Hyperphosphatemia, Phosphoprotein Phosphatases, and Microparticle Release in Vascular Endothelial Cells.
Hyperphosphatemia in patients with advanced CKD is thought to be an important contributor to cardiovascular risk, in part because of endothelial cell (EC) dysfunction induced by inorganic phosphate (Pi). Such patients also have an elevated circulating concentration of procoagulant endothelial microparticles (MPs), leading to a prothrombotic state, which may contribute to acute occlusive events. We hypothesized that hyperphosphatemia leads to MP formation from ECs through an elevation of intracellular Pi concentration, which directly inhibits phosphoprotein phosphatases, triggering a global increase in phosphorylation and cytoskeletal changes. In cultured human ECs (EAhy926), incubation with elevated extracellular Pi (2.5 mM) led to a rise in intracellular Pi concentration within 90 minutes. This was mediated by PiT1/slc20a1 Pi transporters and led to global accumulation of tyrosine- and serine/threonine-phosphorylated proteins, a marked increase in cellular Tropomyosin-3, plasma membrane blebbing, and release of 0.1- to 1-μm-diameter MPs. The effect of Pi was independent of oxidative stress or apoptosis. Similarly, global inhibition of phosphoprotein phosphatases with orthovanadate or fluoride yielded a global protein phosphorylation response and rapid release of MPs. The Pi-induced MPs expressed VE-cadherin and superficial phosphatidylserine, and in a thrombin generation assay, they displayed significantly more procoagulant activity than particles derived from cells incubated in medium with a physiologic level of Pi (1 mM). These data show a mechanism of Pi-induced cellular stress and signaling, which may be widely applicable in mammalian cells, and in ECs, it provides a novel pathologic link between hyperphosphatemia, generation of MPs, and thrombotic risk.
Departments of Infection, Immunity and Inflammation and.
Nima
N
Abbasian
Departments of Infection, Immunity and Inflammation and John Walls Renal Unit, University Hospitals of Leicester, Leicester, United Kingdom; and.
James O
JO
Burton
Cardiovascular Sciences, University of Leicester, Leicester, United Kingdom; Leicester National Institute for Health Research Cardiovascular Biomedical Research Unit Cardiovascular Sciences, University of Leicester, United Kingdom.
Karl E
KE
Herbert
Departments of Infection, Immunity and Inflammation and.
Barbara-Emily
BE
Tregunna
Departments of Infection, Immunity and Inflammation and John Walls Renal Unit, University Hospitals of Leicester, Leicester, United Kingdom; and.
Jeremy R
JR
Brown
Cardiovascular Sciences, University of Leicester, Leicester, United Kingdom; Leicester National Institute for Health Research Cardiovascular Biomedical Research Unit Cardiovascular Sciences, University of Leicester, United Kingdom.
Maryam
M
Ghaderi-Najafabadi
Departments of Infection, Immunity and Inflammation and John Walls Renal Unit, University Hospitals of Leicester, Leicester, United Kingdom; and.
Nigel J
NJ
Brunskill
Cardiovascular Sciences, University of Leicester, Leicester, United Kingdom; Leicester National Institute for Health Research Cardiovascular Biomedical Research Unit Cardiovascular Sciences, University of Leicester, United Kingdom.
Alison H
AH
Goodall
Departments of Infection, Immunity and Inflammation and ab74@leicester.ac.uk.
Alan
A
Bevington
D002457:Cell Extracts / Q000737:chemistry; D055252:Cell-Derived Microparticles / Q000378:metabolism*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000201:enzymology / Q000378:metabolism*; D005459:Fluorides / Q000494:pharmacology; D006801:Humans; D054559:Hyperphosphatemia / Q000201:enzymology / Q000378:metabolism*; D028061:Phosphate Transport Proteins / Q000378:metabolism; D010710:Phosphates / Q000032:analysis / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010766:Phosphorylation / Q000187:drug effects; D015398:Signal Transduction; D014335:Tropomyosin / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D002457:Cell Extracts; D028061:Phosphate Transport Proteins; D010710:Phosphates; C498783:TPM3 protein, human; D014335:Tropomyosin; D014638:Vanadates; D010749:Phosphoprotein Phosphatases; D005459:Fluorides
CKD; cardiovascular disease; cell signaling; endothelial cells; hyperphosphatemia; microparticle