Urszula Rykaczewska1, Bianca E Suur1, Samuel Röhl1, Anton Razuvaev1, Mariette Lengquist1, Maria Sabater-Lleal2,3, Sander W van der Laan4, Clint L Miller5,6, Robert C Wirka6, Malin Kronqvist1, Maria Gonzalez Diez2, Mattias Vesterlund7, Peter Gillgren8, Jacob Odeberg2,9, Jan H Lindeman10, Fabrizio Veglia11, Steve E Humphries12, Ulf de Faire13, Damiano Baldassarre11,14, Elena Tremoli11, Janne Lehtiö7, Göran K Hansson2, Gabrielle Paulsson-Berne2, Gerard Pasterkamp15, Thomas Quertermous6, Anders Hamsten2, Per Eriksson2, Ulf Hedin1, Ljubica Matic1. 1. From the Department of Molecular Medicine and Surgery, Karolinska Institute and Karolinska University Hospital Solna, Stockholm, Sweden (U.R., B.E.S., S.R., A.R., M.L., M.K., U.H., L.M.). 2. Department of Medicine Solna, Karolinska Institute and Karolinska University Hospital Solna, Stockholm, Sweden (M.S.-L., M.G.D., G.P.-B., G.K.H., A.H., P.E., J.O.). 3. Unit of Genomics of Complex Diseases, Institut de Recerca Hospital de Sant Pau (IIB-Sant Pau), Barcelona, Spain (M.S.-L.). 4. Central Diagnostics Laboratory, Laboratories, Pharmacy, and Biomedical Genetics, University Medical Center Utrecht, Utrecht University, The Netherlands (S.v.d.L.). 5. Center for Public Health Genomics, Department of Public Health Sciences, University of Virginia, Charlottesville (C.L.M.). 6. Division of Cardiovascular Medicine, Stanford University School of Medicine, CA (C.L.M., R.C.W., T.Q.). 7. Science for Life Laboratory, Department of Oncology-Pathology, Karolinska Institute, Sweden (M.V., J.L.). 8. Department of Clinical Science and Education, Södersjukhuset, Karolinska Institutet, and Department of Vascular Surgery, Södersjukhuset, Stockholm, Sweden (P.G.). 9. Science for Life Laboratory, Department of Proteomics, School of Chemistry Biotechnology and Health (CBH), KTH, Stockholm, Sweden (J.O.). 10. Department of Vascular Surgery, Leiden University Medical Center, The Netherlands (J.H.N.L.). 11. Centro Cardiologico Monzino, IRCCS, Milan, Italy (F.V., D.B., E.T.). 12. Cardiovascular Genetics, Institute Cardiovascular Science, University College of London, Department of Medicine, Rayne Building, United Kingdom (S.E.H.). 13. Division of Cardiovascular Epidemiology, Institute of Environmental Medicine, Karolinska Institutet, Sweden (H.d.F.). 14. Department of Medical Biotechnology and Translational Medicine, Università di Milano, Milan, Italy (D.B.). 15. Laboratory of Experimental Cardiology, Division Heart & Lungs, University Medical Center Utrecht, The Netherlands (G.P.).
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. Visual Overview: An online visual overview is available for this article.
RATIONALE: PCSKs (Proprotein convertase subtilisins/kexins) are a protease family with unknown functions in vasculature. Previously, we demonstrated PCSK6 upregulation in humanatherosclerotic 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. Visual Overview: An online visual overview is available for this article.
Authors: Tiffany R Bellomo; William P Bone; Brian Y Chen; Katerina A B Gawronski; David Zhang; Joseph Park; Michael Levin; Noah Tsao; Derek Klarin; Julie Lynch; Themistocles L Assimes; J Michael Gaziano; Peter W Wilson; Kelly Cho; Marijana Vujkovic; Christopher J O'Donnell; Kyong-Mi Chang; Philip S Tsao; Daniel J Rader; Marylyn D Ritchie; Scott M Damrauer; Benjamin F Voight Journal: Front Genet Date: 2022-02-02 Impact factor: 4.772
Authors: Bianca E Suur; Melody Chemaly; Moritz Lindquist Liljeqvist; Djordje Djordjevic; Markus Stenemo; Otto Bergman; Eva Karlöf; Mariette Lengquist; Jacob Odeberg; Eva Hurt-Camejo; Per Eriksson; Daniel F J Ketelhuth; Joy Roy; Ulf Hedin; Michael Nyberg; Ljubica Matic Journal: Front Pharmacol Date: 2022-09-15 Impact factor: 5.988