Background-Calcific aortic valve stenosis is a common disease in the elderly and is characterized by progressive calcification and fibrous thickening of the valve, but the cellular and molecular mechanisms are not fully understood. We hypothesized that human valve interstitial cells (ICs) are able to differentiate into osteoblast-like cells through the influence of defined mediators and that this process can be modulated pharmacologically. Methods and Results-To

Background-Calcific aortic valve disease is a common condition and is associated with inflammatory changes and expression of osteoblast-like cell phenotypes, but the cellular mechanisms are unclear. Recent studies identified extracellular ATP and P2Y receptor cascade as important regulators of bone remodeling, whereas its breakdown product, adenosine, is known to have anti-inflammatory properties. We hypothesize that extracellular ATP and adenosine have important roles in regulating osteoblast differentiation in human valve interstitial cells, and that this can be a potential target for therapy. Method and Results-Primary cultures of human valve interstitial cells (ICs) treated for 21 days with osteogenic media, ATP, and ATP-␥-S (a stable agonist of the P2Y receptor) revealed a significant increase in alkaline phosphatase (ALP) (an osteoblast marker) activity and expression as measured using spectrophotometric assay and immunocytochemistry staining. Valve ICs treated with adenosine alone did not cause an increase in ALP activity; however, adenosine treatment decreased the ALP activity and expression induced by osteogenic media after 21 days of incubation. In addition, atorvastatin inhibited the activity of ALP induced by ATP in human valve ICs, and enzyme studies revealed that atorvastatin upregulated the breakdown of extracellular ATP into adenosine in human valve ICs after 24-hour treatment. Conclusion-These

Background-Aortic valve calcification is a progressive process resembling ossification. Recent evidence indicates that the sympathetic nervous system plays an important role in regulating bone deposition and resorption through the ␤ 2 -adrenergic receptors (␤ 2 -ARs). The aim of this study is to determine the level and pattern of expression of

We recently discovered new nucleotides (4-pyridone-3-carboxamide-1-beta -D-ribonucleoside phosphates) in human erythrocytes. To establish the precursor compound and pathways of nucleotide derivative formation and breakdown, human erythrocytes were incubated for 3 hours with 0.3 mM 4-pyridone-3-carboxamide-1-beta-D-ribonucleoside (4PYR) and erythrocyte concentrations of 4PYR and adenine nucleotides were followed. 4PYR triphosphate increased from 16.1 +/- 0.6 micro M to 74.9 +/- 9.17 and 4PYR monophosphate increased from 5 micro M to 254.7 +/- 13.9 micro M. Conversely, incubation with 0.3 mM 4-pyridone-3-carboxamide (4PY) did not lead to additional 4PYR nucleotide formation. 4PYR nucleotides were catabolized to 4PYR. We conclude that 4PYR nucleotides are formed in erythrocytes by nucleoside kinase-mediated 4PYR phosphorylation and catabolized by 5'nucleotidase-mediated dephosphorylation.

Following discovery of NAD(+)-dependent reactions that control gene expression, cytoprotection, and longevity, there has been a renewed therapeutic interest in precursors, such as nicotinamide and its derivatives. We tested 20 analogues of nicotinamide for their ability to protect endothelial cells from peroxynitrite stress and their effect on poly (ADP-ribose) polymerase (PARP) activity. Several nicotinamide derivatives protected endothelial cells from peroxynitrite-induced depletion of cellular NAD(+) and ATP concentrations, but only some of these compounds inhibited PARP. We conclude that some nicotinamide derivatives provide protection of endothelial cells against peroxynitrite-induced injury independent of inhibition of PARP activity. Preservation of the NAD(+) pool was a common effect of these compounds.

Endothelial degradation of extracellular nucleotides is known to be an important mechanism in regulation of thrombosis, inflammation and immune response. It is possible that this pathway is a target for pleiotropic drugs such as atorvastatin. We studied therefore the effect of atorvastatin on extracellular nucleotide degradation in human endothelial cells. Atorvastatin treatment of human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC-1) resulted in significant increase in ATP breakdown and adenosine formation both if analysed in intact cell studies and as enzyme activity in cell lysates. We conclude that one of the beneficial effects of atorvastatin may include acceleration of extracellular nucleotide breakdown. This will attenuate nucleotide mediated pro-inflammatory effect and stimulate protective mechanisms of adenosine.

HMG-CoA reductase inhibitors (statins) exert pleiotropic effects in the cardiovascular system beyond its cholesterol-lowering action. We aimed to investigate how atorvastatin affects extracellular nucleotide degradation in human endothelial cells, as increased activity of this pathway would facilitate conversion of pro-inflammatory nucleotides into anti-inflammatory adenosine. Primary cultures of human endothelial cells were treated with 1 microM, 10 microM and 100 microM atorvastatin for 24 h. Enzyme assays were performed as well as intact cell studies, to evaluate capacity of cells to degrade ATP to adenosine. Atorvastatin significantly increased ATP breakdown and adenosine formation in the medium of intact cells in a dose-dependent manner. The activities of ATPase, ADPase and ecto-5'-nucleotidase (eN) in cell homogenates following Atorvastatin treatment were also increased while no change was observed in the lactate dehydrogenase activity. We suggest a new mechanism of protective effect of atorvastatin by activation of endothelial enzymes involved in extracellular nucleotide degradation in human endothelial cells.

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