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The role of nitric oxide (NO) in tumour progression and metastasis is not clear, therefore the present work aimed to better characterise the effects of nitric oxide synthase (NOS) inhibition by L-Nω -nitroarginine methyl ester (L-NAME) on primary tumour growth, pulmonary metastasis, inflammatory state and prostacyclin (PGI2)/thromboxane A2 (TXA2) balance in a 4T1 murine model of breast cancer. To distinguish effects of NO deficiency on disease development, 4T1 cancer cells were administered orthotopically or intravenously to Balb/c mice. The systemic NO bioavailability, pulmonary inflammation and plasma levels of thromboxane B2 (TXB2) and 6-keto-prostaglandin F1α (6-keto-PGF1α ) were assessed. The study shows that, in the orthotopic model of 4T1 breast cancer, L-NAME hampered primary tumour growth, reduced pulmonary metastases, delayed inflammatory response but did not alter biosynthesis of TXB2 and 6- keto-PGF1α as well as PGI2/TXA2 ratio in cancer-bearing mice. Interestingly, in the intravenous model of 4T1 breast cancer, NOS inhibition did not influence metastasis nor inflammation, but it increased both TXB2 and 6-keto-PGF1α biosynthesis without affecting PGI2/TXA2 ratio. In conclusion, in a 4T1 murine model of metastatic breast cancer, NO plays a major role in primary tumour development, while NO is not the key mediator of cancer cell extravasation to the lungs. Furthermore, NO-deficiency activates a PGI2-dependent compensatory mechanism only in the intravenous model of 4T1 breast cancer. [ABSTRACT FROM AUTHOR]

Background: Patients with cancer develop endothelial dysfunction and subsequently display a higher risk of cardiovascular events. The aim of the present work was to examine changes in nitric oxide (NO)- and prostacyclin (PGI2)-dependent endothelial function in the systemic conduit artery (aorta), in relation to the formation of lung metastases and to local and systemic inflammation in a murine orthotopic model of metastatic breast cancer.Methods: BALB/c female mice were orthotopically inoculated with 4T1 breast cancer cells. Development of lung metastases, lung inflammation, changes in blood count, systemic inflammatory response (e.g. SAA, SAP and IL-6), as well as changes in NO- and PGI2-dependent endothelial function in the aorta, were examined 2, 4, 5 and 6 weeks following cancer cell transplantation.Results: As early as 2 weeks following transplantation of breast cancer cells, in the early metastatic stage, lungs displayed histopathological signs of inflammation, NO production was impaired and nitrosylhemoglobin concentration in plasma was decreased. After 4 to 6 weeks, along with metastatic development, progressive leukocytosis and systemic inflammation (as seen through increased SAA, SAP, haptoglobin and IL-6 plasma concentrations) were observed. Six weeks following cancer cell inoculation, but not earlier, endothelial dysfunction in aorta was detected; this involved a decrease in basal NO production and a decrease in NO-dependent vasodilatation, that was associated with a compensatory increase in cyclooxygenase-2 (COX-2)- derived PGI2 production.Conclusions: In 4 T1 metastatic breast cancer in mice early pulmonary metastasis was correlated with lung inflammation, with an early decrease in pulmonary as well as systemic NO availability. Late metastasis was associated with robust, cancer-related, systemic inflammation and impairment of NO-dependent endothelial function in the aorta that was associated with compensatory upregulation of the COX-2-derived PGI2 pathway. [ABSTRACT FROM AUTHOR]

Phylloquinon (PK) and menaquinones (MK) are both naturally occurring compounds belonging to vitamin K group. Present study aimed to comprehensively analyze the influence of PK in several models of vascular dysfunction to determine whether PK has vasoprotective properties, similar to those previously described for MK. Effects of PK and MK on endothelial dysfunction were studied in ApoE/LDLR -/- mice in vivo, in the isolated aorta incubated with TNF, and in vascular cells as regard inflammation and cell senescence (including replicative and stress-induced models of senescence). Moreover, the vascular conversion of exogenous vitamins to endogenous MK-4 was analyzed. PK, as well as MK, given for 8 weeks in diet (10 mg/kg) resulted in comparable improvement in endothelial function in the ApoE/LDLR -/- mice. Similarly, PK and MK prevented TNF-induced impairment of endothelium-dependent vasorelaxation in the isolated aorta. In in vitro studies in endothelial and vascular smooth muscle cells, we identified that both PK and MK displayed anti-senescence effects via decreasing DNA damage while in endothelial cells anti-inflammatory activity was ascribed to the modulation of NFκB activation. The activity of PK and MK was comparable in terms of their effect on senescence and inflammation. Presence of endogenous synthesis of MK-4 from PK in aorta and endothelial and smooth muscle cells suggests a possible involvement of MK in vascular effects of PK. In conclusion, PK and MK display comparable vasoprotective effects, which may be ascribed, at least in part, to the inhibition of cell senescence and inflammation. The vasoprotective effect of PK in the vessel wall can be related to the direct effects of PK, as well as to the action of MK formed from PK in the vascular wall., (© 2024. The Author(s).)

Ageing is a major risk factor for cancer metastasis but the underlying mechanisms remain unclear. Here, we characterised ageing effects on cancer-induced endothelial-mesenchymal transition (EndMT) in the pulmonary circulation of female BALB/c mice in a metastatic 4T1 breast cancer model. The effect of intravenously injected 4T1 cells on pulmonary endothelium, pulmonary metastasis, lung tissue architecture, and systemic endothelium was compared between 40-week-old and 20-week-old mice. The 40-week-old mice showed features of ongoing EndMT in their lungs before 4T1 breast cancer cell injection. Moreover, they had preexisting endothelial dysfunction in the aorta detected by in vivo magnetic resonance imaging (MRI) compared to 20-week-old mice. The injection of 4T1 breast cancer cells into 40-week-old mice resulted in rapid EndMT progression in their lungs. In contrast, injection of 4T1 breast cancer cells into 20-week-old mice resulted in initiation and less pronounced EndMT progression. Although the number of metastases did not differ significantly between 20-week-old and 40-week-old mice, the lungs of older mice displayed altered lung tissue architecture and biochemical content, reflected in higher Amide II/Amide I ratio, higher fibronectin levels, and hypoxia-inducible factor 1 subunit alpha (HIF1α) levels as well as lower nitric oxide (NO) production. Our results indicate that age-dependent pre-existing endothelial dysfunction in the pulmonary endothelium of 40-week-old mice predisposed them to rapid EndMT progression in the presence of circulating 4T1 breast cancer cells what might contribute to a more severe metastatic breast cancer phenotype in these ageing mice compared to younger mice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Smeda, Jasztal, Maleki, Bar, Sternak, Kwiatkowski, Suraj-Prażmowska, Proniewski, Kieronska-Rudek, Wojnar-Lason, Skrzypek, Majka, Chrabaszcz, Malek and Chlopicki.)

Aims: Endothelial dysfunction (ED) and red blood cell distribution width (RDW) are both prognostic factors in heart failure (HF), but the relationship between them is not clear. In this study, we used a unique mouse model of chronic HF driven by cardiomyocyte-specific overexpression of activated Gαq protein (Tgαq*44 mice) to characterize the relationship between the development of peripheral ED and the occurrence of structural nanomechanical and biochemical changes in red blood cells (RBCs)., Methods and Results: Systemic ED was detected in vivo in 8-month-old Tgαq*44 mice, as evidenced by impaired acetylcholine-induced vasodilation in the aorta and increased endothelial permeability in the brachiocephalic artery. ED in the aorta was associated with impaired nitric oxide (NO) production in the aorta and diminished systemic NO bioavailability. ED in the aorta was also characterized by increased superoxide and eicosanoid production. In 4- to 6-month-old Tgαq*44 mice, RBC size and membrane composition displayed alterations that did not result in significant changes in their nanomechanical and functional properties. However, 8-month-old Tgαq*44 mice presented greatly accentuated structural and size changes and increased RBC stiffness. In 12-month-old Tgαq*44 mice, the erythropathy was featured by severely altered RBC shape and elasticity, increased RDW, impaired RBC deformability, and increased oxidative stress (gluthatione (GSH)/glutathione disulfide (GSSG) ratio). Moreover, RBCs taken from 12-month-old Tgαq*44 mice, but not from 12-month-old FVB mice, coincubated with aortic rings from FVB mice, induced impaired endothelium-dependent vasodilation and this effect was partially reversed by an arginase inhibitor [2(S)-amino-6-boronohexanoic acid]., Conclusion: In the Tgαq*44 murine model of HF, systemic ED accelerates erythropathy and, conversely, erythropathy may contribute to ED. These results suggest that erythropathy may be regarded as a marker and a mediator of systemic ED in HF. RBC arginase and possibly other RBC-mediated mechanisms may represent novel therapeutic targets for systemic ED in HF., Competing Interests: Conflict of interest: none declared., (© The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Angiotensin II (Ang II) induces hypertension and endothelial dysfunction, but the involvement of thrombin in these responses is not clear. Here, we assessed the effects of the inhibition of thrombin activity by dabigatran on Ang II-induced hypertension and endothelial dysfunction in mice with a particular focus on NO- and 20-HETE-dependent pathways. As expected, dabigatran administration significantly delayed thrombin generation (CAT assay) in Ang II-treated hypertensive mice, and interestingly, it prevented endothelial dysfunction development, but it did not affect elevated blood pressure nor excessive aortic wall thickening. Dabigatran's effects on endothelial function in Ang II-treated mice were evidenced by improved NO-dependent relaxation in the aorta in response to acetylcholine in vivo (MRI measurements) and increased systemic NO bioavailability (NO 2 - quantification) with a concomitant increased ex vivo production of endothelium-derived NO (EPR analysis). Dabigatran treatment also contributed to the reduction in the endothelial expression of pro-inflammatory vWF and ICAM-1. Interestingly, the fall in systemic NO bioavailability in Ang II-treated mice was associated with increased 20-HETE concentration in plasma (UPLC-MS/MS analysis), which was normalised by dabigatran treatment. Taking together, the inhibition of thrombin activity in Ang II-induced hypertension in mice improves the NO-dependent function of vascular endothelium and normalises the 20-HETE-depedent pathway without affecting the blood pressure and vascular remodelling.

Hyperglycemia linked to diabetes results in endothelial dysfunction. In the present work, we comprehensively characterized effects of short-term hyperglycemia induced by administration of an insulin receptor antagonist, the S961 peptide, on endothelium and perivascular adipose tissue (PVAT) in mice. Endothelial function of the thoracic and abdominal aorta in 12-week-old male C57Bl/6Jrj mice treated for two weeks with S961 infusion via osmotic pumps was assessed in vivo using magnetic resonance imaging and ex vivo by detection of nitric oxide (NO) production using electron paramagnetic resonance spectroscopy. Additional methods were used to analyze PVAT, aortic segments and endothelial-specific plasma biomarkers. Systemic disruption of insulin signaling resulted in severe impairment of NO-dependent endothelial function and a loss of vasoprotective function of PVAT affecting the thoracic as well as abdominal parts of the aorta, however a fall in adiponectin expression and decreased uncoupling protein 1-positive area were more pronounced in the thoracic aorta. Results suggest that dysfunctional PVAT contributes to vascular pathology induced by altered insulin signaling in diabetes, in the absence of fat overload and obesity.

Background Long-term feeding with a high-fat diet (HFD) induces endothelial dysfunction in mice, but early HFD-induced effects on endothelium have not been well characterized. Methods and Results Using an magnetic resonance imaging-based methodology that allows characterization of endothelial function in vivo, we demonstrated that short-term (2 weeks) feeding with a HFD to C57BL/6 mice or to E3L.CETP mice resulted in the impairment of acetylcholine-induced response in the abdominal aorta (AA), whereas, in the thoracic aorta (TA), the acetylcholine-induced response was largely preserved. Similarly, HFD resulted in arterial stiffness in the AA, but not in the TA. The difference in HFD-induced response was ascribed to distinct characteristics of perivascular adipose tissue in the TA and AA, related to brown- and white-like adipose tissue, respectively, as assessed by histology, immunohistochemistry, and Raman spectroscopy. In contrast, short-term HFD-induced endothelial dysfunction could not be linked to systemic insulin resistance, changes in plasma concentration of nitrite, or concentration of biomarkers of glycocalyx disruption (syndecan-1 and endocan), endothelial inflammation (soluble form of vascular cell adhesion molecule 1, soluble form of intercellular adhesion molecule 1 and soluble form of E-selectin), endothelial permeability (soluble form of fms-like tyrosine kinase 1 and angiopoietin 2), and hemostasis (tissue plasminogen activator and plasminogen activator inhibitor 1). Conclusions Short-term feeding with a HFD induces endothelial dysfunction in the AA but not in the TA, which could be ascribed to a differential response of perivascular adipose tissue to a HFD in the AA versus TA. Importantly, early endothelial dysfunction in the AA is not linked to elevation of classical systemic biomarkers of endothelial dysfunction.

Long-term administration of acetylsalicylic acid (ASA) was effective in prevention of colorectal cancer, whereas the efficacy of this compound in other cancer types, including breast cancer, has been less convincingly documented. Indeed, the antimetastatic effect of low-dose ASA was observed only in the early intravascular phase of metastasis of breast cancer. In the present work, we characterized the effects of long-term treatment with ASA on the late phase of pulmonary metastasis in a mouse orthotopic 4T1 breast cancer model. Mice were treated with ASA at a dose of 12 mg·kg-1 of body weight daily starting one week prior to inoculation of 4T1 breast cancer cells, and the treatment was continued throughout progression of the disease. ASA administration decreased platelet TXB2 production in ex vivo assays but did not change thrombin-induced platelet reactivity. Although the number of metastases in the lungs remained unchanged in ASA-treated mice, infiltration of inflammatory cells was increased concomitantly with higher G-CSF and serotonin concentrations in the lungs. Pulmonary NO production was compromised compared to control 4T1 mice. ASA treatment also evoked an increase in platelet and granulocyte counts and decreased systemic NO bioavailability along with increased markers of systemic oxidant stress such as higher GSSG/lower GSH concentrations in RBC. Analysis of eicosanoids in stirred blood demonstrated that administration of ASA at a dose of 12 mg·kg-1 to cancer-bearing mice had an effect beyond inhibition of platelet COX-1, suggesting long-term treatment with low-dose aspirin is not a selective murine platelet COX-1/TXA2 pathway inhibitor in cancer-bearing mice. In summary, quite surprisingly, long-term treatment with low-dose ASA administered until the advanced phase of breast cancer in a murine orthotopic model of 4T1 breast cancer negatively affected the phenotype of the disease., Competing Interests: The authors have declared that no competing interests exist.

Premature senescence, a death escaping pathway for cells experiencing stress, is conducive to aging and cardiovascular diseases. The molecular switch between senescent and apoptotic fate remains, however, poorly recognized. Nrf2 is an important transcription factor orchestrating adaptive response to cellular stress. Here, we show that both human primary endothelial cells (ECs) and murine aortas lacking Nrf2 signaling are senescent but unexpectedly do not encounter damaging oxidative stress. Instead, they exhibit markedly increased S-nitrosation of proteins. A functional role of S-nitrosation is protection of ECs from death by inhibition of NOX4-mediated oxidative damage and redirection of ECs to premature senescence. S-nitrosation and senescence are mediated by Keap1, a direct binding partner of Nrf2, which colocalizes and precipitates with nitric oxide synthase (NOS) and transnitrosating protein GAPDH in ECs devoid of Nrf2. We conclude that the overabundance of this "unrestrained" Keap1 determines the fate of ECs by regulation of S-nitrosation and propose that Keap1/GAPDH/NOS complex may serve as an enzymatic machinery for S-nitrosation in mammalian cells., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Detection of free radicals in tissues is challenging. Most approaches rely on incubating excised sections or homogenates with reagents, typically at supraphysiologic oxygen tensions, to finally detect surrogate, nonspecific end products. In the present work, we explored the potential of using intravenously (i.v.) injected dihydroethidine (DHE) to detect superoxide radical (O 2 ∙- ) abundance in vivo by quantification of the superoxide-specific DHE oxidation product, 2-hydroxyethidium (2-OH-E + ), as well as ethidium (E + ) and DHE in multiple tissues in a murine model of endotoxemia induced by lipopolysaccharide (LPS). LPS was injected intraperitoneally (i.p.), while DHE was delivered via the tail vein one hour before sacrifice. Tissues (kidney, lung, liver, and brain) were harvested and subjected to HPLC/fluorescent analysis of DHE and its monomeric oxidation products. In parallel, electron spin resonance (EPR) spin trapping was used to measure nitric oxide ( ∙ NO) production in the aorta, lung, and liver isolated from the same mice. Endotoxemic inflammation was validated by analysis of plasma biomarkers. The concentration of 2-OH-E + varied in the liver, lung, and kidney; however, the ratios of 2-OH-E + /E + and 2-OH-E + /DHE were increased in the liver and kidney but not in the lung or the brain. An LPS-induced robust level of ∙ NO burst was observed in the liver, whereas the lung demonstrated a moderate yet progressive increase in the rate of ∙ NO production. Interestingly, endothelial dysfunction was observed in the aorta, as evidenced by decreased ∙ NO production 6 hours post-LPS injection that coincided with the inflammatory burden of endotoxemia (e.g. elevated serum amyloid A and prostaglandin E 2 ). Combined, these data demonstrate that systemic delivery of DHE affords the capacity to specifically detect O 2 ∙- production in vivo . Furthermore, the ratio of 2-OH-E + /E + oxidation products in tissues provides a tool for comparative insight into the oxidative environments in various organs. Based on our findings, we demonstrate that the endotoxemic liver is susceptible to both O 2 ∙- -mediated and nonspecific oxidant stress as well as nitrosative stress. Oxidant stress in the lung was detected to a lesser extent, thus underscoring a differential response of liver and lung to endotoxemic injury induced by intraperitoneal LPS injection.

Background The impairment of endothelium-dependent vasodilation, increased endothelial permeability, and glycocalyx degradation are all important pathophysiological components of endothelial dysfunction. However, it is still not clear whether in atherosclerosis, glycocalyx injury precedes other features of endothelial dysfunction or these events coincide. Methods and Results Herein, we demonstrate that in 4- to 8-week-old apolipoprotein E/low-density lipoprotein receptor-deficient mice, at the stage before development of atherosclerotic plaques, impaired acetylcholine-induced vasodilation, reduced NO production in aorta, and increased endothelial permeability were all observed; however, flow-mediated dilation in the femoral artery was fully preserved. In 4-week-old mice, glycocalyx coverage was reduced and endothelial stiffness was increased, whereas glycocalyx length was significantly decreased at 8 weeks of age. Early changes in endothelial function were also featured by increased plasma concentration of biomarkers of glycocalyx disruption (endocan), biomarkers of endothelial inflammation (soluble vascular cell adhesion molecule 1), increased vascular permeability (angiopoietin 2), and alterations in hemostasis (tissue plasminogen activator and plasminogen activator inhibitor 1). In 28-week-old mice, at the stage of advanced atherosclerotic plaque development, impaired NO production and nearly all other features of endothelial dysfunction were changed to a similar extent, compared with the preatherosclerotic plaque phase. The exceptions were the occurrence of acetylcholine-induced vasoconstriction in the aorta and brachiocephalic artery, impaired flow-mediated vasodilation in the femoral artery, and further reduction of glycocalyx length and coverage with a concomitant further increase in endothelial permeability. Conclusions In conclusion, even at the early stage before the development of atherosclerotic plaques, endothelial dysfunction is a complex multifactorial response that has not been previously appreciated.

The role of nitric oxide (NO) in tumour progression and metastasis is not clear, therefore the present work aimed to better characterise the effects of nitric oxide synthase (NOS) inhibition by L-N ω -nitroarginine methyl ester (L-NAME) on primary tumour growth, pulmonary metastasis, inflammatory state and prostacyclin (PGI 2 )/thromboxane A 2 (TXA 2 ) balance in a 4T1 murine model of breast cancer. To distinguish effects of NO deficiency on disease development, 4T1 cancer cells were administered orthotopically or intravenously to Balb/c mice. The systemic NO bioavailability, pulmonary inflammation and plasma levels of thromboxane B 2 (TXB 2 ) and 6-keto-prostaglandin F 1α (6-keto-PGF 1α ) were assessed. The study shows that, in the orthotopic model of 4T1 breast cancer, L-NAME hampered primary tumour growth, reduced pulmonary metastases, delayed inflammatory response but did not alter biosynthesis of TXB 2 and 6-keto-PGF 1α as well as PGI 2 /TXA 2 ratio in cancer-bearing mice. Interestingly, in the intravenous model of 4T1 breast cancer, NOS inhibition did not influence metastasis nor inflammation, but it increased both TXB 2 and 6-keto-PGF 1α biosynthesis without affecting PGI 2 /TXA 2 ratio. In conclusion, in a 4T1 murine model of metastatic breast cancer, NO plays a major role in primary tumour development, while NO is not the key mediator of cancer cell extravasation to the lungs. Furthermore, NO-deficiency activates a PGI 2 -dependent compensatory mechanism only in the intravenous model of 4T1 breast cancer.

Background: Mesenchymal transformation of pulmonary endothelial cells contributes to the formation of a metastatic microenvironment, but it is not known whether this precedes or follows early metastasis formation. In the present work, we characterize the development of nitric oxide (NO) deficiency and markers of endothelial-mesenchymal transition (EndMT) in the lung in relation to the progression of 4T1 metastatic breast cancer injected orthotopically in mice., Methods: NO production, endothelial nitric oxide synthase (eNOS) phosphorylation status, markers of EndMT in the lung, pulmonary endothelium permeability, and platelet activation/reactivity were analyzed in relation to the progression of 4T1 breast cancer metastasis to the lung, as well as to lung tissue remodeling, 1-5 weeks after 4T1 cancer cell inoculation in Balb/c mice., Results: Phosphorylation of eNOS and NO production in the lungs of 4T1 breast cancer-bearing mice was compromised prior to the development of pulmonary metastasis, and was associated with overexpression of Snail transcription factor in the pulmonary endothelium. These changes developed prior to the mesenchymal phenotypic switch in the lungs evidenced by a decrease in vascular endothelial-cadherin (VE-CAD) and CD31 expression, and the increase in pulmonary endothelial permeability, phenomena which coincided with early pulmonary metastasis. Increased activation of platelets was also detected prior to the early phase of metastasis and persisted to the late phase of metastasis, as evidenced by the higher percentage of unstimulated platelets binding fibrinogen without changes in von Willebrand factor and fibrinogen binding in response to ADP stimulation., Conclusions: Decreased eNOS activity and phosphorylation resulting in a low NO production state featuring pulmonary endothelial dysfunction was an early event in breast cancer pulmonary metastasis, preceding the onset of its phenotypic switch toward a mesenchymal phenotype (EndMT) evidenced by a decrease in VE-CAD and CD31 expression. The latter coincided with development of the first metastatic nodules in the lungs. These findings suggest that early endothelial dysfunction featured by NO deficiency rather than EndMT, might represent a primary regulatory target to prevent early pulmonary metastasis.

Introduction: An acute bout of strenuous exercise in humans results in transient impairment of nitric oxide (NO)-dependent function, but it remains unknown whether this phenomenon is associated with increased risk of thrombotic events after exercise. This study aimed to evaluate effects of a single bout of exhaustive running in mice on the balance of vascular NO/reactive oxygen species production, and on thrombogenicity., Methods: At different time points (0, 2, and 4 h) after exercise and in sedentary C57BL/6 mice, the production of NO and superoxide (O2) in aorta was measured by electron paramagnetic resonance spin trapping and by dihydroethidium/high-performance liquid chromatography-based method, respectively, whereas collagen-induced thrombus formation was analyzed in a microchip-based flow-chamber system (total thrombus-formation analysis system). We also measured pre- and postexercise plasma concentration of nitrite/nitrate and 6-keto-PGF1α., Results: An acute bout of exhaustive running in mice resulted in decreased production of NO and increased production of O2 in aorta, with maximum changes 2 h after completion of exercise when compared with sedentary mice. However, platelet thrombus formation was not changed by exercise as evidenced by unaltered time to start of thrombus formation, capillary occlusion time, and total thrombogenicity (area under the flow pressure curve) as measured in a flow-chamber system. Strenuous exercise increased the plasma concentration of nitrite but did not affect nitrate and 6-keto-PGF1α concentrations., Conclusion: An acute bout of strenuous exercise in mice reduced NO and in parallel increased O2 production in aorta. This response was most pronounced 2 h after exercise. Surprisingly, the reduced NO and increased O2 production in mice after exercise did not result in increased platelet-dependent thrombogenicity. These results show that transient reduction in NO bioavailability does not modify thromboresistance in healthy mice after exercise.

Recent studies suggest both beneficial and detrimental role of increased reactive oxygen species and oxidative stress in heart failure (HF). However, it is not clear at which stage oxidative stress and oxidative modifications occur in the endothelium in relation to cardiomyocytes in non-ischemic HF. Furthermore, most methods used to date to study oxidative stress are either non-specific or require tissue homogenization. In this study, we used immuno-spin trapping (IST) technique with fluorescent microscopy-based detection of DMPO nitrone adducts to localize and quantify oxidative modifications of the hearts from Tgαq*44 mice; a murine model of HF driven by cardiomyocyte-specific overexpression of Gαq* protein. Tgαq*44 mice and age-matched FVB controls at early, transition, and late stages of HF progression were injected with DMPO in vivo and analyzed ex vivo for DMPO nitrone adducts signals. Progressive oxidative modifications in cardiomyocytes, as evidenced by the elevation of DMPO nitrone adducts, were detected in hearts from 10- to 16-month-old, but not in 8-month-old Tgαq*44 mice, as compared with age-matched FVB mice. The DMPO nitrone adducts were detected in left and right ventricle, septum, and papillary muscle. Surprisingly, significant elevation of DMPO nitrone adducts was also present in the coronary endothelium both in large arteries and in microcirculation simultaneously, as in cardiomyocytes, starting from 10-month-old Tgαq*44 mice. On the other hand, superoxide production in heart homogenates was elevated already in 6-month-old Tgαq*44 mice and progressively increased to high levels in 14-month-old Tgαq*44 mice, while the enzymatic activity of catalase, glutathione reductase, and glutathione peroxidase was all elevated as early as in 4-month-old Tgαq*44 mice and stayed at a similar level in 14-month-old Tgαq*44. In summary, this study demonstrates that IST represents a unique method that allows to quantify oxidative modifications in cardiomyocytes and coronary endothelium in the heart. In Tgαq*44 mice with slowly developing HF, driven by cardiomyocyte-specific overexpression of Gαq* protein, an increase in superoxide production, despite compensatory activation of antioxidative mechanisms, results in the development of oxidative modifications not only in cardiomyocytes but also in coronary endothelium, at the transition phase of HF, before the end-stage disease.

Platelet inhibition has been considered an effective strategy for combating cancer metastasis and compromising disease malignancy although recent clinical data provided evidence that long-term platelet inhibition might increase incidence of cancer deaths in initially cancer-free patients. In the present study we demonstrated that dual anti-platelet therapy based on aspirin and clopidogrel (ASA+Cl), a routine regiment in cardiovascular patients, when given to cancer-bearing mice injected orthotopically with 4T1 breast cancer cells, promoted progression of the disease and reduced mice survival in association with induction of vascular mimicry (VM) in primary tumour. In contrast, treatment with ASA+Cl or platelet depletion did reduce pulmonary metastasis in mice, if 4T1 cells were injected intravenously. In conclusion, distinct platelet-dependent mechanisms inhibited by ASA+Cl treatment promoted cancer malignancy and VM in the presence of primary tumour and afforded protection against pulmonary metastasis in the absence of primary tumour. In view of our data, long-term inhibition of platelet function by dual anti-platelet therapy (ASA+Cl) might pose a hazard when applied to a patient with undiagnosed and untreated malignant cancer prone to undergo VM., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Carbon monoxide-releasing molecules (CO-RMs) induce nitric oxide (NO) release (which requires NADPH), and Ca 2+ -dependent signalling; however, their contribution in mediating endothelial responses to CO-RMs is not clear. Here, we studied the effects of CO liberated from CORM-401 on NO production, calcium signalling and pentose phosphate pathway (PPP) activity in human endothelial cell line (EA.hy926). CORM-401 induced NO production and two types of calcium signalling: a peak-like calcium signal and a gradual increase in cytosolic calcium. CORM-401-induced peak-like calcium signal, originating from endoplasmic reticulum, was reduced by thapsigargin, a SERCA inhibitor, and by dantrolene, a ryanodine receptors (RyR) inhibitor. In contrast, the phospholipase C inhibitor U73122 did not significantly affect peak-like calcium signalling, but a slow and progressive CORM-401-induced increase in cytosolic calcium was dependent on store-operated calcium entrance. CORM-401 augmented coupling of endoplasmic reticulum and plasmalemmal store-operated calcium channels. Interestingly, in the presence of NO synthase inhibitor (l-NAME) CORM-401-induced increases in NO and cytosolic calcium were both abrogated. CORM-401-induced calcium signalling was also inhibited by superoxide dismutase (poly(ethylene glycol)-SOD). Furthermore, CORM-401 accelerated PPP, increased NADPH concentration and decreased the ratio of reduced to oxidized glutathione (GSH/GSSG). Importantly, CORM-401-induced NO increase was inhibited by the PPP inhibitor 6-aminonicotinamide (6-AN), but neither by dantrolene nor by an inhibitor of large-conductance calcium-regulated potassium ion channel (paxilline). The results identify the primary role of CO-induced NO increase in the regulation of endothelial calcium signalling, that may have important consequences in controlling endothelial function., (© 2018 Federation of European Biochemical Societies.)

It has been repeatedly shown that regular aerobic exercise exerts beneficial effects on incidence and progression of cancer. However, the data regarding effects of exercise on metastatic dissemination remain conflicting. Therefore, in the present study the possible preventive effects of voluntary wheel running on primary tumor growth and metastases formation in the model of spontaneous pulmonary metastasis were analyzed after orthotopic injection of 4T1 breast cancer cells into mammary fat pads of female Balb/C mice. This study identified that in the mice injected with 4T1 breast cancer cells and running on the wheels (4T1 ex) the volume and size of the primary tumor were not affected, but the number of secondary nodules formed in the lungs was significantly increased compared to their sedentary counterparts (4T1 sed). This effect was associated with decreased NO production in the isolated aorta of exercising mice (4T1 ex), suggesting deterioration of endothelial function that was associated with lower platelet count without their overactivation. This was evidenced by comparable selectin P, active GPIIb/IIIa expression, fibrinogen and vWF binding on the platelet surface. In conclusion, voluntary wheel running appeared to impair, rather than improve endothelial function, and to promote, but not decrease metastasis in the murine orthotopic model of metastatic breast cancer. These results call for revising the notion of the persistent beneficial effects of voluntary exercise on breast cancer progression, though further studies are needed to elucidate mechanisms involved in pro-metastatic effects of voluntary exercise., Competing Interests: None.