Management of neurological symptoms in Lesch-Nyhan disease: A systematic review.

Lesch-Nyhan Disease (LND) is an X-linked recessive genetic disorder arising from hypoxanthine phosphoribosyltransferase 1 gene mutations, leading to a complete deficiency. LND presents a complex neurological profile characterized by generalized dystonia, motor dysfunctions and self-injurious behavior, which management is challenging. We conducted a systematic review of studies assessing the efficacy of pharmacological and non-pharmacological interventions in management of neurological symptoms in LND (PROSPERO registration number:CRD42023446513). Among 34 reviewed full-text papers; 22 studies were rated as having a high risk of bias. Considerable heterogeneity was found in studies regarding the timing of treatment implementation, adjunctive treatments and outcome assessment. Single-patient studies and clinical trials often showed contradictory results, while therapeutic failures were underreported. S-Adenosylmethionine and Deep Brain Stimulation were the most studied treatment methods and require further research to address inconsistencies. The evidence from levodopa studies underlines that optimal timing of treatment implementation should be thoroughly investigated. Standardized study design and reducing publication bias are crucial to overcome current limitations of assessing intervention efficacy in LND.

Effects of lactate dehydrogenase A and GLUT1 inhibition on human endothelial cell migration in relation to their intracellular nucleotide pool.

The expression of both lactate dehydrogenase A (LDH-A) and glucose transporter type 1 (GLUT1) is high in pancreatic, thoracic and many other types of cancer. GLUT1 is also highly expressed in endothelial cells (EC), that play an important role in tumor metastasis. We investigated the effect of inhibition of LDH-A by NHI-2 and GLUT1 by PGL14 on cellular migration, a hallmark of metastasis, in relation to changes in intracellular purine nucleotide and nicotinamide adenine dinucleotide pools in a human microvascular endothelial cell line (HMEC-1). HMEC-1 were treated with NHI-2 and PGL14 alone or in combination. Cell migration was tested by the wound healing assay. The intracellular purine nucleotides and NAD+/NADH concentrations were measured using Reversed-Phase High Performance Liquid Chromatography (RP-HPLC). Both NHI-2 at 15 µM and 45 µM and PGL14 at 10 µM and 30 µM inhibited migration by 5 to 28% while the combination led to 46% inhibition. The drugs also decreased intracellular nucleotide pools, but only 45 µM NHI-2 altered energy charge and redox status in HMEC-1 cells. Inhibitors of glycolysis attenuated migration and the energy charge of EC and support further development of LDH-A and GLUT1 inhibitors to target cancer aggressiveness and metastasis.

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.

Evaluation of the combination of gemcitabine, carboplatin, and lactate dehydrogenase A inhibitor on malignant mesothelioma.

OBJECTIVES: Lactate dehydrogenase A (LDH-A) catalyzes the last step of glycolysis: supplying cells rapidly but inefficiently with ATP. Many tumors, including malignant mesothelioma (MM), have a high expression of LDH-A, which is associated with cancer aggressiveness. We aimed to determine whether the efficacy of the gemcitabine/carboplatin (Gem + Carbo) combination, widely used to treat this disease, could be increased by inhibition of LDH-A (by NHI-2). To this aim, we analyzed the growth inhibition of pleural and peritoneal MM by multiple combinations. METHODS: The 72 h sulforhodamine B assay (SRB) was used to test the cytotoxicity of the combination of gemcitabine (in the range 0.1 - 400 nM) and carboplatin (0.01 - 40 µM) with a fixed concentration of NHI-2 (at IC25). We used pleural (H2452) and primary peritoneal (STO, MESO-II) MM cell lines, cultured at normoxic conditions. RESULTS: NHI-2 did not increase the cytotoxicity of the combination of 100 nM gemcitabine and 10 µM carboplatin in peritoneal MM cell lines. The cell growth inhibition was 10% smaller after the triple combination than the Gem + Carbo treatment. CONCLUSIONS: Inhibition of LDH-A did not increase the efficacy of gemcitabine and carboplatin in MM under normoxic conditions.

Chain-length dependent effects of inorganic polyphosphate on endothelial function and nucleotide pool.

Endothelial cells (ECs) are the first line that comes into contact with blood pathogens, pathogen-derived molecules, and factors that stimulate coagulation and inflammation. Inorganic polyphosphate (polyP) - a polymer of orthophosphate units synthesized by bacteria under stress and released by platelets upon their activation is among these factors. Bacterial and platelet polyPs differ in length, and both variants elicit different effects in eukaryotes. This study aimed to investigate how bacterial-like long-chain polyP (Lc-polyP) and platelet-like short-chain polyP (Sc-polyP) affect the functionality of cultured endothelial cells. Murine immortalized heart endothelial cells (H5V) were exposed to polyP of different chain lengths to assess the effects of these stimuli on intracellular energetics, permeability, and endothelial adhesion. We observed varying effects between Lc-polyP and Sc-polyP treatments. Lc-polyP more potently disturbs the intracellular ATP pool, a parameter strongly connected with vascular injury, whereas Sc-polyP robustly stimulates cellular adhesion to the endothelium. Both polymers similarly enhance endothelial permeability, suggesting potent immunomodulatory properties. This study provides evidence that polyP elicits profound cellular responses in endothelium depending on the polymer's length.

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.

Elevated Plasma Concentration of 4-Pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR) Highlights Malignancy of Renal Cell Carcinoma.

Nicotinamide (NA) derivatives play crucial roles in various biological processes, such as inflammation, regulation of the cell cycle, and DNA repair. Recently, we proposed that 4-pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR), an unusual derivative of NA, could be classified as an oncometabolite in bladder, breast, and lung cancer. In this study, we investigated the relations between NA metabolism and the progression, recurrence, metastasis, and survival of patients diagnosed with different histological subtypes of renal cell carcinoma (RCC). We identified alterations in plasma NA metabolism, particularly in the clear cell RCC (ccRCC) subtype, compared to papillary RCC, chromophobe RCC, and oncocytoma. Patients with ccRCC also exhibited larger tumor sizes and elevated levels of diagnostic serum biomarkers, such as hsCRP concentration and ALP activity, which were positively correlated with the plasma 4PYR. Notably, 4PYR levels were elevated in advanced stages of ccRCC cancer and were associated with a highly aggressive phenotype of ccRCC. Additionally, elevated concentrations of 4PYR were related to a higher likelihood of mortality, recurrence, and particularly metastasis in ccRCC. These findings are consistent with other studies, suggesting that NA metabolism is accelerated in RCC, leading to abnormal concentrations of 4PYR. This supports the concept of 4PYR as an oncometabolite and a potential prognostic factor in the ccRCC subtype.

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.

Accelerated Extracellular Nucleotide Metabolism in Brain Microvascular Endothelial Cells in Experimental Hypercholesterolemia.

Hypercholesterolemia affects the neurovascular unit, including the cerebral blood vessel endothelium. Operation of this system, especially in the context of energy metabolism, is controlled by extracellular concentration of purines, regulated by ecto-enzymes, such as e-NTPDase-1/CD39, ecto-5'-NT/CD73, and eADA. We hypothesize that hypercholesterolemia, via modulation of the activity of nucleotide metabolism-regulating ecto-enzymes, deteriorates glycolytic efficiency and energy metabolism of endothelial cells, which may potentially contribute to development of neurodegenerative processes. We aimed to determine the effect of hypercholesterolemia on the concentration of purine nucleotides, glycolytic activity, and activity of ecto-enzymes in the murine brain microvascular endothelial cells (mBMECs). We used 3-month-old male LDLR-/-/Apo E-/- double knockout mice to model hypercholesterolemia and atherosclerosis. The age-matched wild-type C57/BL6 mice were a control group. The intracellular concentration of ATP and NAD and extracellular activity of the ecto-enzymes were measured by HPLC. The glycolytic function of mBMECs was assessed by means of the extracellular acidification rate (ECAR) using the glycolysis stress test. The results showed an increased activity of ecto-5'-NT and eADA in mBMECs of the hypercholesterolemic mice, but no differences in intracellular concentration of ATP, NAD, and ECAR between the hypercholesterolemic and control groups. The changed activity of ecto-5'-NT and eADA leads to increased purine nucleotides turnover and a shift in their concentration balance towards adenosine and inosine in the extracellular space. However, no changes in the energetic metabolism of the mBMECs are reported. Our results confirm the influence of hypercholesterolemia on regulation of purine nucleotides metabolism, which may impair the function of the cerebral vascular endothelium. The effect of hypercholesterolemia on the murine brain microvascular endothelial cells (mBMECs). An increased activity of ecto-5'-NT and eADA in mBMECs of the LDLR-/-/Apo E-/- mice leads to a shift in the concentration balance towards adenosine and inosine in the extracellular space with no differences in intracellular concentration of ATP. Figure was created with Biorender.com.

Role of exosomes in transferring chemoresistance through modulation of cancer glycolytic cell metabolism.

Chemoresistance constitute a major obstacle in cancer treatment, leading to limited options and decreased patient survival. Recent studies have revealed a novel mechanism of chemoresistance acquisition: the transfer of information via exosomes, small vesicles secreted by various cells. Exosomes play a crucial role in intercellular communication by carrying proteins, nucleic acids, and metabolites, influencing cancer cell behavior and response to treatment. One crucial mechanism of resistance is cancer metabolic reprogramming, which involves alterations in the cellular metabolic pathways to support the survival and proliferation of drug-resistant cancer cells. This metabolic reprogramming often includes increased glycolysis, providing cancer cells with the necessary energy and building blocks to evade the effects of chemotherapy. Notably, exosomes have been found to transport glycolytic enzymes, as identified in proteomic profiling, leading to the reprogramming of metabolic pathways, facilitating altered glucose metabolism and increased lactate production. As a result, they profoundly impact the tumor microenvironment, promoting tumor progression, survival, immune evasion, and drug resistance.Understanding the complexities of such exosome-mediated cell-to-cell communication might open new therapeutic avenues and facilitate biomarker development in managing cancers characterized by aggressive glycolytic features. Moreover, given the intricate nature of metabolic abnormalities combining future exosome-based-targeted therapies with existing treatments like chemotherapy, immunotherapy, and targeted therapies holds promise for achieving synergistic effects to overcome resistance and improve cancer treatment outcomes.

Target Therapy in Malignant Pleural Mesothelioma: Hope or Mirage?

Malignant Pleural Mesothelioma (MPM) is a rare neoplasm that is typically diagnosed in a locally advanced stage, making it not eligible for radical surgery and requiring systemic treatment. Chemotherapy with platinum compounds and pemetrexed has been the only approved standard of care for approximately 20 years, without any relevant therapeutic advance until the introduction of immune checkpoint inhibitors. Nevertheless, the prognosis remains poor, with an average survival of only 18 months. Thanks to a better understanding of the molecular mechanisms underlying tumor biology, targeted therapy has become an essential therapeutic option in several solid malignancies. Unfortunately, most of the clinical trials evaluating potentially targeted drugs for MPM have failed. This review aims to present the main findings of the most promising targeted therapies in MPM, and to explore possible reasons leading to treatments failures. The ultimate goal is to determine whether there is still a place for continued preclinical/clinical research in this area.

Metabolic Effects of New Glucose Transporter (GLUT-1) and Lactate Dehydrogenase-A (LDH-A) Inhibitors against Chemoresistant Malignant Mesothelioma.

Malignant mesothelioma (MM) is a highly aggressive and resistant tumor. The prognostic role of key effectors of glycolytic metabolism in MM prompted our studies on the cytotoxicity of new inhibitors of glucose transporter type 1 (GLUT-1) and lactate dehydrogenase-A (LDH-A) in relation to ATP/NAD+ metabolism, glycolysis and mitochondrial respiration. The antiproliferative activity of GLUT-1 (PGL13, PGL14) and LDH-A (NHI-1, NHI-2) inhibitors, alone and in combination, were tested with the sulforhodamine-B assay in peritoneal (MESO-II, STO) and pleural (NCI-H2052 and NCI-H28) MM and non-cancerous (HMEC-1) cells. Effects on energy metabolism were measured by both analysis of nucleotides using RP-HPLC and evaluation of glycolysis and respiration parameters using a Seahorse Analyzer system. All compounds reduced the growth of MM cells in the µmolar range. Interestingly, in H2052 cells, PGL14 decreased ATP concentration from 37 to 23 and NAD+ from 6.5 to 2.3 nmol/mg protein. NHI-2 reduced the ATP/ADP ratio by 76%. The metabolic effects of the inhibitors were stronger in pleural MM and in combination, while in HMEC-1 ATP reduction was 10% lower compared to that of the H2052 cells, and we observed a minor influence on mitochondrial respiration. To conclude, both inhibitors showed cytotoxicity in MM cells, associated with a decrease in ATP and NAD+, and were synergistic in the cells with the highest metabolic modulation. This underlines cellular energy metabolism as a potential target for combined treatments in selected cases of MM.

Hidden Pool of Cardiac Adenine Nucleotides That Controls Adenosine Production.

Myocardial ischemic adenosine production decreases in subsequent events that may blunt its protective functions. To test the relation between total or mitochondrial cardiac adenine nucleotide pool (TAN) on the energy status with adenosine production, Langendorff perfused rat hearts were subjected to three protocols: 1 min ischemia at 40 min, 10 min ischemia at 50 min, and 1 min ischemia at 85 min in Group I; additional infusion of adenosine (30 µM) for 15 min after 10 min ischemia in Group I-Ado, and 1 min ischemia at 40 and 85 min in the controls (Group No I). A 31P NMR and an HPLC were used for the analysis of nucleotide and catabolite concentrations in the heart and coronary effluent. Cardiac adenosine production in Group I measured after 1 min ischemia at 85 min decreased to less than 15% of that at 40 min in Group I, accompanied by a decrease in cardiac ATP and TAN to 65% of the initial results. Adenosine production at 85 min was restored to 45% of that at 40 min in Group I-Ado, accompanied by a rebound of ATP and TAN by 10% vs. Group I. Mitochondrial TAN and free AMP concentrations paralleled that of total cardiac TAN. Changes in energy equilibrium or mitochondrial function were minor. This study highlights that only a fraction of the cardiac adenine nucleotide pool is available for adenosine production, but further studies are necessary to clarify its nature.

The Influence of Mitochondrial Energy and 1C Metabolism on the Efficacy of Anticancer Drugs: Exploring Potential Mechanisms of Resistance.

Mitochondria are the main energy factory in living cells. To rapidly proliferate and metastasize, neoplastic cells increase their energy requirements. Thus, mitochondria become one of the most important organelles for them. Indeed, much research shows the interplay between cancer chemoresistance and altered mitochondrial function. In this review, we focus on the differences in energy metabolism between cancer and normal cells to better understand their resistance and how to develop drugs targeting energy metabolism and nucleotide synthesis. One of the differences between cancer and normal cells is the higher nicotinamide adenine dinucleotide (NAD+) level, a cofactor for the tricarboxylic acid cycle (TCA), which enhances their proliferation and helps cancer cells survive under hypoxic conditions. An important change is a metabolic switch called the Warburg effect. This effect is based on the change of energy harvesting from oxygen-dependent transformation to oxidative phosphorylation (OXPHOS), adapting them to the tumor environment. Another mechanism is the high expression of one-carbon (1C) metabolism enzymes. Again, this allows cancer cells to increase proliferation by producing precursors for the synthesis of nucleotides and amino acids. We reviewed drugs in clinical practice and development targeting NAD+, OXPHOS, and 1C metabolism. Combining novel drugs with conventional antineoplastic agents may prove to be a promising new way of anticancer treatment.

Nucleotide metabolism during experimental preservation for transplantation with Transmedium Transplant Fluid (TTF) in comparison to Histidine-Tryptophan-Ketoglutarate (HTK).

Organ preservation solutions are essential to diminish ischemic/hypoxic injury during cold storage and to improve graft survival. In our experiments, we investigated novel solutions that target such mechanisms as Transmedium Transplant Fluid (TTF) in comparison to PlegiStore solution (HTK). Rat hearts were infused with TTF or HTK and then subjected to 4 hours of 4 °C preservation followed by 25 minutes of reperfusion in the Langendorff system. Assessment of purine release from the heart, mechanical function, and cardiac nucleotide content in the heart homogenates was done. A significant increase in the uric acid, hypoxanthine, inosine, and total purine metabolite concentrations were observed in the HTK hearts when compared to TTF. The TTF group had lower left ventricular systolic pressure and left ventricular end-diastolic pressure when compared to the HTK. Left ventricular diastolic pressure, minimal dp/dt, and maximal dp/dt in both groups were similar. The concentration of ADP in the heart homogenates of the HTK group was increased when compared to the TTF group. ATP and GTP concentration showed a tendency to increase in the homogenates of TTF hearts when NAD, AMP, GDP, GMP, and ADPR were similar in both groups of rats. TTF provided enhanced cardioprotection as evidenced by inhibiting the purine nucleotide metabolites released from the rat hearts during reperfusion and enhanced systolic and diastolic mechanical function recovery. In particular, better preservation of GTP and ATP concentrations may translate into enhanced protection of endothelium and the cytoskeleton, which are not adequately protected with current preservation techniques.

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.

Lactate Dehydrogenase and its clinical significance in pancreatic and thoracic cancers.

The energy metabolism of tumor cells is considered one of the hallmarks of cancer because it is different from normal cells and mainly consists of aerobic glycolysis, fatty acid oxidation, and glutaminolysis. It is about one hundred years ago since Warburg observed that cancer cells prefer aerobic glycolysis even in normoxic conditions, favoring their high proliferation rate. A pivotal enzyme driving this phenomenon is lactate dehydrogenase (LDH), and this review describes prognostic and therapeutic opportunities associated with this enzyme, focussing on tumors with limited therapeutic strategies and life expectancy (i.e., pancreatic and thoracic cancers). Expression levels of LDH-A in pancreatic cancer tissues correlate with clinicopathological features: LDH-A is overexpressed during pancreatic carcinogenesis and showed significantly higher expression in more aggressive tumors. Similarly, LDH levels are a marker of negative prognosis in patients with both adenocarcinoma or squamous cell lung carcinoma, as well as in malignant pleural mesothelioma. Additionally, serum LDH levels may play a key role in the clinical management of these diseases because they are associated with tissue damage induced by tumor burden. Lastly, we discuss the promising results of strategies targeting LDH as a treatment strategy, reporting recent preclinical and translational studies supporting the use of LDH-inhibitors in combinations with current/novel chemotherapeutics that can synergistically target the oxygenated cells present in the tumor.

Hepatocyte Nuclear Factor 1α Proinflammatory Effect Linked to the Overexpression of Liver Nuclear Factor-κB in Experimental Model of Chronic Kidney Disease.

Chronic kidney disease (CKD) is associated with low-grade inflammation that activates nuclear factor-κB (NF-κB), which upregulates the expression of numerous NF-κB responsive genes, including the genes encoding IL-6, ICAM-1, VCAM-1, and MCP-1. Herein, we found the coordinated overexpression of genes encoding RelA/p65 (a subunit of NF-κB) and HNF1α in the livers of chronic renal failure (CRF) rats-an experimental model of CKD. The coordinated overexpression of RelA/p65 and HNF1α was associated with a significant increase in IL-6, ICAM-1, VCAM-1, and MCP-1 gene expressions. A positive correlation between liver RelA/p65 mRNA levels and a serum concentration of creatinine and BUN suggest that RelA/p65 gene transcription is tightly related to the progression of renal failure. The knockdown of HNF1α in the HepG2 cell line by siRNA led to a decrease in Rel A/p65 mRNA levels. This was associated with a decrease in IL-6, ICAM-1, VCAM-1, and MCP-1 gene expressions. The simultaneous repression of HNF-1α and RelA/p65 by clofibrate is tightly associated with the downregulation of IL-6, ICAM-1, VCAM-1, and MCP-1 gene expression. In conclusion, our findings suggest that NF-κB could be a downstream component of the HNF1α-initiated signaling pathway in the livers of CRF rats.

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.