Adenosine Triphosphate Metabolism Research Articles

A birdcage transmit, 24-channel conformal receive array coil for sensitive 31P magnetic resonance spectroscopic imaging of the human brain at 7 T.

Phosphorus (31P) magnetic resonance spectroscopic imaging (MRSI) can serve as a critical tool for more direct quantification of brain energy metabolism, tissue pH, and cell membrane turnover. However, the low concentration of 31P metabolites in biological tissue may result in low signal-to-noise ratio (SNR) in 31P MRS images. In this work, we present an innovative design and construction of a 31P radiofrequency coil for whole-brain MRSI at 7 T. Our coil builds on current literature in ultra-high field 31P coil design and offers complete coverage of the brain, including the cerebellum and brainstem. The coil consists of an actively detunable volume transmit (Tx) resonator and a custom 24-channel receive (Rx) array. The volume Tx resonator is a 16-rung high-pass birdcage coil. The Rx coil consists of a 24-element phased array composed of catered loop shapes and sizes built onto a custom, close-fitting, head-shaped housing. The Rx array was designed to provide complete coverage of the head, while minimizing mutual coupling. The Rx configuration had a mean reflection coefficient better than -20 decibels (dB) when the coil was loaded with a human head. The mean mutual coupling ( ) among Rx elements, when loaded with a human head, was -16 dB. In phantom imaging, the phased array produced a central SNR that was 4.4-fold higher than the corresponding central SNR when operating the 31P birdcage as a transceiver. The peripheral SNR was 12-fold higher when applying the optimized phased array. In vivo 3D 31P MRSI experiments produced high-quality spectra in the cerebrum gray and white matter, as well as in the cerebellum. Characteristic phosphorus metabolites related to adenosine triphosphate metabolism and cell membrane turnover were distinguishable across all brain regions. In summary, our results demonstrate the potential of our novel coil for accurate, whole-brain 31P metabolite quantification.

Impact of Zero-Valent Iron Nanoparticles and Ampicillin on Adenosine Triphosphate and Lactate Metabolism in the Cyanobacterium Fremyella diplosiphon.

In cyanobacteria, the interplay of ATP and lactate dynamics underpins cellular energetics; their pronounced shifts in response to zero-valent iron (nZVI) nanoparticles and ampicillin highlight the nuanced metabolic adaptations to environmental challenges. In this study, we investigated the impact of nZVIs and ampicillin on Fremyella diplosiphon cellular energetics as determined by adenosine triphosphate (ATP) content, intracellular and extracellular lactate levels, and their impact on cell morphology as visualized by transmission electron microscopy. While a significant increase in ATP concentration was observed in 0.8 mg/L ampicillin-treated cells compared to the untreated control, a significant decline was noted in cells treated with 3.2 mg/L nZVIs. ATP levels in the combination regimen of 0.8 mg/L ampicillin and 3.2 mg/L nZVIs were significantly elevated (p < 0.05) compared to the 3.2 mg/L nZVI treatment. Intracellular and extracellular lactate levels were significantly higher in 0.8 mg/L ampicillin, 3.2 mg/L nZVIs, and the combination regimen compared to the untreated control; however, extracellular lactate levels were the highest in cells treated with 3.2 mg/L nZVIs. Visualization of morphological changes indicated increased thylakoid membrane stacks and inter-thylakoidal distances in 3.2 mg/L nZVI-treated cells. Our findings demonstrate a complex interplay of nanoparticle and antibiotic-induced responses, highlighting the differential impact of these stressors on F. diplosiphon metabolism and cellular integrity.

The Warburg Effect Reinterpreted 100 yr on: A First-Principles Stoichiometric Analysis and Interpretation from the Perspective of ATP Metabolism in Cancer Cells.

The Warburg Effect is a longstanding enigma in cancer biology. Despite the passage of 100 yr since its discovery, and the accumulation of a vast body of research on the subject, no convincing biochemical explanation has been given for the original observations of aerobic glycolysis in cancer cell metabolism. Here, we have worked out a first-principles quantitative analysis of the problem from the principles of stoichiometry and available electron balance. The results have been interpreted using Nath's unified theory of energy coupling and adenosine triphosphate (ATP) synthesis, and the original data of Warburg and colleagues have been analyzed from this new perspective. Use of the biomass yield based on ATP per unit substrate consumed, [Formula: see text], or the Nath-Warburg number, NaWa has been shown to excellently model the original data on the Warburg Effect with very small standard deviation values, and without employing additional fitted or adjustable parameters. Based on the results of the quantitative analysis, a novel conservative mechanism of synthesis, utilization, and recycling of ATP and other key metabolites (eg, lactate) is proposed. The mechanism offers fresh insights into metabolic symbiosis and coupling within and/or among proliferating cells. The fundamental understanding gained using our approach should help in catalyzing the development of more efficient metabolism-targeting anticancer drugs.

Suppressing the Hypoxia‐Adenosinergic Axis by a Tailored Nanoreactor for Enhanced Photothermal Immunotherapy

Cell metabolite adenosine can induce extensive and persistent immunosuppression by binding to adenosine receptors on immune cells. Seriously, the hypoxia‐driven adenosinergic axis aggravates adenosine accumulation via dephosphorizing immune‐activating adenosine triphosphate (ATP) released during immunogenic cell death (ICD). Different from direct adenosine clearance or adenosine receptor blockade or directly using ecto‐enzyme (CD39/CD73) antagonist, it is hoped to use an innovative small science engineering to regulate the upstream hypoxia/HIF‐1α signal of the hypoxia‐adenosinergic axis, thereby reducing the immunosuppressive extracellular adenosine and enhancing ICD‐triggered antitumor immunity. PM@Mn is constructed by gradually integrating metformin and MnO2 on polydopamine (PDA) nanoparticles. PM@Mn can effectively suppress hypoxia‐adenosinergic axis via combining catalytic oxygen production with reduced endogenous oxygen consumption. Such motif of hypoxia relief suppresses the metabolism of ATP to adenosine via down‐regulating the expression of HIF‐1α, CD39, and CD73. Meanwhile, PDA in PM@Mn can induce local tumor ablation and trigger the “vaccine effect” of ICD under near‐infrared radiation. In a mouse breast cancer model with low immunogenicity, our strategy can effectively reduce adenosine accumulation, PM@Mn group exhibits 4.51‐fold cytotoxic T lymphocyte infiltration and tumor inhibition rate of 75.4%. This study provides a new strategy to advance ICD‐triggered antitumor immunity through supressing hypoxia‐adenosinergic axis.

Correlation of serum uric acid levels with certain anthropometric parameters in prediabetic and drug-naive diabetic subjects.

Uric acid is produced during the metabolism of nucleotide and adenosine triphosphate and contains the final product of human purine metabolism. It acts both as an antioxidant and pro-inflammatory marker and has a positive association with visceral fat in overweight subjects. The aim of the present study is to find an association of uric acid level with certain anthropometric parameters in subjects having type 2 diabetes. The study included 124 urban drug-naive diabetic Indian subjects above 18 years of age from the general population of the city of North India. Uric acid concentrations were estimated by the uricase method. Fasting plasma glucose (FPG) concentrations were estimated by the glucose oxidase-peroxidase method. Anthropometric measurements and information on lifestyle factors and disease history were collected through in-person meeting. All participants of the study subjects had a body mass index (BMI) of more than 23.5. BMI, waist-to-hip ratio (WHR), waist-to-height ratio, waist circumference, neck circumference, weight, age, sagittal abdominal diameter (SAD), skinfold thickness, and body roundness index were positively correlated with the serum uric acid level. The correlation of weight, BMI, SAD, and WHR was statistically significant. We found that serum uric acid level increases as body fat content increases. Statistical data show remarkable results for a significant correlation of uric acid level with BMI, WHR, SAD, and FPG. Hypertrophy occurs as a result of inflammatory processes and oxidative stress when the supply of energy starts to exceed the storage capacity of adipocytes, as a result, adipokines such as interleukin (IL)-1, IL-6, and tumor-necrosis factor-alpha are released more frequently which lead to low-grade chronic inflammation. Uric acid levels are much lean toward visceral obesity than overall body fat content.

A nanozipper structured efficient FRET probe for ratiometric detection of ATP based on target-induced cycling amplification

In the realm of disease diagnostics and prognostics, accurate quantification of adenosine triphosphate (ATP) has become crucial, as the intricate interplay between ATP and cellular metabolism is a fundamental factor in the pathogenesis and progression of many pathological conditions. Herein, a ratiometric fluorescence DNA nanozipper is developed, which enables highly sensitive detection of ATP by employing catalytic hairpin assembly. This innovative approach utilizes target-induced conformational changes in the nanozipper structure to tune the distance between the carboxyfluorescein as donor and tetramethylrhodamine as acceptor around the Förster radius, thereby enhancing the efficiency of fluorescence resonance energy transfer (FRET). In the state of nanozipper tension, the rigid and steric hindrance Y-shaped configuration leads to a considerable distance between two fluorophores, resulting in FRET low efficiency. However, the presence of ATP triggers allosteric configuration of the DNA nanozipper, switching the nanozipper to a relaxed state, which results in a visible fluorescent ratiometric signal. The sensing strategy exhibits a linear response to ATP concentrations in the range of 30 − 600 nM and can detect concentrations as low as 9.6 nM. Additionally, this detection method introduces a novel idea for the design of efficient FRET probes and demonstrates promising application potential in biological samples.

Differential Influences of Endogenous and Exogenous Sensory Neuropeptides on the ATP Metabolism by Soluble Ectonucleotidases in the Murine Bladder Lamina Propria.

Bladder urothelium and suburothelium/lamina propria (LP) have prominent sensory and transducer functions with the active participation of afferent neurons and urothelium-derived purine mediators such as adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine (ADO). Effective concentrations of purines at receptor targets depend significantly on the extracellular degradation of ATP by ectonucleotidases (ENTDs). We recently reported the regulated release of soluble ENTDs (s-ENTDs) in the LP and the consequent degradation of ATP to ADP, AMP, and ADO. Afferent neurons in the LP can be activated by urothelial ATP and release peptides and other transmitters that can alter the activity of cells in their vicinity. Using a murine decentralized ex vivo detrusor-free bladder model, 1,N6-etheno-ATP (eATP) as substrate, and sensitive HPLC-FLD methodologies, we found that exogenous neuropeptides calcitonin gene-related peptide (CGRP), substance P (Sub P), neurokinin A (NKA), and pituitary adenylate cyclase-activating polypeptide [PACAP (1-38)] all increased the degradation of eATP by s-ENTDs that were released in the LP spontaneously and/or during bladder filling. Using antagonists of neuropeptide receptors, we observed that endogenous NKA did not modify the ATP hydrolysis by s-ENTDs, whereas endogenous Sub P increased both the constitutive and distention-induced release of s-ENTDs. In contrast, endogenous CGRP and PACAP (1-38) increased the distention-induced, but not the spontaneous, release of s-ENTDs. The present study puts forward the novel idea that interactions between peptidergic and purinergic signaling mechanisms in the LP have an impact on bladder excitability and functions by regulating the effective concentrations of adenine purines at effector cells in the LP.

Triggered metabolism of adenosine triphosphate as an explanation for the chemical heterogeneity of heterotopic ossification

Heterotopic ossification (HO), the pathological formation of bone in soft tissues, is a debilitating condition, as well as one of the few instances of de novo bone formation in adults. Chemical mapping of HO tissue showed distinct islands of calcium phosphate within phosphate-deficient, calcium-rich regions, suggesting a transition to apatitic bone mineral from a non-phosphatic precursor. The transition of amorphous calcium carbonate (ACC), a generally suggested bone-mineral precursor, in physiological conditions was thus investigated. Here, we show that adenosine triphosphate (ATP), present in high amounts in forming bone, stabilised ACC for weeks in physiological conditions and that enzymatic degradation of ATP triggered rapid crystallisation into apatite, through an amorphous calcium phosphate phase. It is suggested that this localised enzymatic degradation could explain the chemical heterogeneity seen in HO and may also represent a pathway to physiological bone mineralisation.

Locomotor function of skeletal muscle is regulated by vitamin D via adenosine triphosphate metabolism

ObjectivesDuring musculoskeletal development, the vitamin D endocrine system is crucial, because vitamin D–dependent calcium absorption is a major regulator of bone growth. Because exercise regimens depend on bone mass, the direct action of active vitamin D (1,25-dihydroxyvitamin D3 [1,25(OH)2D3]) on musculoskeletal performance should be determined. MethodsTo evaluate the effect of 1,25(OH)2D3 on muscle tissue, the vitamin D receptor (Vdr) gene was genetically inactivated in mouse skeletal muscle and the role of 1,25(OH)2D3-VDR signaling on locomotor function was assessed. The direct action of 1,25(OH)2D3 on muscle development was determined using cultured C2C12 cells with myogenic differentiation. ResultsThe lack of Vdr activity in skeletal muscle decreased spontaneous locomotor activity, suggesting that the skeletal muscle performance depended on 1,25(OH)2D3-VDR signaling. Bone phenotypes, reduced femoral bone mineral density, and accelerated osteoclast bone resorption were confirmed in mice lacking skeletal muscle Vdr activity. In vitro study revealed that the treatment with 1,25(OH)2D3 decreased the cellular adenosine triphosphate (ATP)–to–adenosine monophosphate ratio without reducing ATP production. Remarkably, protein expressions of connexin 43, an ATP releaser to extracellular space, and ATP metabolizing enzyme ectonucleotide pyrophosphatase phosphodiesterase 1 were increased responding to 1,25(OH)2D3 treatment. Furthermore, the concentration of pyrophosphate in the culture medium, which inhibits tissue calcification, was increased with 1,25(OH)2D3 treatment. In the presence of 1,25(OH)2D3-VDR signaling, calcium accumulation was suppressed in both muscle samples isolated from mice and in cultured C2C12 cells. ConclusionsThis study dissected the physiological functions of 1,25(OH)2D3-VDR signaling in muscle and revealed that regulation of ATP dynamics is involved in sustaining locomotor function.

Molecular characterization and immunological properties of Echinococcus granulosus sensu stricto (G1) ADK1 and ADK8.

Adenylate kinases (ADKs) are one of the important enzymes regulating adenosine triphosphate (ATP) metabolism in Echinococcus granulosus sensu lato. The objective of the present study was to explore the molecular characteristics and immunological properties of E. granulosus sensu stricto (G1) adenylate kinase 1 (EgADK1) and adenylate kinase 8 (EgADK8). EgADK1 and EgADK8 were cloned and expressed, and the molecular characteristics of EgADK1 and EgADK8 were analyzed through different bioinformatics tools. Western blotting was used to examine the reactogenicity of recombinant adenylate kinase 1 (rEgADK1) and recombinant adenylate kinase 8 (rEgADK8) and to evaluate their diagnostic value. The expression profiles of EgADK1 and EgADK8 in 18-day-old strobilated worms and protoscoleces were analyzed by quantitative real-time PCR, and their distribution in 18-day-old strobilated worms, the germinal layer, and protoscoleces was determined by immunofluorescence localization. EgADK1 and EgADK8 were successfully cloned and expressed. Bioinformatics analysis predicted that EgADK1 and EgADK8 have multiple phosphorylation sites and B-cell epitopes. Compared with EgADK8, EgADK1 and other parasite ADKs have higher sequence similarity. In addition, both cystic echinococcosis (CE)-positive sheep sera and Cysticercustenuicollis-infected goat sera could recognize rEgADK1 and rEgADK8. EgADK1 and EgADK8 were localized in protoscoleces, the germinal layer, and 18-day-old strobilated worms. EgADK1 and EgADK8 showed no significant difference in their transcription level in 18-day-old strobilated worms and protoscoleces, suggesting that EgADK1 and EgADK8 may play an important role in the growth and development of E. granulosus sensu lato. Since EgADK1 and EgADK8 can be recognized by other parasite-positive sera, they are not suitable as candidate antigens for the diagnosis of CE.

Endothelial P2X7 promotes venous thromboembolism

Venous thromboembolism (VTE) affects 117 people per 100,000 each year and is an important cause of morbidity and mortality. VTE can lead to 1) death through pulmonary embolism, 2) the post-thrombotic syndrome, or 3) pulmonary sequelae that dramatically impair quality of life. Thrombosis has long been characterized by the Virchow's triad encompassing hypercoagulability, venous stasis and vascular wall damage. However, inflammatory pathways are now well recognized mechanisms involved in the physiopathology of venous thromboembolism. Endothelial cells play critical roles in regulating immune functions in response to PAMPs and DAMPs. During inflammation, adenosine triphosphate (ATP) is released in the extracellular compartment and is recognized as a danger signal by endothelial cells. ATP can interact with the CD39/CD73 system involved in the metabolism of ATP into AMP. Recent data indicated that the CD39/CD73 system might protect against venous thrombosis by downregulating the NLRP3 inflammasome. ATP can also interact with the P2X7 receptor involved in a wide range of responses including NLRP3 inflammasome activation leading to IL1β production. However, the role of P2X7 receptor in VTE is unknown. To determine how the endothelial P2X7 receptor contributes to venous thromboembolism. After pretreatment with TNFα, HUVECs were incubated with BzATP alone or with thrombin. Immunofluorescence, western blot and real-time quantitative PCR analyses were used to study P2X7 expression in endothelial cells, activation of p38 and NFκB signaling pathways and gene expression, respectively. We confirmed that HUVECs expressed P2X7 receptor in vitro and in vivo after induction of venous thrombosis in an experimental model. BzATP and thrombin induced the activation of p38 and NFκB signaling pathways. Interestingly, this was associated with inflammasome assembly and increased IL1β expression. In addition, ICAM-1 and VCAM-1 expression was increased, while thrombomodulin expression was decreased. Our data suggest that ATP released in the extracellular space during venous thrombosis induced inflammasome activation in endothelial cell through P2X7 activation. P2X7 might have a pro-thrombotic role exacerbating venous thromboembolism.

Tongue sole creatine kinases function as DAMP and activate antimicrobial immunity via TLR2.

Creatine kinase (CK) is an enzyme that regulates adenosine triphosphate (ATP) metabolism to maintain energy homeostasis. Although CK has been reported to be involved in pathogen infection, the immune function of CK remains elusive. In this study, we identified two muscle-type CK from the teleost tongue sole Cynoglossus semilaevis (designated CsCKM-1 and CsCKM-2). Bacterial infection modulated CsCKM-1/2 expression in tongue sole tissues and induced the release of CsCKM-1/2 into serum. Recombinant CsCKM-1/2 (rCsCKM-1/2) exhibited robust kinase activity and bound to bacterial pathogens and pathogen-associated molecular patterns. rCsCKM-1/2 also bound to tongue sole peripheral blood leukocytes (PBLs) and promoted PBLs to uptake bacterial pathogens, inhibit bacterial proliferation, and express proinflammatory cytokines. When co-expressed in HEK293T cells, CsCKM-1/2 were found to interact with the leucine rich domain of toll-like receptor 2 (TLR2). The presence of TLR2 antagonist significantly reduced CsCKM-1/2-induced immune response and antibacterial effect. Taken together, these results indicated that tongue sole creatine kinases function as damage-associated molecular pattern (DAMP) molecules and play an important role in antimicrobial immunity via TLR2.

Purinoreceptors and ectonucleotidases control ATP-induced calcium waveforms and calcium-dependent responses in microglia: Roles of P2 receptors and CD39 in ATP-stimulated microglia.

Adenosine triphosphate (ATP) and its metabolites drive microglia migration and cytokine production by activating P2X- and P2Y- class purinergic receptors. Purinergic receptor activation gives rise to diverse intracellular calcium (Ca2+ signals, or waveforms, that differ in amplitude, duration, and frequency. Whether and how these characteristics of diverse waveforms influence microglia function is not well-established. We developed a computational model trained with data from published primary murine microglia studies. We simulate how purinoreceptors influence Ca2+ signaling and migration, as well as, how purinoreceptor expression modifies these processes. Our simulation confirmed that P2 receptors encode the amplitude and duration of the ATP-induced Ca2+ waveforms. Our simulations also implicate CD39, an ectonucleotidase that rapidly degrades ATP, as a regulator of purinergic receptor-induced Ca2+ responses. Namely, it was necessary to account for CD39 metabolism of ATP to align the model's predicted purinoreceptor responses with published experimental data. In addition, our modeling results indicate that small Ca2+ transients accompany migration, while large and sustained transients are needed for cytokine responses. Lastly, as a proof-of-principal, we predict Ca2+ transients and cell membrane displacements in a BV2 microglia cell line using published P2 receptor mRNA data to illustrate how our computer model may be extrapolated to other microglia subtypes. These findings provide important insights into how differences in purinergic receptor expression influence microglial responses to ATP.

Metabolic adaptation and ATP homeostasis in Pseudomonas fluorescens exposed to phosphate stress.

Phosphate (Pi) is essential for life as it is an integral part of the universal chemical energy adenosine triphosphate (ATP), and macromolecules such as, DNA, RNA proteins and lipids. Despite the core roles and the need of this nutrient in living cells, some bacteria can grow in environments that are poor in Pi. The metabolic mechanisms that enable bacteria to proliferate in a low phosphate environment are not fully understood. In this study, the soil microbe Pseudomonas (P.) fluorescens was cultured in a control and a low Pi (stress) medium in order to delineate how energy homeostasis is maintained. Although there was no significant variation in biomass yield in these cultures, metabolites like isocitrate, oxaloacetate, pyruvate and phosphoenolpyruvate (PEP) were markedly increased in the phosphate-starved condition. Components of the glycolytic, glyoxylate and tricarboxylic acid cycles operated in tandem to generate ATP by substrate level phosphorylation (SLP) as NADH-producing enzymes were impeded. The α-ketoglutarate (KG) produced when glutamine, the sole carbon nutrient was transformed into phosphoenol pyruvate (PEP) and succinyl-CoA (SC), two high energy moieties. The metabolic reprogramming orchestrated by isocitrate lyase (ICL), phosphoenolpyruvate synthase (PEPS), pyruvate phosphate dikinase (PPDK), and succinyl-CoA synthetase fulfilled the ATP budget. Cell free extract experiments confirmed ATP synthesis in the presence of such substrates as PEP, oxaloacetate and isocitrate respectively. Gene expression profiling revealed elevated transcripts associated with numerous enzymes including ICL, PEPS, and succinyl-CoA synthetase (SCS). This microbial adaptation will be critical in promoting biological activity in Pi-poor ecosystems.

Recent Advances in Single-Cell Metabolomics Based on Mass Spectrometry

Recent Advances in Single-Cell Metabolomics Based on Mass Spectrometry

ATP Secretion and Metabolism in Regulating Pancreatic Beta Cell Functions and Hepatic Glycolipid Metabolism.

Diabetes (DM), especially type 2 diabetes (T2DM) has become one of the major diseases severely threatening public health worldwide. Islet beta cell dysfunctions and peripheral insulin resistance including liver and muscle metabolic disorder play decisive roles in the pathogenesis of T2DM. Particularly, increased hepatic gluconeogenesis due to insulin deficiency or resistance is the central event in the development of fasting hyperglycemia. To maintain or restore the functions of islet beta cells and suppress hepatic gluconeogenesis is crucial for delaying or even stopping the progression of T2DM and diabetic complications. As the key energy outcome of mitochondrial oxidative phosphorylation, adenosine triphosphate (ATP) plays vital roles in the process of almost all the biological activities including metabolic regulation. Cellular adenosine triphosphate participates intracellular energy transfer in all forms of life. Recently, it had also been revealed that ATP can be released by islet beta cells and hepatocytes, and the released ATP and its degraded products including ADP, AMP and adenosine act as important signaling molecules to regulate islet beta cell functions and hepatic glycolipid metabolism via the activation of P2 receptors (ATP receptors). In this review, the latest findings regarding the roles and mechanisms of intracellular and extracellular ATP in regulating islet functions and hepatic glycolipid metabolism would be briefly summarized and discussed.

Empagliflozin attenuates cardiac microvascular ischemia/reperfusion injury through improving mitochondrial homeostasis

BackgroundEmpagliflozin has been reported to protect endothelial cell function, regardless of diabetes status. However, the role of empagliflozin in microvascular protection during myocardial ischemia reperfusion injury (I/R) has not been fully understood.MethodsElectron microscopy, western blots, immunofluorescence, qPCR, mutant plasmid transfection, co-immunoprecipitation were employed to explore whether empagliflozin could alleviate microvascular damage and endothelial injury during cardiac I/R injury.ResultsIn mice, empagliflozin attenuated I/R injury-induced microvascular occlusion and microthrombus formation. In human coronary artery endothelial cells, I/R injury led to adhesive factor upregulation, endothelial nitric oxide synthase inactivation, focal adhesion kinase downregulation, barrier dysfunction, cytoskeletal degradation and cellular apoptosis; however, empagliflozin treatment diminished these effects. Empagliflozin improved mitochondrial oxidative stress, mitochondrial respiration and adenosine triphosphate metabolism in I/R-treated human coronary artery endothelial cells by preventing the phosphorylation of dynamin-related protein 1 (Drp1) and mitochondrial fission 1 protein (Fis1), thus repressing mitochondrial fission. The protective effects of empagliflozin on mitochondrial homeostasis and endothelial function were abrogated by the re-introduction of phosphorylated Fis1, but not phosphorylated Drp1, suggesting that Fis1 dephosphorylation is the predominant mechanism whereby empagliflozin inhibits mitochondrial fission during I/R injury. Besides, I/R injury induced Fis1 phosphorylation primarily by activating the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) pathway, while empagliflozin inactivated this pathway by exerting anti-oxidative effects.ConclusionsThese results demonstrated that empagliflozin can protect the microvasculature by inhibiting the DNA-PKcs/Fis1/mitochondrial fission pathway during myocardial I/R injury.

Endothelial P2X7 promotes venous thromboembolism

Venous thromboembolism (VTE) affects 117 people per 100,000 each year and is an important cause of morbidity and mortality. VTE can lead to 1) death through pulmonary embolism, 2) the post-thrombotic syndrome, or 3) chronic pulmonary hypertension resulting in significant chronic respiratory compromise. Inflammatory pathways are intricately involved in thrombus formation and therefore in VTE mechanisms. In pathological conditions, adenosine triphosphate (ATP) is released in the extracellular compartment and is recognized as a danger signal by several cell types including endothelial cells. Recent data indicated that the CD39/CD73 system involved in the metabolism of ATP into AMP might protects against thrombosis by downregulating the pro-inflammatory pathway of the inflammasome. ATP can also interact with the P2X7 receptor involved in inflammation causing a wide range of responses. To determine how the endothelial P2X7 receptor contributes to venous thromboembolism. HUVECs were incubated with BzATP, thrombin or both. HUVECs were primed with TNF-alpha prior to stimulation. We used immunofluorescence, western blot and real-time quantitative PCR analyses to assess P2X7 expression by endothelial cells, activation of p38 and NFκB activation and expression of pro-inflammatory and pro-coagulant genes. We confirmed that endothelial cells expressed P2X7 in vitro in HUVECs and in vivo after induction of venous thrombosis by ligation of the inferior vena cava. Treatment of endothelial cells with BzATP and thrombin induced the activation of p38 and NFκB signaling pathways. This appears to be associated with an increased expression of IL-1β and downregulation of thrombomodulin expression. The expression of adhesion molecules, ICAM-1 and VCAM-1, tends to increase. Our data suggest that ATP released in the extracellular space following cell damage or activation induced a pro-inflammatory response in endothelial cells through P2X7 activation. P2X7 might have a pro-thrombotic role exacerbating venous thromboembolism.

The functional activity of donor kidneys is negatively regulated by microribonucleic acid-451 in different perfusion methods to inhibit adenosine triphosphate metabolism and the proliferation of HK2 cells

ABSTRACT This study explored the regulation of different perfusion methods on ischemia-reperfusion injury in donor kidneys. In this study, renal cortical/medullary tissue specimens were collected from porcine kidneys donors using different perfusion methods at various time points. Hematoxylin and eosin (H&E) staining was used to test the histological differences. Differentially expressed micro-ribonucleic acids (miRNAs) were identified by miRNA transcriptome sequencing. Reverse transcription-polymerase chain reaction (RT-PCR) tests were used to verify the changes in miRNAs in the kidney tissue taken from different perfusion groups. The related signaling pathways and the changes in the cell functions of different perfusion groups were analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) /Gene Ontology (GO) bioinformatics analyses. The effects of miRNA overexpression on the metabolism and proliferation of HK2 cells were detected by ATP kit and MTT assay. The H&E staining results showed that there were essentially no differences in the tissue samples among different perfusion groups at and before 12 h compared with a control group. The quantitative PCR results revealed that there was essentially no change in the expression of ssc-miR-451, ssc-miR-1285, and ssc-miR-486 in the cis infusion or joint infusion kidney groups, and their expression was significantly down-regulated over time in the trans-infusion kidney group. The bioinformatics analysis showed that the cellular component, molecular function, and biological processes of the kidney tissue, which had been perfused using three methods, had been consistently affected. The most significant changes after perfusion occurred in the intracellular metabolism signaling pathways. Furthermore, the energy metabolism and proliferation of the HK2 cells were significantly inhibited after the overexpression of miR-451. Specific miRNA markers, such as miR-451, may play a negative regulatory role in cell metabolism following the perfusion of kidney transplants using different methods.

Endothelial P2X7 promotes venous thromboembolism

Venous thromboembolism (VTE) afflicts 117 people per 100,000 each year and is an important cause of morbidity and mortality. VTE can lead to 1) death through pulmonary embolism, 2) the post-thrombotic syndrome, or 3) chronic pulmonary hypertension resulting in significant chronic respiratory compromise. Inflammatory pathways are intricately involved in venous thromboembolism mechanisms. In pathological conditions, adenosine triphosphate (ATP) is released in the extracellular compartment and is recognized as a danger signal. Recent data indicated that the CD39/CD73 system involved in the metabolism of ATP into AMP might protect against thrombosis by downregulating the pro-inflammatory pathway of the inflammasome. ATP can also interact with the P2X7 receptor involved in inflammation causing a wide range of responses. Determining how the endothelial P2X7 receptor contribute to venous thromboembolism. HUVEC were incubated with BzATP, thrombin or both. In some experiment, HUVEC were primed with TNFaprior to stimulation. We used immunofluorescence, western blot and real-time quantitative PCR analyses to assess P2X7 expression by endothelial cells, activation of p38 and NFkB activation and expression of pro-inflammatory and pro- coagulant genes. We confirmed that endothelial cells expressed P2X7 in vitro in HUVECs and in vivo after induction of venous thrombosis by ligation of the inferior vena cava. Treatment of endothelial cells with BzATP and thrombin induced the activation of p38 and NFkB signaling pathways. This was associated with an increased expression of IL-1b and tissue factor and downregulation of thrombomodulin expression. Our data suggest that ATP released in the extracellular space following cell damage or activation induced the inflammasome in endothelial through P2X7 activation. P2X7 might have a pro-thrombotic role exacerbating venous thromboembolism.