Maria Giovanna Lupo1, Alessandro Bressan1, Maristella Donato2, Paola Canzano3, Marina Camera4, Paolo Poggio3, Maria Francesca Greco5, Mariangela Garofalo2, Sara De Martin2, Giovanni Panighel2, Massimiliano Ruscica5, Andrea Baragetti5, Valentina Bollati6, Elisabetta Faggin1, Marcello Rattazzi7, Alberico L Catapano8, Nicola Ferri9. 1. Dipartimento di Medicina, Università degli Studi di Padova, Padua, Italy. 2. Dipartimento di Scienze del Farmaco, Università degli Studi di Padova, Padua, Italy. 3. Centro Cardiologico Monzino IRCCS, Milan, Italy. 4. Centro Cardiologico Monzino IRCCS, Milan, Italy; Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Milan, Italy. 5. Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan, Italy. 6. EPIGET Department of Clinical Sciences and Co mMunity Health, Università degli Studi di Milano, Milan, Italy; Dipartimento di Scienze Cliniche e di Comunità, Università degli Studi di Milano, Milan, Italy. 7. Dipartimento di Medicina, Università degli Studi di Padova, Padua, Italy; Medicina Interna I^, Cà Foncello Hospital, Treviso, Italy. 8. Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan, Italy; IRCCS Multimedica Hospital, Milan, Italy. Electronic address: alberico.catapano@unimi.it. 9. Dipartimento di Medicina, Università degli Studi di Padova, Padua, Italy. Electronic address: nicola.ferri@unipd.it.
Abstract
BACKGROUND AND AIMS: A complex interplay among chronic kidney disease (CKD), lipid metabolism and aortic calcification has been recognized. Here we investigated the influence of kidney function on PCSK9 levels and its potential direct action on smooth muscle cells (SMCs) calcification. METHODS AND RESULTS: In a cohort of 594 subjects, a negative association between glomerular filtration rate and plasma PCSK9 was found. Atherosclerotic cardiovascular disease, as co-morbidity, further increased PCSK9 plasma levels. Diet-induced uremic condition in rats led to aortic calcification and increased total cholesterol and Pcsk9 levels in plasma, livers, and kidneys. Both human and rat SMCs overexpressing human PCSK9 (SMCsPCSK9), cultured in a pro-calcific environment (2.0 mM or 2.4 mM inorganic phosphate, Pi) showed a significantly higher extracellular calcium (Ca2+) deposition compared to control SMCs. The addition of recombinant human PCSK9 did not increase the extracellular calcification of SMCs, suggesting the involvement of intracellular PCSK9. Accordingly, the further challenge with evolocumab did not affect calcium deposition in hSMCsPCSK9. Under pro-calcific conditions, SMCsPCSK9 released a higher number of extracellular vesicles (EVs) positive for three tetraspanin molecules, such as CD63, CD9, and CD81. EVs derived from SMCsPCSK9 tended to be more enriched in calcium and alkaline phosphatase (ALP), compared to EVs from control SMCs. In addition, PCSK9 has been detected in SMCsPCSK9-derived EVs. Finally, SMCsPCSK9 showed an increase in pro-calcific markers, namely bone morphogenetic protein 2 and ALP, and a decrease in anti-calcific mediator matrix GLA protein and osteopontin. CONCLUSIONS: Our study reveals a direct role of PCSK9 on vascular calcification induced by higher inorganic phosphate levels associated with renal impairment. This effect appears to be mediated by a switch towards a pro-calcific phenotype of SMCs associated with the release of EVs containing Ca2+ and ALP.
BACKGROUND AND AIMS: A complex interplay among chronic kidney disease (CKD), lipid metabolism and aortic calcification has been recognized. Here we investigated the influence of kidney function on PCSK9 levels and its potential direct action on smooth muscle cells (SMCs) calcification. METHODS AND RESULTS: In a cohort of 594 subjects, a negative association between glomerular filtration rate and plasma PCSK9 was found. Atherosclerotic cardiovascular disease, as co-morbidity, further increased PCSK9 plasma levels. Diet-induced uremic condition in rats led to aortic calcification and increased total cholesterol and Pcsk9 levels in plasma, livers, and kidneys. Both human and rat SMCs overexpressing human PCSK9 (SMCsPCSK9), cultured in a pro-calcific environment (2.0 mM or 2.4 mM inorganic phosphate, Pi) showed a significantly higher extracellular calcium (Ca2+) deposition compared to control SMCs. The addition of recombinant human PCSK9 did not increase the extracellular calcification of SMCs, suggesting the involvement of intracellular PCSK9. Accordingly, the further challenge with evolocumab did not affect calcium deposition in hSMCsPCSK9. Under pro-calcific conditions, SMCsPCSK9 released a higher number of extracellular vesicles (EVs) positive for three tetraspanin molecules, such as CD63, CD9, and CD81. EVs derived from SMCsPCSK9 tended to be more enriched in calcium and alkaline phosphatase (ALP), compared to EVs from control SMCs. In addition, PCSK9 has been detected in SMCsPCSK9-derived EVs. Finally, SMCsPCSK9 showed an increase in pro-calcific markers, namely bone morphogenetic protein 2 and ALP, and a decrease in anti-calcific mediator matrix GLA protein and osteopontin. CONCLUSIONS: Our study reveals a direct role of PCSK9 on vascular calcification induced by higher inorganic phosphate levels associated with renal impairment. This effect appears to be mediated by a switch towards a pro-calcific phenotype of SMCs associated with the release of EVs containing Ca2+ and ALP.
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