Abstract
Extracellular nucleotide catabolism contributes to immunomodulation, cell differentiation and tissue mineralization by controlling nucleotide and adenosine concentrations and its purinergic effects. Disturbances of purinergic signaling in valves may lead to its calcification. This study aimed to investigate the side-specific changes in extracellular nucleotide and adenosine metabolism in the aortic valve during calcific aortic valve disease (CAVD) and to identify the individual enzymes that are involved in these pathways as well as their cellular origin.
Stenotic aortic valves were characterized by reduced levels of extracellular ATP removal and impaired production of adenosine. Respectively, already reduced levels of extracellular adenosine were immediately degraded further due to the elevated rate of adenosine deamination. For the first time, we revealed that this metabolic pattern was observed only on the fibrosa surface of stenotic valve that is consistent with the mineral deposition on the aortic side of the valve. Furthermore, we demonstrated that non-stenotic valves expressed mostly ecto-nucleoside triphosphate diphosphohydrolase 1 (eNTPD1) and ecto-5’nucleotidase (e5NT), while stenotic valves ecto-nucleotide pyrophosphatase/ phosphodiesterase 1, alkaline phosphatase and ecto-adenosine deaminase (eADA). On the surface of endothelial cells, isolated from non-stenotic valves, high activities of eNTPD1 and e5NT were found. Whereas, in valvular interstitial cells, eNPP1 activity was also detected. Stenotic valve immune infiltrate was an additional source of eADA. We demonstrated the presence of A1, A2a and A2b adenosine receptors in both, non-stenotic and stenotic valves with diminished expression of A2a and A2b in the former.
Extracellular nucleotide and adenosine metabolism that involves complex ecto-enzyme pathways and adenosine receptor signaling were adversely modified in CAVD. In particular, diminished activities of eNTPD1 and e5NT with the increase in eADA that originated from valvular endothelial and interstitial cells as well as from immune inflitrate may affect aortic valve extracellular nucleotide concentrations to favor a proinflammatory milieu, highlighting a potential mechanism and target for CAVD therapy.
Abbreviations
- α-SMA
- smooth muscle cells alpha actin
- A1R
- adenosine A1 receptor
- A2aR
- adenosine A2a receptor
- A2bR
- adenosine A2b receptor
- A3R
- adenosine A3 receptor
- ADP
- adenosine diphosphate
- AMP
- adenosine monophosphate
- AOPCP
- 5’-(α,β-methylene)diphosphate
- AP
- alkaline phosphatase
- ARL67156
- 6-N,N-Diethyl-β-γ-dibromomethylene-D-adenosine-5’-triphosphate trisodium salt hydrate
- ATP
- adenosine triphosphate
- aVIC
- activated valvular interstitial cells
- AVR
- aortic valve replacement
- CAVD
- calcific aortic valve disease
- e5NT
- ecto-5’nucleotidase
- eADA
- ecto-adenosine deaminase
- EHNA
- erythro-9-(2-hydroxy-3-nonyl)adenine
- eNPP1
- ecto-nucleotide pyrophosphatase/phosphodiesterase 1
- eNPPs
- ecto-nucleotide pyrophosphatases/ phosphodiesterases
- eNTPD1
- ecto-nucleoside triphosphate diphosphohydrolase 1
- FBS
- fetal bovine serum
- HBSS
- Hanks Balanced Salt Solution
- HE
- hematoxylin and eosin staining
- HPLC
- high performance liquid chromatography
- VEC
- aortic valve endothelial cells
- hVIC
- aortic valve interstitial cells
- LDL
- low density lipoproteins
- NBTI
- S-(4-Nitrobenzyl)-6-thioinosine
- obVIC
- osteoblast-like valvular interstitial cells
- OMSB
- Orcein Martius Scarlet Blue staining
- PBMC
- peripheral blood mononuclear cells
- PBS
- phosphate buffered saline
- Pi
- inorganic phosphate
- PPADS
- pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt hydrate
- PPi
- inorganic pyrophosphate
- qVIC
- quiescent valvular interstitial cells
- TAVI
- transcatheter aortic valve implantation
- TNAP
- tissue nonspecific alkaline phosphatase
- TR
- Masson’s Trichrome staining
- VSMC
- vascular smooth muscle cells
- vWF
- von Wilebrant factor