Nucleotide depletion in hypoxia experimental models of mouse myocardial slices.

OBJECTIVES: Experimental models to test the effective protection against cardiac ischemia injury are still challenging in pre-clinical studies. The use of myocardial slices creates a special link between testing isolated cardiomyocytes and whole-heart research. In this work, we investigated the effects of oxygen deprivation in a hypoxic chamber and treatment with cobalt chloride (CoCl2) on the nucleotide profile in isolated mouse myocardial slices. METHODS: 200 µm-thick left ventricle myocardial slices were obtained from 3-month-old male C57Bl/6J mice using an oscillatory microtome. Slices were then exposed to 1% O2 atmosphere or 100 µM CoCl2 at 37 °C for 45 min and used for nucleotide measurements using ultra-high-performance liquid chromatography. The effects of two short-term experimental models of hypoxia were compared to 2'-deoxyglucose with oligomycin (2-DG + OLIGO) treatment, which inhibited both glycolysis and mitochondrial ATP synthesis. KEY FINDINGS: A significant effect of hypoxia with 1% O2 was observed on adenosine triphosphate (ATP) and total adenine nucleotide (TAN) concentrations as well as on adenylate energy charge (AEC), ATP/ADP and ATP/AMP ratios. Oxygen deprivation caused changes almost as profound as 2-DG + OLIGO, emphasizing the critical role of mitochondrial oxidative phosphorylation in the energy metabolism of cultured heart slices. CoCl2 treatment that elicits hypoxia-like responses via HIF-1α stabilization only slightly affected nucleotide levels. This suggests that mechanisms induced by cobalt ions require more time to change the cardiac energy metabolism. CONCLUSIONS: A short-term culture of myocardial slices in a hypoxic chamber seems to be an appropriate model of cardiac ischemia for testing new pharmacological approaches based on modulating the energy metabolism of cardiac cells.

Editorial for the Special Issue Titled "Adenosine Metabolism: Key Targets in Cardiovascular Pharmacology".

Adenine nucleotides and adenosine maintain cardiovascular homeostasis, producing diverse effects by intracellular and extracellular mechanisms [...].

Red Blood Cell Adenylate Energetics Is Related to Endothelial and Microvascular Function in Long COVID.

Adenine nucleotides play a critical role in maintaining essential functions of red blood cells (RBCs), including energy metabolism, redox status, shape fluctuations and RBC-dependent endothelial and microvascular functions. Recently, it has been shown that infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) might lead to morphological and metabolic alterations in erythrocytes in both mild and severe cases of coronavirus disease (COVID-19). However, little is known about the effects of COVID-19 on the nucleotide energetics of RBCs nor about the potential contribution of nucleotide metabolism to the long COVID syndrome. This study aimed to analyze the levels of adenine nucleotides in RBCs isolated from patients 12 weeks after mild SARS-CoV-2 infection who suffered from long COVID symptoms and to relate them with the endothelial and microvascular function parameters as well as the rate of peripheral tissue oxygen supply. Although the absolute quantities of adenine nucleotides in RBCs were rather slightly changed in long COVID individuals, many parameters related to the endothelial and microcirculatory function showed significant correlations with RBC adenosine triphosphate (ATP) and total adenine nucleotide (TAN) concentration. A particularly strong relationship was observed between ATP in RBCs and the serum ratio of arginine to asymmetric dimethylarginine-an indicator of endothelial function. Consistently, a positive correlation was also observed between the ATP/ADP ratio and diminished reactive hyperemic response in long COVID patients, assessed by the flow-mediated skin fluorescence (FMSF) technique, which reflected decreased vascular nitric oxide bioavailability. In addition, we have shown that patients after COVID-19 have significantly impaired ischemic response parameters (IR max and IR index), examined by FMSF, which revealed diminished residual bioavailability of oxygen in epidermal keratinocytes after brachial artery occlusion. These ischemic response parameters revealed a strong positive correlation with the RBC ATP/ADP ratio, confirming a key role of RBC bioenergetics in peripheral tissue oxygen supply. Taken together, the outcomes of this study indicate that dysregulation of metabolic processes in erythrocytes with the co-occurring endothelial and microvascular dysfunction is associated with diminished intracellular oxygen delivery, which may partly explain long COVID-specific symptoms such as physical impairment and fatigue.

Endothelial Effects of Simultaneous Expression of Human HO-1, E5NT, and ENTPD1 in a Mouse.

The vascular endothelium is key target for immune and thrombotic responses that has to be controlled in successful xenotransplantation. Several genes were identified that, if induced or overexpressed, help to regulate the inflammatory response and preserve the transplanted organ function and metabolism. However, few studies addressed combined expression of such genes. The aim of this work was to evaluate in vivo the effects of the simultaneous expression of three human genes in a mouse generated using the multi-cistronic F2A technology. Male 3-month-old mice that express human heme oxygenase 1 (hHO-1), ecto-5'-nucleotidase (hE5NT), and ecto-nucleoside triphosphate diphosphohydrolase 1 (hENTPD1) (Transgenic) were compared to wild-type FVB mice (Control). Background analysis include extracellular nucleotide catabolism enzymes profile on the aortic surface, blood nucleotide concentration, and serum L-arginine metabolites. Furthermore, inflammatory stress induced by LPS in transgenic and control mice was used to characterize interleukin 6 (IL-6) and adhesion molecules endothelium permeability responses. Transgenic mice had significantly higher rates of extracellular adenosine triphosphate and adenosine monophosphate hydrolysis on the aortic surface in comparison to control. Increased levels of blood AMP and adenosine were also noticed in transgenics. Moreover, transgenic animals demonstrated the decrease in serum monomethyl-L-arginine level and a higher L-arginine/monomethyl-L-arginine ratio. Importantly, significantly decreased serum IL-6, and adhesion molecule levels were observed in transgenic mice in comparison to control after LPS treatment. Furthermore, reduced endothelial permeability in the LPS-treated transgenic mice was noted as compared to LPS-treated control. The human enzymes (hHO-1, hE5NT, hENTPD1) simultaneously encoded in transgenic mice demonstrated benefits in several biochemical and functional aspects of endothelium. This is consistent in use of this approach in the context of xenotransplantation.

Changes in Adenosine Deaminase Activity and Endothelial Dysfunction after Mild Coronavirus Disease-2019.

Endothelial cells are a preferential target for SARS-CoV-2 infection. Previously, we have reported that vascular adenosine deaminase 1 (ADA1) may serve as a biomarker of endothelial activation and vascular inflammation, while ADA2 plays a critical role in monocyte and macrophage function. In this study, we investigated the activities of circulating ADA isoenzymes in patients 8 weeks after mild COVID-19 and related them to the parameters of inflammation and microvascular/endothelial function. Post-COVID patients revealed microvascular dysfunction associated with the changes in circulating parameters of endothelial dysfunction and inflammatory activation. Interestingly, serum total ADA and ADA2 activities were diminished in post-COVID patients, while ADA1 remained unchanged in comparison to healthy controls without a prior diagnosis of SARS-CoV-2 infection. While serum ADA1 activity tended to positively correspond with the parameters of endothelial activation and inflammation, sICAM-1 and TNFα, serum ADA2 activity correlated with IL-10. Simultaneously, post-COVID patients had lower circulating levels of ADA1-anchoring protein, CD26, that may serve as an alternative receptor for virus binding. This suggests that after the infection CD26 is rather maintained in cell-attached form, enabling ADA1 complexing. This study points to the possible role of ADA isoenzymes in cardiovascular complications after mild COVID-19.

Drugs targeting adenosine signaling pathways: A current view.

Adenosine is an endogenous nucleoside that regulates many physiological and pathological processes. It is derived from either the intracellular or extracellular dephosphorylation of adenosine triphosphate and interacts with cell-surface G-protein-coupled receptors. Adenosine plays a substantial role in protecting against cell damage in areas of increased tissue metabolism and preventing organ dysfunction in pathological states. Targeting adenosine metabolism and receptor signaling may be an effective therapeutic approach for human diseases, including cardiovascular and central nervous system disorders, rheumatoid arthritis, asthma, renal diseases, and cancer. Several lines of evidence have shown that many drugs exert their beneficial effects by modulating adenosine signaling pathways but this knowledge urgently needs to be summarized, and most importantly, actualized. The present review collects pharmaceuticals and pharmacological or diagnostic tools that target adenosine signaling in their primary or secondary mode of action. We overviewed FDA-approved drugs as well as those currently being studied in clinical trials. Among them are already used in clinic A2A adenosine receptor modulators like istradefylline or regadenoson, but also plenty of anti-platelet, anti-inflammatory, or immunosuppressive, and anti-cancer drugs. On the other hand, we investigated dozens of specific adenosine pathway regulators that are tested in clinical trials to treat human infectious and noninfectious diseases. In conclusion, targeting purinergic signaling represents a great therapeutic challenge. The actual knowledge of the involvement of adenosinergic signaling as part of the mechanism of action of old drugs has open a path not only for drug-repurposing but also for new therapeutic strategies.

Cardiac Mitochondria Dysfunction in Dyslipidemic Mice.

Dyslipidemia triggers many severe pathologies, including atherosclerosis and chronic inflammation. Several lines of evidence, including our studies, have suggested direct effects of dyslipidemia on cardiac energy metabolism, but details of these effects are not clear. This study aimed to investigate how mild dyslipidemia affects cardiac mitochondria function and vascular nucleotide metabolism. The analyses were performed in 3- and 6-month-old knock-out mice for low-density lipoprotein receptor (Ldlr-/-) and compared to wild-type C57Bl/6J mice (WT). Cardiac isolated mitochondria function was analyzed using Seahorse metabolic flux analyzer. The mechanical function of the heart was measured using echocardiography. The levels of fusion, fission, and mitochondrial biogenesis proteins were determined by ELISA kits, while the cardiac intracellular nucleotide concentration and vascular pattern of nucleotide metabolism ecto-enzymes were analyzed using reverse-phase high-performance liquid chromatography. We revealed the downregulation of mitochondrial complex I, together with a decreased activity of citrate synthase (CS), reduced levels of nuclear respiratory factor 1 and mitochondrial fission 1 protein, as well as lower intracellular adenosine and guanosine triphosphates' pool in the hearts of 6-month Ldlr-/- mice vs. age-matched WT. The analysis of vascular ecto-enzyme pattern revealed decreased rate of extracellular adenosine monophosphate hydrolysis and increased ecto-adenosine deaminase activity (eADA) in 6-month Ldlr-/- vs. WT mice. No changes were observed in echocardiography parameters in both age groups of Ldlr-/- mice. Younger hyperlipidemic mice revealed no differences in cardiac mitochondria function, CS activity, intracellular nucleotides, mitochondrial biogenesis, and dynamics but exhibited minor changes in vascular eADA activity vs. WT. This study revealed that dysfunction of cardiac mitochondria develops during prolonged mild hyperlipidemia at the time point corresponding to the formation of early vascular alterations.

The impaired distribution of adenosine deaminase isoenzymes in multiple sclerosis plasma and cerebrospinal fluid.

Background: Adenosine deaminase (ADA) via two isoenzymes, ADA1 and ADA2, regulates intra- and extracellular adenosine concentrations by converting it to inosine. In the central nervous system (CNS), adenosine modulates the processes of neuroinflammation and demyelination that together play a critical role in the pathophysiology of multiple sclerosis (MS). Except for their catalytic activities, ADA isoenzymes display extra-enzymatic properties acting as an adhesion molecule or a growth factor. Aims: This study aimed to explore the distribution and activity of ADA1 and ADA2 in the plasma and the CSF of MS patients as well as in the human brain microvascular endothelial cells (HBMEC), human brain vascular pericytes and human astrocytes. Methods and results: The enzyme assay following reverse phase-high performance liquid chromatography (HPLC) analysis was used to detect the ADA1 and ADA2 activities and revealed an increased ratio of ADA1 to ADA2 in both the plasma and the CSF of MS patients. Plasma ADA1 activity was significantly induced in MS, while ADA2 was decreased in the CSF, but significance was not reached. The brain astrocytes, pericytes and endothelial cells revealed on their surface the activity of ADA1, with its basal level being five times higher in the endothelial cells than in the astrocytes or the pericytes. In turn, ADA2 activity was only observed in pericytes and endothelial cells. Stimulation of the cells with pro-inflammatory cytokines TNFα/IL17 for 18 h decreased intracellular nucleotide levels measured by HPLC only in pericytes. The treatment with TNFα/IL17 did not modulate cell-surface ATP and AMP hydrolysis nor adenosine deamination in pericytes or astrocytes. Whereas in endothelial cells it downregulated AMP hydrolysis and ADA2 activity and upregulated the ADA1, which reflects the ADA isoenzyme pattern observed here in the CSF of MS patients. Conclusion: In this study, we determined the impaired distribution of both ADA isoenzymes in the plasma and the CSF of patients with MS. The increased ADA1 to ADA2 ratio in the CSF and plasma may translate to unfavorable phenotype that triggers ADA1-mediated pro-inflammatory mechanisms and decreases ADA2-dependent neuroprotective and growth-promoting effects in MS.

CoCl2-Mimicked Endothelial Cell Hypoxia Induces Nucleotide Depletion and Functional Impairment That Is Reversed by Nucleotide Precursors.

Chronic hypoxia drives vascular dysfunction by various mechanisms, including changes in mitochondrial respiration. Although endothelial cells (ECs) rely predominantly on glycolysis, hypoxia is known to alter oxidative phosphorylation, promote oxidative stress and induce dysfunction in ECs. Our work aimed to analyze the effects of prolonged treatment with hypoxia-mimetic agent CoCl2 on intracellular nucleotide concentration, extracellular nucleotide breakdown, mitochondrial function, and nitric oxide (NO) production in microvascular ECs. Moreover, we investigated how nucleotide precursor supplementation and adenosine deaminase inhibition protected against CoCl2-mediated disturbances. Mouse (H5V) and human (HMEC-1) microvascular ECs were exposed to CoCl2-mimicked hypoxia for 24 h in the presence of nucleotide precursors: adenine and ribose, and adenosine deaminase inhibitor, 2'deoxycoformycin. CoCl2 treatment decreased NO production by ECs, depleted intracellular ATP concentration, and increased extracellular nucleotide and adenosine catabolism in both H5V and HMEC-1 cell lines. Diminished intracellular ATP level was the effect of disturbed mitochondrial phosphorylation, while nucleotide precursors effectively restored the ATP pool via the salvage pathway and improved endothelial function under CoCl2 treatment. Endothelial protective effects of adenine and ribose were further enhanced by adenosine deaminase inhibition, that increased adenosine concentration. This work points to a novel strategy for protection of hypoxic ECs by replenishing the adenine nucleotide pool and promoting adenosine signaling.

4-Pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR)-A Novel Oncometabolite Modulating Cancer-Endothelial Interactions in Breast Cancer Metastasis.

The accumulation of specific metabolic intermediates is known to promote cancer progression. We analyzed the role of 4-pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR), a nucleotide metabolite that accumulates in the blood of cancer patients, using the 4T1 murine in vivo breast cancer model, and cultured cancer (4T1) and endothelial cells (ECs) for in vitro studies. In vivo studies demonstrated that 4PYR facilitated lung metastasis without affecting primary tumor growth. In vitro studies demonstrated that 4PYR affected extracellular adenine nucleotide metabolism and the intracellular energy status in ECs, shifting catabolite patterns toward the accumulation of extracellular inosine, and leading to the increased permeability of lung ECs. These changes prevailed over the direct effect of 4PYR on 4T1 cells that reduced their invasive potential through 4PYR-induced modulation of the CD73-adenosine axis. We conclude that 4PYR is an oncometabolite that affects later stages of the metastatic cascade by acting specifically through the regulation of EC permeability and metabolic controls of inflammation.

The effect of lactate dehydrogenase-A inhibition on intracellular nucleotides and mitochondrial respiration in pancreatic cancer cells.

Pancreatic cancer (PC) is one of the most lethal malignancies. PC is characterized by a high expression of the glucose transporter GLUT-1 and of lactate dehydrogenase A (LDH-A). The novel LDH-A inhibitor NHI-Glc-2 was designed for a better uptake via GLUT-1 and was shown to be cytotoxic against the PC cell line PANC-1. Using RP-HPLC we investigated its effect on adenine nucleotides and NADH/NAD+, while the Seahorse analyzer was used to determine its effect on glycolysis and mitochondrial function. A 24 hour exposure to 10 µM NHI-Glc-2 (around the IC50) decreased the ATP concentration by about 10%, but at 25 µM this decrease was 38%, while NAD+ decreased by 26%, associated with a 35% decrease in the NADH/NAD+ ratio. A 10 µM NHI-Glc-2 decreased extracellular acidification and oxygen consumption (about 75%), as well as the mitochondrial respiration parameters by 50%. In conclusion, LDH-A inhibition markedly affected the energy supply of PANC-1 cells. The respiration data indicated a dependency of the cells on glycolysis and fatty acid oxidation.Supplemental data for this article is available online at https://doi.org/10.1080/15257770.2022.2031215 .

The decreased serum activity of cytosolic 5'-nucleotidase IA as a potential marker of breast cancer-associated muscle inflammation.

Cytosolic 5'-nucleotidase IA (cN-IA) plays a central role in the regulation of the purine nucleotide pool in skeletal muscle, preferentially converting adenosine monophosphate to adenosine. cN-IA can act as an autoantigen in muscle diseases, including the paraneoplastic syndrome related to breast cancer (BC). As a result of myocyte damage, released cN-IA protein may trigger the production of anti-cN-IA antibodies (anti-NT5C1A). This work aimed to develop an effective method to measure cN-IA activity in the serum and analyze it in BC patients. Our study demonstrated that serum cN-IA activity was decreased in BC patients and we assumed it is due to the presence of specific autoantibodies. We found correlations between cN-IA activity and parameters of inflammatory muscle damage. Thus, cN-IA is worth further attention to clarify its usefulness as a biomarker of BC-associated polymyositis.

An unusual nicotinamide derivative, 4-pyridone-3-carboxamide ribonucleoside (4PYR), is a novel endothelial toxin and oncometabolite.

Our recent studies identified a novel pathway of nicotinamide metabolism that involves 4-pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR) and demonstrated its endothelial cytotoxic effect. This study tested the effects of 4PYR and its metabolites in experimental models of breast cancer. Mice were divided into groups: 4T1 (injected with mammary 4T1 cancer cells), 4T1 + 4PYR (4PYR-treated 4T1 mice), and control, maintained for 2 or 21 days. Lung metastasis and endothelial function were analyzed together with blood nucleotides (including 4PYR), plasma amino acids, nicotinamide metabolites, and vascular ectoenzymes of nucleotide catabolism. 4PYR metabolism was also evaluated in cultured 4T1, MDA-MB-231, MCF-7, and T47D cells. An increase in blood 4PYR in 4T1 mice was observed at 2 days. 4PYR and its metabolites were noticed after 21 days in 4T1 only. Higher blood 4PYR was linked with more lung metastases in 4T1 + 4PYR vs. 4T1. Decreased L-arginine, higher asymmetric dimethyl-L-arginine, and higher vascular ecto-adenosine deaminase were observed in 4T1 + 4PYR vs. 4T1 and control. Vascular relaxation caused by flow-dependent endothelial activation in 4PYR-treated mice was significantly lower than in control. The permeability of 4PYR-treated endothelial cells was increased. Decreased nicotinamide but enhanced nicotinamide metabolites were noticed in 4T1 vs. control. Reduced N-methylnicotinamide and a further increase in Met2PY were observed in 4T1 + 4PYR vs. 4T1 and control. In cultured breast cancer cells, estrogen and progesterone receptor antagonists inhibited the production of 4PYR metabolites. 4PYR formation is accelerated in cancer and induces metabolic disturbances that may affect cancer progression and, especially, metastasis, probably through impaired endothelial homeostasis. 4PYR may be considered a new oncometabolite.

Macrophage-Derived Adenosine Deaminase 2 Correlates with M2 Macrophage Phenotype in Triple Negative Breast Cancer.

Several lines of evidence suggest that altered adenosine deaminase (ADA) activity, especially its ADA2 iso-enzyme, is associated with malignant breast cancer (BC) development. Triple-negative breast cancer (TNBC) is currently the most challenging BC subtype due to its metastatic potential and recurrence. Herein, we analyzed the sources of ADA iso-enzymes in TNBC by investigating the effects of cell-to-cell interactions between TNBC cells, macrophages, lymphocytes, and endothelial cells. We also examined the potential relationship between ADA activity and cancer progression in TNBC patients. In vitro analyses demonstrated that the interactions of immune and endothelial cells with MDA-MB-231 triple negative BC cells modulated their extracellular adenosine metabolism pattern. However, they caused an increase in the ADA1 activity, and did not alter ADA2 activity in cancer cells. In turn, the co-culture of MDA-MB-231 cells with THP-1 monocyte/macrophages, Jurkat cells, and human lung microvascular endothelial cells (HULEC) caused the increase in ADA2 activity on THP-1 cells and ADA1 activity on Jurkat cells and HULEC. Clinical sample analysis revealed that TNBC patients had higher plasma ADA2 activities and lower ADA1/ADA2 ratio at advanced stages of cancer development than in the initial stages, while patients with hormone receptor positive, HER2 negative (HR+HER2-), and triple positive (HR+HER2+) breast cancers at the same stages showed opposite trends. TNBC patients also demonstrated positive associations between plasma ADA2 activity and pro-tumor M2 macrophage markers, as well as between ADA1 activity and endothelial dysfunction or inflammatory parameters. The analysis of TNBC patients, at 6 and 12 months following cancer treatment, did not showed significant changes in plasma ADA activities and macrophage polarization markers, which may be the cause of their therapeutic failure. We conclude that alterations in both ADA iso-enzymes can play a role in breast cancer development and progression by the modulation of extracellular adenosine-dependent pathways. Additionally, the changes in ADA2 activity that may contribute to the differentiation of macrophages into unfavorable pro-tumor M2 phenotype deserve special attention in TNBC.

Therapeutic Perspectives of Adenosine Deaminase Inhibition in Cardiovascular Diseases.

Adenosine deaminase (ADA) is an enzyme of purine metabolism that irreversibly converts adenosine to inosine or 2'deoxyadenosine to 2'deoxyinosine. ADA is active both inside the cell and on the cell surface where it was found to interact with membrane proteins, such as CD26 and adenosine receptors, forming ecto-ADA (eADA). In addition to adenosine uptake, the activity of eADA is an essential mechanism that terminates adenosine signaling. This is particularly important in cardiovascular system, where adenosine protects against endothelial dysfunction, vascular inflammation, or thrombosis. Besides enzymatic function, ADA protein mediates cell-to-cell interactions involved in lymphocyte co-stimulation or endothelial activation. Furthermore, alteration in ADA activity was demonstrated in many cardiovascular pathologies such as atherosclerosis, myocardial ischemia-reperfusion injury, hypertension, thrombosis, or diabetes. Modulation of ADA activity could be an important therapeutic target. This work provides a systematic review of ADA activity and anchoring inhibitors as well as summarizes the perspectives of their therapeutic use in cardiovascular pathologies associated with increased activity of ADA.

Statin treatment of patients with calcific aortic valve disease modulates extracellular adenosine metabolism on the cell surface of the aortic valve.

Statins efficiently prevent cardiovascular events by lipid-dependent and independent mechanisms. We hypothesize that part of these protective effects could be associated with an increased extracellular adenosine signaling. We demonstrated previously that aortic valves obtained from patients with calcific aortic valve disease (CAVD) disclosed disturbances in extracellular adenosine metabolism. This study aimed to analyze the impact of statin treatment on extracellular nucleotides and adenosine metabolism in aortic valves originated from CAVD patients and to elucidate potential mechanisms that are involved in the regulation of ecto-enzyme activities by statins. Aortic valves of CAVD patients treated with statins (n = 45) revealed higher adenosine production and its lower degradation than in non-treated patients (n = 28). Statin treatment was also related to the improvement in pre-operative echocardiographic data indicating milder aortic valve stenosis and a better function of the left ventricle. The rates of aortic valve adenosine conversions correlated with plasma lipid profile parameters, within both statin-treated and non-treated groups. Valvular extracellular AMP hydrolysis correlated negatively, while adenosine deamination positively with plasma total and LDL cholesterol. Atorvastatin treatment of murine heart endothelial cells led to the enhanced ecto-5'nucleotidase (CD73) and decreased ecto-adenosine deaminase (eADA) activity. When endothelial cells were stimulated with thrombin that induces endothelial cell exocytosis, activities of both cell-surface CD73 and eADA were increased, while co-treatment with atorvastatin reversed only thrombin-induced eADA activity. In conclusion, early intervention with statins may provide beneficial effects for CAVD therapy. Here, we presented results showing that these protective outcomes could be mediated via the regulation of extracellular adenosine metabolism pathways.

Nucleotide ecto-enzyme metabolic pattern and spatial distribution in calcific aortic valve disease; its relation to pathological changes and clinical presentation.

BACKGROUND: Extracellular nucleotide metabolism contributes to chronic inflammation, cell differentiation, and tissue mineralization by controlling nucleotide and adenosine concentrations and hence its purinergic effects. This study investigated location-specific changes of extracellular nucleotide metabolism in aortic valves of patients with calcific aortic valve disease (CAVD). Individual ecto-enzymes and adenosine receptors involved were analyzed together with correlation with CAVD severity and risk factors. RESULTS: Nucleotide and adenosine degradation rates were adversely modified on the aortic surface of stenotic valve as compared to ventricular side, including decreased ATP removal (1.25 ± 0.35 vs. 2.24 ± 0.61 nmol/min/cm2) and adenosine production (1.32 ± 0.12 vs. 2.49 ± 0.28 nmol/min/cm2) as well as increased adenosine deamination (1.28 ± 0.31 vs. 0.67 ± 0.11 nmol/min/cm2). The rates of nucleotide to adenosine conversions were lower, while adenosine deamination was higher on the aortic sides of stenotic vs. non-stenotic valve. There were no differences in extracellular nucleotide metabolism between aortic and ventricular sides of non-stenotic valves. Furthermore, nucleotide degradation rates, measured on aortic side in CAVD (n = 62), negatively correlated with echocardiographic and biochemical parameters of disease severity (aortic jet velocity vs. ATP hydrolysis: r = - 0.30, p < 0.05; vs. AMP hydrolysis: r = - 0.44, p < 0.001; valvular phosphate concentration vs. ATP hydrolysis: r = - 0.26, p < 0.05; vs. AMP hydrolysis: r = - 0.25, p = 0.05) while adenosine deamination showed positive correlation trend with valvular phosphate deposits (r = 0.23, p = 0.07). Nucleotide and adenosine conversion rates also correlated with CAVD risk factors, including hyperlipidemia (AMP hydrolysis vs. serum LDL cholesterol: r = - 0.28, p = 0.05; adenosine deamination vs. total cholesterol: r = 0.25, p = 0.05; LDL cholesterol: r = 0.28, p < 0.05; triglycerides: r = 0.32, p < 0.05), hypertension (adenosine deamination vs. systolic blood pressure: r = 0.28, p < 0.05) and thrombosis (ATP hydrolysis vs. prothrombin time: r = - 0.35, p < 0.01). Functional assays as well as histological and immunofluorescence, flow cytometry and RT-PCR studies identified all major ecto-enzymes engaged in nucleotide metabolism in aortic valves that included ecto-nucleotidases, alkaline phosphatase, and ecto-adenosine deaminase. We have shown that changes in nucleotide-converting ecto-enzymes were derived from their altered activities on valve cells and immune cell infiltrate. We have also demonstrated a presence of A1, A2a and A2b adenosine receptors with diminished expression of A2a and A2b in stenotic vs. non-stenotic valves. Finally, we revealed that augmenting adenosine effects by blocking adenosine deamination with deoxycoformycin decreased aortic valve thickness and reduced markers of calcification via adenosine-dependent pathways in a mouse model of CAVD. CONCLUSIONS: This work highlights profound changes in extracellular nucleotide and adenosine metabolism in CAVD. Altered extracellular nucleotide hydrolysis and degradation of adenosine in stenotic valves may affect purinergic responses to support a pro-stenotic milieu and valve calcification. This emphasizes a potential mechanism and target for prevention and therapy. .

Increased plasma concentration of 4-pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR) in lung cancer. Preliminary studies.

4-pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR) is a new nicotinamide derivative, which is potentially toxic to the endothelium. Dysfunction of the endothelium promotes cancer cell proliferation, invasiveness, and inflammatory signaling. The aim of this study was to analyze 4PYR concentration in the plasma of lung cancer patients and its relationship to other known biochemical parameters associated with the endothelium function. The concentration of 4PYR, nicotinamide, 1-methylnicotinamide (MNA), amino acids, and their derivatives were measured in samples obtained from patients with primary squamous cell carcinoma (n = 48) and control group (n = 100). The concentration of 4PYR and 4PYR/MNA ratio were significantly higher in lung cancer patients as compared to controls (0.099 ± 0.009 vs. 0.066 ± 0.006 µmol/L and 1.10 ± 0.08 vs. 1.97 ± 0.15, respectively). The plasma arginine/asymmetric dimethylarginine (Arg/ADMA) ratio was considerably lower in lung cancer patients (253 ± 17 vs. 369 ± 19) as well as plasma MNA (0.057 ± 0.004 vs. 0.069 ± 0.003 µmol/L). There was no difference in the plasma concentrations of nicotinamide and nicotinamide riboside in both groups (0.116 ± 0.019 vs. 0.131 ± 0.014 and 0.102 ± 0.006 vs. 0.113 ± 0.011, respectively). In this study, a higher 4PYR concentration was observed for the first time in patients with squamous cell carcinoma. This change may be related to the endothelial dysfunction that promote cancer progression since 4PYR and its derivatives are known to disrupt glycolytic pathway.

Inhibition of LPS-stimulated ecto-adenosine deaminase attenuates endothelial cell activation.

Vascular inflammation is an important factor in the pathophysiology of cardiovascular diseases, such as atherosclerosis. Changes in the extracellular nucleotide and in particular adenosine catabolism may alter a chronic inflammation and endothelial activation. This study aimed to evaluate the relation between vascular ecto-adenosine deaminase (eADA) activity and endothelial activation in humans and to analyze the effects of LPS-mediated inflammation on this activity as well as mechanisms of its increase. Moreover, we investigated a therapeutic potential of ADA inhibition by deoxycofromycin (dCF) for endothelial activation. We demonstrated a positive correlation of vascular eADA activity and ADA1 mRNA expression with endothelial activation parameters in humans with atherosclerosis. The activation of vascular eADA was also observed under LPS stimulation in vivo along with endothelial activation, an increase in markers of inflammation and alterations in the lipid profile of a rat model. Ex vivo and in vitro studies on human specimen demonstrated that at an early stage of vascular pathology, eADA activity originated from activated endothelial cells, while at later stages also from an inflammatory infiltrate. We proposed that LPS-stimulated increase in endothelial adenosine deaminase activity could be a result of IL-6/JAK/STAT pathway activation, since the lack of IL-6 in mice was associated with lower vascular and plasma eADA activities. Furthermore, the inhibitors of JAK/STAT pathway decreased LPS-stimulated adenosine deaminase activity in endothelial cells. We demonstrated that cell surface eADA activity could be additionally regulated by transcytosis pathways, as exocytosis inhibitors including lipid raft inhibitor, methyl-ß-cyclodextrin decreased LPS-induced eADA activity. This suggests that cholesterol-dependent protein externalization mediated by lipid rafts could be an important factor in the eADA increase. Moreover, endocytosis inhibitors and exocytosis activators increased this activity on the cell surface. Furthermore, the inhibition of adenosine deaminase in endothelial cells in vitro attenuated LPS-mediated IL-6 release and soluble ICAM-1 and VCAM-1 concentration in the incubation medium through the restoration of the extracellular adenosine pool and adenosine receptor-dependent pathways. This study demonstrated that the vascular endothelial eADA activity remains under control of inflammatory mediators acting through JAK/STAT pathway that could be further modified by dyslipidemic-dependent exocytosis and transcytosis pathways. Inhibition of eADA blocked endothelial activation suggesting a crucial role of this enzyme in the control of vascular inflammation. This supports the concept of eADA targeted vascular protection therapy.

The effects of pro- and anti-atherosclerotic factors on intracellular nucleotide concentration in murine endothelial cells.

Endothelial cell activation and dysfunction could lead to endothelial injury that is an important factor in the development of vascular diseases. Vascular injury is strongly associated with disturbed endothelial cell energetics and pyridine nucleotide pool. This study aimed to evaluate the effects of inflammatory stimuli (IL-6, LPS), uric acid, hyperglycemia, fatty acids, flavonoids, statins and nonsteroidal anti-inflammatory drugs on cellular concentration of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and nicotinamide adenine dinucleotide (NAD+) in cultured endothelial cells. Murine-immortalized heart endothelial cells (H5V cells) were treated with different concentrations of pro- and anti-atherosclerotic factors and intracellular concentration of nucleotides were measured using high performance liquid chromatography. Intracellular ATP concentration in H5V cells was not changed by inflammatory stimuli (IL-6 and LPS), uric acid, glucose, atorvastatin, acetylsalicylic acid, monounsaturated and polyunsaturated fatty acids. Only high concentration of palmitic acid (1 mM) and kaempferol (>0.1 mM) decreased intracellular ATP concentration. The concentration of intracellular ADP has not been altered by any of tested compounds. In turn, intracellular NAD+ pool was modified only by polyunsaturated fatty acids and atorvastatin. Linoleic acid, docosahexaenoic acid and atorvastatin increased cellular NAD+ concentration. Tested compounds have a small influence on murine endothelial cell energetics, but polyunsaturated fatty acids and atorvastatin increased intracellular NAD+ concentration that could be an important protective mechanism against endothelial cell injury.