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.

Functional deterioration of vascular mitochondrial and glycolytic capacity in the aortic rings of aged mice.

Vascular ageing is associated with increased arterial stiffness and cardiovascular mortality that might be linked to altered vascular energy metabolism. The aim of this study was to establish a Seahorse XFe96 Analyzer-based methodology for the reliable, functional assessment of mitochondrial respiration and glycolysis in single murine aortic rings and to validate this functional assay by characterising alterations in vascular energy metabolism in aged mice. Healthy young and old C57BL/6 mice were used for the analyses. An optimised setup consisting of the Seahorse XFe96 Analyzer and Seahorse Spheroid Microplates was applied for the mitochondrial stress test and the glycolysis stress test on the isolated murine aortic rings, supplemented with analysis of NAD content in the aorta. To confirm the age-dependent stiffness of the vasculature, pulse wave velocity was measured in vivo. In addition, the activity of vascular nitric oxide synthase and vascular wall morphology were analysed ex vivo. The vascular ageing phenotype in old mice was confirmed by increased aortic stiffness, vascular wall remodelling, and nitric oxide synthase activity impairment. The rings of the aorta taken from old mice showed changes in vascular energy metabolism, including impaired spare respiratory capacity, maximal respiration, glycolysis, and glycolytic capacity, as well as a fall in the NAD pool. In conclusion, optimised Seahorse XFe96-based analysis to study energy metabolism in single aortic rings of murine aorta revealed a robust impairment of functional vascular respiratory and glycolytic capacity in old mice linked to NAD deficiency that coincided with age-related aortic wall remodelling and stiffness.

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.

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.

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.

Serum fatty acid profiles in breast cancer patients following treatment.

BACKGROUND: Breast cancer is associated with alterations in lipid metabolism. The treatment of breast cancer can also affect serum lipid composition. The purpose of this study was the examination of serum fatty acids (FAs) profiles in breast cancer survivors to assess if the FA levels normalize. METHODS: Serum levels of FAs were determined by gas chromatography-mass spectrometry in a group of breast cancer patients at baseline (before treatment, n = 28), at two follow-up visits at 12 months (n = 27) and 24 months (n = 19) after the breast cancer resection, and in the group of healthy controls (n = 25). Multivariate analysis was performed to assess how FA serum profile changes following treatment. RESULTS: Breast cancer patients' serum FA profiles at follow-ups did not normalize to the levels of control group. The greatest differences were found for levels of branched-chain (BCFA), odd-chain (OCFA) and polyunsaturated (PUFAs) FAs, all of which were significantly increased 12 months after the surgery. CONCLUSIONS: After treatment for breast cancer, the patients' serum FA profile differs from the profile before treatment and from controls, especially 12 months after treatment. Some changes may be beneficial - increased BCFA and OCFA levels, and improved n-6/n-3 PUFA ratio. This may reflect lifestyle changes in breast cancer survivors and have an impact on the risk of recurrence.

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.

Rosiglitazone Ameliorates Cardiac and Skeletal Muscle Dysfunction by Correction of Energetics in Huntington's Disease.

Huntington's disease (HD) is a rare neurodegenerative disease that is accompanied by skeletal muscle atrophy and cardiomyopathy. Tissues affected by HD (central nervous system [CNS], skeletal muscle, and heart) are known to suffer from deteriorated cellular energy metabolism that manifests already at presymptomatic stages. This work aimed to test the effects of peroxisome proliferator-activated receptor (PPAR)-γ agonist-rosiglitazone on grip strength and heart function in an experimental HD model-on R6/1 mice and to address the mechanisms. We noted that rosiglitazone treatment lead to improvement of R6/1 mice grip strength and cardiac mechanical function. It was accompanied by an enhancement of the total adenine nucleotides pool, increased glucose oxidation, changes in mitochondrial number (indicated as increased citric synthase activity), and reduction in mitochondrial complex I activity. These metabolic changes were supported by increased total antioxidant status in HD mice injected with rosiglitazone. Correction of energy deficits with rosiglitazone was further indicated by decreased accumulation of nucleotide catabolites in HD mice serum. Thus, rosiglitazone treatment may not only delay neurodegeneration but also may ameliorate cardio- and myopathy linked to HD by improvement of cellular energetics.

Novel Targets for a Combination of Mechanical Unloading with Pharmacotherapy in Advanced Heart Failure.

LVAD therapy is an effective rescue in acute and especially chronic cardiac failure. In several scenarios, it provides a platform for regeneration and sustained myocardial recovery. While unloading seems to be a key element, pharmacotherapy may provide powerful tools to enhance effective cardiac regeneration. The synergy between LVAD support and medical agents may ensure satisfying outcomes on cardiomyocyte recovery followed by improved quality and quantity of patient life. This review summarizes the previous and contemporary strategies for combining LVAD with pharmacotherapy and proposes new therapeutic targets. Regulation of metabolic pathways, enhancing mitochondrial biogenesis and function, immunomodulating treatment, and stem-cell therapies represent therapeutic areas that require further experimental and clinical studies on their effectiveness in combination with mechanical unloading.

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.

Enhanced Muscle Strength in Dyslipidemic Mice and Its Relation to Increased Capacity for Fatty Acid Oxidation.

Dyslipidemia is commonly linked to skeletal muscle dysfunction, accumulation of intramyocellular lipids, and insulin resistance. However, our previous research indicated that dyslipidemia in apolipoprotein E and low-density lipoprotein receptor double knock-out mice (ApoE/LDLR -/-) leads to improvement of exercise capacity. This study aimed to investigate in detail skeletal muscle function and metabolism in these dyslipidemic mice. We found that ApoE/LDLR -/- mice showed an increased grip strength as well as increased troponins, and Mhc2 levels in skeletal muscle. It was accompanied by the increased skeletal muscle mitochondria numbers (judged by increased citrate synthase activity) and elevated total adenine nucleotides pool. We noted increased triglycerides contents in skeletal muscles and increased serum free fatty acids (FFA) levels in ApoE/LDLR -/- mice. Importantly, Ranolazine mediated inhibition of FFA oxidation in ApoE/LDLR -/- mice led to the reduction of exercise capacity and total adenine nucleotides pool. Thus, this study demonstrated that increased capacity for fatty acid oxidation, an adaptive response to dyslipidemia leads to improved cellular energetics that translates to increased skeletal muscle strength and contributes to increased exercise capacity in ApoE/LDLR -/- mice.

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.