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.

BackgroundMesenchymal 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.MethodsNO 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.ResultsPhosphorylation 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.ConclusionsDecreased 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.Electronic supplementary materialThe online version of this article (10.1186/s13058-018-1013-z) contains supplementary material, which is available to authorized users.

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.

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 (O2∙-) 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 E2). Combined, these data demonstrate that systemic delivery of DHE affords the capacity to specifically detect O2∙- 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 O2∙--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.

BackgroundPatients 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.MethodsBALB/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.ResultsAs 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.ConclusionsIn 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.

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.

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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 characterise 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 characterised 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 (GSH/GSSH ratio). Moreover, RBCs taken from 12-month-old Tgαq*44 mice, but not from 12-month-old FVB mice, co-incubated with aortic rings from FVB mice, induced impaired endothelium-dependent vasodilation and this effect was partially reversed by an arginase inhibitor (ABH, 2(S)-amino-6-boronohexanoic acid). Conclusion In the Tgαq*44 murine model of HF, systemic endothelial dysfunction accelerates erythropathy and, conversely, erythropathy may contribute to endothelial dysfunction. These results suggest that erythropathy may be regarded as a marker and a mediator of systemic endothelial dysfunction in HF. In particular, targeting RBC arginase may represent a novel treatment strategy for systemic endothelial dysfunction in HF. RBC arginase and possibly other RBC-mediated mechanisms may represent novel therapeutic targets for systemic endothelial dysfunction in HF. Translational perspective Endothelial dysfunction (ED) and red blood cell distribution width (RDW) both have prognostic value for heart failure (HF), but it is not known whether these pathologies are related. We comprehensively characterized endothelial and RBC functional status in a unique murine model of chronic heart failure with a prolonged time course of HF progression. Our results suggest that ED accelerates erythropathy and, conversely, erythropathy may contribute to ED. Accordingly, erythropathy in HF reflects ED and involves various changes (in functional, structural, nanomechanical, and biochemical levels) that could have diagnostic and therapeutic significance for HF.