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- Rykaczewska, Urszula
(author)
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Novel markers for smooth muscle cell modulation in vascular injury and disease
- 2022
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Doctoral thesis (other academic/artistic)abstract
- 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).
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| 2. |
- Rykaczewska, Urszula, et al.
(author)
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PCSK6 Is a Key Protease in the Control of Smooth Muscle Cell Function in Vascular Remodeling
- 2020
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In: Circulation Research. - : LIPPINCOTT WILLIAMS & WILKINS. - 0009-7330 .- 1524-4571. ; 126:5, s. 571-585
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Journal article (peer-reviewed)abstract
- Rationale: PCSKs (Proprotein convertase subtilisins/kexins) are a protease family with unknown functions in vasculature. Previously, we demonstrated PCSK6 upregulation in human atherosclerotic plaques associated with smooth muscle cells (SMCs), inflammation, extracellular matrix remodeling, and mitogens. Objective: Here, we applied a systems biology approach to gain deeper insights into the PCSK6 role in normal and diseased vessel wall. Methods and Results: Genetic analyses revealed association of intronic PCSK6 variant rs1531817 with maximum internal carotid intima-media thickness progression in high-cardiovascular risk subjects. This variant was linked with PCSK6 mRNA expression in healthy aortas and plaques but also with overall plaque SMA+ cell content and pericyte fraction. Increased PCSK6 expression was found in several independent human cohorts comparing atherosclerotic lesions versus healthy arteries, using transcriptomic and proteomic datasets. By immunohistochemistry, PCSK6 was localized to fibrous cap SMA+ cells and neovessels in plaques. In human, rat, and mouse intimal hyperplasia, PCSK6 was expressed by proliferating SMA+ cells and upregulated after 5 days in rat carotid balloon injury model, with positive correlation to PDGFB (platelet-derived growth factor subunit B) and MMP (matrix metalloprotease) 2/MMP14. Here, PCSK6 was shown to colocalize and cointeract with MMP2/MMP14 by in situ proximity ligation assay. Microarrays of carotid arteries from Pcsk6(-/-) versus control mice revealed suppression of contractile SMC markers, extracellular matrix remodeling enzymes, and cytokines/receptors. Pcsk6(-/-) mice showed reduced intimal hyperplasia response upon carotid ligation in vivo, accompanied by decreased MMP14 activation and impaired SMC outgrowth from aortic rings ex vivo. PCSK6 silencing in human SMCs in vitro leads to downregulation of contractile markers and increase in MMP2 expression. Conversely, PCSK6 overexpression increased PDGFBB (platelet-derived growth factor BB)-induced cell proliferation and particularly migration. Conclusions: PCSK6 is a novel protease that induces SMC migration in response to PDGFB, mechanistically via modulation of contractile markers and MMP14 activation. This study establishes PCSK6 as a key regulator of SMC function in vascular remodeling.
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| 3. |
- Röhl, Samuel, et al.
(author)
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Transcriptomic profiling of experimental arterial injury reveals new mechanisms and temporal dynamics in vascular healing response
- 2020
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In: JVS-Vascular Science. - : Elsevier BV. - 2666-3503. ; 315, s. E14-E14
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Journal article (peer-reviewed)abstract
- Objective: Endovascular interventions cause arterial injury and induce a healing response to restore vessel wall homeostasis. Complications of defective or excessive healing are common and result in increased morbidity and repeated interventions. Experimental models of intimal hyperplasia are vital for understanding the vascular healing mechanisms and resolving the clinical problems of restenosis, vein graft stenosis, and dialysis access failure. Our aim was to systematically investigate the transcriptional, histologic, and systemic reaction to vascular injury during a prolonged time. Methods: Balloon injury of the left common carotid artery was performed in male rats. Animals (n = 69) were euthanized before or after injury, either directly or after 2 hours, 20 hours, 2 days, 5 days, 2 weeks, 6 weeks, and 12 weeks. Both injured and contralateral arteries were subjected to microarray profiling, followed by bioinformatic exploration, histologic characterization of the biopsy specimens, and plasma lipid analyses. Results: Immune activation and coagulation were key mechanisms in the early response, followed by cytokine release, tissue remodeling, and smooth muscle cell modulation several days after injury, with reacquisition of contractile features in later phases. Novel pathways related to clonal expansion, inflammatory transformation, and chondro-osteogenic differentiation were identified and immunolocalized to neointimal smooth muscle cells. Analysis of uninjured arteries revealed a systemic component of the reaction after local injury, underlined by altered endothelial signaling, changes in overall tissue bioenergy metabolism, and plasma high-density lipoprotein levels. Conclusions: We demonstrate that vascular injury induces dynamic transcriptional landscape and metabolic changes identifiable as early, intermediate, and late response phases, reaching homeostasis after several weeks. This study provides a temporal “roadmap” of vascular healing as a publicly available resource for the research community.
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