Novel markers for smooth muscle cell modulation in vascular injury and disease

Smooth muscle cells (SMCs) are major constituents of the vascular wall, indispensable for basic physiological functions of a healthy vessel, such as regulating vascular tone and blood pressure, but also critical during disease development. With remarkable plasticity, SMCs act as early responders to vessel wall injury, where by activating molecular mechanisms, including phenotypic modulation and transdifferentiation, they counteract detrimental stimuli and aim to restore vascular homeostasis. The nature of SMC response to injury constitutes a major determinant of cardiovascular pathologies, including atherosclerosis, restenosis and aortic aneurysms, however, despite extensive progress in understanding the biology behind SMC phenotypic modulation, its many aspects remain elusive. With this perspective, the presented thesis aimed to identify and comprehensively characterize novel molecular signatures demarcating SMC phenotypic modulation, with a particular focus on transcriptional and cytoskeletal regulation of various SMC transitions.

Study I identified muscle contraction and actin cytoskeleton among the most downregulated pathways in atherosclerosis, while cytoskeleton-related leiomodin 1 (LMOD1), synaptopodin 2 (SYNPO2), PDZ And LIM Domain 7 (PDLIM7), phospholamban (PLN) and synemin (SYNM) emerged as the top molecular signatures repressed in atherosclerotic carotid plaques in comparison to control arteries. These genes positively correlated to classical contractility markers and showed abundant expression in SMCs in healthy arteries, but were largely absent from end-stage lesions. Subcellularly, the majority of the proteins localized to the SMC cytoskeleton and was significantly downregulated in response to atherosclerosis-relevant stimuli. Mechanistically, repression of PDLIM7 resulted in downregulation of SMC markers, and impaired cell spreading, but increased proliferation. Altogether, this study identifies a panel of novel sensitive SMC markers, which could serve as early indicators of SMC phenotypic modulation in vascular disease.

Study II investigated the role of proprotein convertase subtilisin/kexin 6 (PCSK6), previously identified as one of the top molecules upregulated in human atherosclerotic plaques. PCSK6 localized to fibrous cap and neovessels in carotid lesions as well as to injuryinduced intimal hyperplasia, where it was expressed by proliferating smooth muscle alphaactin (SMA) + cells and shown to colocalize and co-interact with matrix metalloproteinases (MMPs) 2 and 14. Pcsk6-/- mice were characterized by the repression of SMC contractility markers and extracellular matrix (ECM) remodeling transcripts, displayed reduced intimal hyperplasia formation upon carotid ligation in vivo and impaired outgrowth of SMCs from aortic rings ex vivo, the latter two likely attributable to decreased MMP14 activity. In summary, this study establishes PCSK6 as a molecule of crucial importance for the SMC function in vascular remodeling.

Study III focused on key molecular signatures in carotid plaques stratified by ultrasoundassessed echogenicity. BCL2 Associated Transcription Factor 1 (BCLAF1) emerged as a top molecule downregulated in relation to plaque echolucency, abundantly expressed in SMA+ SMCs in the normal arteries, strongly repressed early during atheroprogression, however restored in cluster of differentiation 68 (CD68) + cells in advanced lesions, where it was also shown to co-interact with pro-survival B-Cell CLL/Lymphoma 2 (BCL2). Repression of BCLAF1 resulted in suppression of SMC contractility markers, decreased cell viability, as well as partially prevented oxLDL-induced SMC transdifferentiation into macrophage-like cells by preserving higher MYH11 expression and reducing levels of CD36 and CD68 scavenger receptors. Overall, BCLAF1 emerged as a molecule indispensable for SMC survival and transdifferentiation into CD68+ macrophage-like cells.

Study IV aimed to identify key transcription factors (TFs) in the control of SMC phenotype and function in human atherosclerosis. Forkhead Box C1 (FOXC1) emerged as a master upstream regulator of genes differentially expressed in carotid plaques compared to control arteries and in relation to patient symptomatology, involved in the regulation of cell cycle, response to T3 hormone and cell adhesion. It was abundantly expressed in SMA+ cells in the control arteries and plaques, strongly downregulated in early phases of vascular wall healing, with its expression gradually restored concomitantly with SMCs regaining their contractile properties. Silencing of FOXC1 resulted in significant repression of SMC contractility markers, increased migration and proliferation, as well as partially abolished T3-induced SMC phenotypic modulation. Altogether, these results provide compelling evidence for FOXC1 being an important TF in the control of SMC quiescence vs. activation, especially in response to T3.

Collectively, by unraveling the intricacies of various aspects of SMC phenotypic modulation, this thesis contributes to a better understanding of molecular mechanisms underlying cardiovascular disease (CVD).

List of scientific papers

I. Perisic L, Rykaczewska U, Razuvaev A, Sabater-Lleal M, Lengquist M, Miller CL, Ericsson I, Röhl S, Kronqvist M, Aldi S, Gonzalez Diez M, Roy J, Baldassarre D, Veglia F, Humphries SE, de Faire U, Tremoli E, on behalf of the IMPROVE study group, Odeberg J, Vukojević V, Paulsson-Berne G, Hansson GK, Lindeman JHN, Eriksson P, Quertermous T, Hamsten A, Hedin U. Phenotypic modulation of smooth muscle cells in atherosclerosis is associated with downregulation of LMOD1, SYNPO2, PDLIM7, PLN and SYNM. Arteriosclerosis, Thrombosis, and Vascular Surgery (ATVB). 2016 Sep;36(9):1947-61.
https://doi.org/10.1161/ATVBAHA.116.307893

II. Rykaczewska U*, Suur BE*, Röhl S*, Razuvaev A, Lengquist M, Sabater-Lleal M, van der Laan SW, Miller CL, Wirka R, Kronqvist M, Gonzalez Diez M, Vesterlund M, Gillgren P, Odeberg J, Lindeman J, Veglia F, Humphries SE, de Faire U, Baldassarre D, Tremoli E, on behalf of the IMPROVE study group; Lehtiö J, Hansson GK, Paulsson- Berne G, Pasterkamp G, Quertermous T, Hamsten A, Eriksson P, Hedin U# and Matic L#. PCSK6 is a key protease in the control of smooth muscle cell function in vascular remodeling. Circulation Research. 2020 Feb 28;126(5):571-585. *,#Authors contributed equally.
https://doi.org/10.1161/CIRCRESAHA.119.316063

III. Rykaczewska U, Zhao Q, Saliba-Gustafsson P, Lengquist M, Kronqvist M, Bergman O, Huang Z, Lund K, Waden K, Pons Vila Z, Caidahl K, Skogsberg J, Vukojevic V, Lindeman JHN, Roy J, Hansson GK, Treuter E, Leeper NJ, Eriksson P, Ehrenborg E, Razuvaev A, Hedin U* and Matic L*. Plaque evaluation by ultrasound and transcriptomics reveals BCLAF1 as a regulator of smooth muscle cell lipid transdifferentiation in atherosclerosis. Arteriosclerosis, Thrombosis, and Vascular Surgery (ATVB). 2022 May;42(5):659-676. *Authors contributed equally.
https://doi.org/10.1161/ATVBAHA.121.317018

IV. Rykaczewska U, Suur BE, Zhao Q, Narayanan S, Lengquist M, Kronqvist, Razuvaev A, Eriksson P, Quertermous T, Hedin U, Matic L. Transcription factor FOXC1 controls smooth muscle cell phenotype and function in atherosclerosis. [Manuscript]