title: Pharmacological inhibition of PHOSPHO1 suppresses vascular smooth muscle cell calcification.
abstract: Medial vascular calcification (MVC) is common in patients with chronic kidney disease, obesity, and aging. MVC is an actively regulated process that resembles skeletal mineralization, resulting from chondro-osteogenic transformation of vascular smooth muscle cells (VSMCs). Here, we used mineralizing murine VSMCs to study the expression of PHOSPHO1, a phosphatase that participates in the first step of matrix vesicles-mediated initiation of mineralization during endochondral ossification. Wild-type (WT) VSMCs cultured under calcifying conditions exhibited increased Phospho1 gene expression and Phospho1(-/-) VSMCs failed to mineralize in vitro. Using natural PHOSPHO1 substrates, potent and specific inhibitors of PHOSPHO1 were identified via high-throughput screening and mechanistic analysis and two of these inhibitors, designated MLS-0390838 and MLS-0263839, were selected for further analysis. Their effectiveness in preventing VSMC calcification by targeting PHOSPHO1 function was assessed, alone and in combination with a potent tissue-nonspecific alkaline phosphatase (TNAP) inhibitor MLS-0038949. PHOSPHO1 inhibition by MLS-0263839 in mineralizing WT cells (cultured with added inorganic phosphate) reduced calcification in culture to 41.8% ± 2.0% of control. Combined inhibition of PHOSPHO1 by MLS-0263839 and TNAP by MLS-0038949 significantly reduced calcification to 20.9% ± 0.74% of control. Furthermore, the dual inhibition strategy affected the expression of several mineralization-related enzymes while increasing expression of the smooth muscle cell marker Acta2. We conclude that PHOSPHO1 plays a critical role in VSMC mineralization and that "phosphatase inhibition" may be a useful therapeutic strategy to reduce MVC.
authors.affiliation: Sanford Children's Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA.
authors.forename: Tina
authors.initials: T
authors.lastname: Kiffer-Moreira
authors.forename: Manisha C
authors.initials: MC
authors.lastname: Yadav
authors.forename: Dongxing
authors.initials: D
authors.lastname: Zhu
authors.forename: Sonoko
authors.initials: S
authors.lastname: Narisawa
authors.forename: Campbell
authors.initials: C
authors.lastname: Sheen
authors.forename: Boguslaw
authors.initials: B
authors.lastname: Stec
authors.forename: Nicholas D
authors.initials: ND
authors.lastname: Cosford
authors.forename: Russell
authors.initials: R
authors.lastname: Dahl
authors.forename: Colin
authors.initials: C
authors.lastname: Farquharson
authors.forename: Marc F
authors.initials: MF
authors.lastname: Hoylaerts
authors.forename: Vicky E
authors.initials: VE
authors.lastname: Macrae
authors.forename: José Luis
authors.initials: JL
authors.lastname: Millán
mesh_terms: D053799:2-Pyridinylmethylsulfinylbenzimidazoles / Q000494:pharmacology; D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D002113:Calcification, Physiologic / Q000187:drug effects; D020134:Catalytic Domain; D003198:Computer Simulation; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D020128:Inhibitory Concentration 50; D064747:Lansoprazole; D051379:Mice; D008958:Models, Molecular; D009131:Muscle, Smooth, Vascular / Q000473:pathology*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology*; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000172:deficiency / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000187:drug effects; D061205:Vascular Calcification / Q000201:enzymology* / Q000473:pathology*
chemical_list: D053799:2-Pyridinylmethylsulfinylbenzimidazoles; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D064747:Lansoprazole; C552022:PHOSPHO1 protein, mouse; D000469:Alkaline Phosphatase; D010744:Phosphoric Monoester Hydrolases