scholarly journals Hydrolysis of Extracellular ATP by Vascular Smooth Muscle Cells Transdifferentiated into Chondrocytes Generates Pi but Not PPi

2021 ◽  
Vol 22 (6) ◽  
pp. 2948
Author(s):  
Rene Buchet ◽  
Camille Tribes ◽  
Valentine Rouaix ◽  
Bastien Doumèche ◽  
Michele Fiore ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Atp Hydrolysis ◽  
Muscle Cells ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Extracellular Medium ◽  
Inorganic Pyrophosphate

(1) Background: Tissue non-specific alkaline phosphatase (TNAP) is suspected to induce atherosclerosis plaque calcification. TNAP, during physiological mineralization, hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PPi). Since atherosclerosis plaques are characterized by the presence of necrotic cells that probably release supraphysiological concentrations of ATP, we explored whether this extracellular adenosine triphosphate (ATP) is hydrolyzed into the mineralization inhibitor PPi or the mineralization stimulator inorganic phosphate (Pi), and whether TNAP is involved. (2) Methods: Murine aortic smooth muscle cell line (MOVAS cells) were transdifferentiated into chondrocyte-like cells in calcifying medium, containing ascorbic acid and β-glycerophosphate. ATP hydrolysis rates were determined in extracellular medium extracted from MOVAS cultures during their transdifferentiation, using 31P-NMR and IR spectroscopy. (3) Results: ATP and PPi hydrolysis by MOVAS cells increased during transdifferentiation. ATP hydrolysis was sequential, yielding adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine without any detectable PPi. The addition of levamisole partially inhibited ATP hydrolysis, indicating that TNAP and other types of ectonucleoside triphoshatediphosphohydrolases contributed to ATP hydrolysis. (4) Conclusions: Our findings suggest that high ATP levels released by cells in proximity to vascular smooth muscle cells (VSMCs) in atherosclerosis plaques generate Pi and not PPi, which may exacerbate plaque calcification.

scholarly journals Autocrine ATP release coupled to extracellular pyrophosphate accumulation in vascular smooth muscle cells

2009 ◽  
Vol 296 (4) ◽  
pp. C828-C839 ◽  
Author(s):  
Domenick A. Prosdocimo ◽  
Dezmond C. Douglas ◽  
Andrea M. Romani ◽  
W. Charles O'Neill ◽  
George R. Dubyak
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Atp Release ◽  
Brefeldin A ◽  
Muscle Cells ◽  
Rat Aorta ◽  
Inorganic Pyrophosphate ◽  
Inhibition Of Proliferation

Extracellular inorganic pyrophosphate (PPi) is a potent suppressor of physiological calcification in bone and pathological calcification in blood vessels. Ectonucleotide pyrophosphatase/phosphodiesterases (eNPPs) generate PPi via the hydrolysis of ATP released into extracellular compartments by poorly understood mechanisms. Here we report that cultured vascular smooth muscle cells (VSMC) from rat aorta generate extracellular PPi via an autocrine mechanism that involves ATP release tightly coupled to eNPP activity. The nucleotide analog β,γ-methylene ATP (MeATP or AMPPCP) was used to selectively suppress ATP metabolism by eNPPs but not the CD39-type ecto-ATPases. In the absence of MeATP, VSMC generated extracellular PPi to accumulate ≥600 nM within 2 h while steadily maintaining extracellular ATP at 1 nM. Conversely, the presence of MeATP completely suppressed PPi accumulation while increasing ATP accumulation. Probenecid, which inhibits PPi efflux dependent on ANK, a putative PPi transporter or transport regulator, reduced extracellular PPi accumulation by approximately twofold. This indicates that autocrine ATP release coupled to eNPP activity comprises ≥50% of the extracellular PPi-generating capacity of VSMC. The accumulation of extracellular PPi and ATP was markedly attenuated by reduced temperature but was insensitive to brefeldin A, which suppresses constitutive exocytosis of Golgi-derived secretory vesicles. The magnitude of extracellular PPi accumulation in VSMC cultures increased with time postplating, suggesting that ATP release coupled to PPi generation is upregulated as cultured VSMC undergo contact-inhibition of proliferation or deposit extracellular matrix.

Abstract P292: ENPP1-Fc Protein Inhibits Proliferation of Human Vascular Smooth Muscle Cells

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Yan Yan ◽  
Anumeha Shah ◽  
Ashmita Saigal ◽  
Susan Faas ◽  
Zhiliang Cheng ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Cellular Proliferation ◽  
Fold Increase ◽  
Muscle Cells ◽  
Arterial Stenosis ◽  
Arterial Calcification ◽  
Inorganic Pyrophosphate

Background: Arterial stenosis leading to hypertension or heart failure is common in patients with Generalized Arterial Calcification of Infancy (GACI), an ultra-rare disease associated with loss of function mutations in ENPP1, an ectonucleotide pyrophosphatase that hydrolyzes extracellular ATP. GACI is characterized by accelerated calcification and severe myointimal proliferation. The role of ENPP1 in myoinitimal proliferation is unknown. Here, we examined the effect of ENPP1 on proliferation of human vascular smooth muscle cells (hVSMCs). Methods: ENPP1 expression was assessed in primary hVSMCs using qRT-PCR. ENPP1 expression was silenced using siRNA and its activity was verified by a cell based assay or inorganic pyrophosphate (PPi) assay and its effect on VSMC proliferation was determined by 3H-thymidine incorporation. Results: Treatment of primary hVSMCs with siRNA specific to the hENPP1 resulted in ~90% decrease in ENPP1 levels. Cellular enzyme activity correlated with ENPP1 expression in VSMCs. Silencing ENPP1 in hVSMCs led to 1-3 fold increase in proliferation relative to that of cells transfected with control siRNA in 2 out of 2 donors. Peak cell proliferation was observed at 5 days post-transfection. Human iPSC derived VSMCs (iVSMCs) expressed higher levels of ENPP1 than primary hVSMCs. Silencing ENPP1 in iVSMCs resulted in 3-5 fold increase in proliferation relative to that of cells transfected with negative control siRNA in 2 out of 2 donors. Addition of recombinant ENPP1-Fc protein restored ENPP1- associated proliferation in all donors (8099.75 ±134.32 (untreated) vs 1478 ± 55.34 (5ug/ml ENPP1-Fc treated, P<0.001)). In contrast, bisphosphonates, a current off-label treatment for patients with GACI, had no effect on cellular proliferation. Increased levels of PPi were also detected in culture supernatants obtained from cells treated with the ENPP1-Fc protein. Conclusion: For the first time, we have demonstrated that ENPP1 knockdown promotes proliferation of human VSMCs, and treatment with a functionally active ENPP1-Fc protein significantly inhibits ENPP1-associated proliferation. These results suggest that ENPP1 enzyme replacement may be a potential strategy to treat myointimal proliferation in patients with GACI disease.

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