout (Ccn2{Delta}/{Delta}) mice in normo- and hyper-lipidemic settings. Induction of hyperlipidemia by single intravenous injection of 1{middle dot}1011 viral genomes of rAAV8-D377Y-mPcsk9 combined with 24 weeks of western type diet resulted in severe enlargement (3-5-fold increase of relative aorta mass compared to wildtype littermates, p < 0.0001) and whitening of Ccn2{Delta}/{Delta} aortas. Oil Red O-staining of en face prepared thoracic aortas showed a marked increase in atherosclerosis in Ccn2{Delta}/{Delta} mice as compared to wildtype littermates (75% vs. 10% Oil Red O-positive aortic area, p < 0.0001). Transcriptomic profiling of cultivated SMCs derived from aortas of normolipidemic mice showed signatures of dedifferentiation (reduced expression of e.g. Myocd, Acta2 and Myh11) and modulation toward a synthetic, pro-inflammatory phenotype of Ccn2{Delta}/{Delta} SMCs. These effects were verified in vivo and in CCN2-silenced human aortic SMCs. Taken together, we find that CCN2 plays a critical athero-protective role in artery tissues, likely through maintaining SMCs in a differentiated, contractile phenotype.

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Larsen, J. H., Stubbe, J., Beck, H. C., Overgaard, M., Lindegaard, C. A., Hansen, D. R., Goldschmeding, R., Rodriguez-Diez, R. R., Ruiz-Ortega, M., Pyke, C., Wirka, R., Lindholt, J. S., Rasmussen, L. M., Steffensen, L. B.. 2023-01-19. Ccn2 deficiency causes smooth muscle cell de-differentiation and severe atherosclerosis in hyperlipidemic mice. https://doi.org/10.1101/2023.01.16.524161

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