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. 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. 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. 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.

Small, gaseous molecules such as nitric oxide, carbon monoxide and hydrogen sulfide are produced as signalling molecules in mammalian cells. Here, we show that low concentrations of cyanide are generated endogenously in various mammalian tissues and cells. We detect cyanide in several cellular compartments of human cells and in various tissues and the blood of mice. Cyanide production is stimulated by glycine, occurs at the low pH of lysosomes and requires peroxidase activity. When generated at a specific rate, cyanide exerts stimulatory effects on mitochondrial bioenergetics, cell metabolism and cell proliferation, but impairs cellular bioenergetics at high concentrations. Cyanide can modify cysteine residues via protein S-cyanylation, which is detectable basally in cells and mice, and increases in response to glycine. Low-dose cyanide supplementation exhibits cytoprotective effects in hypoxia and reoxygenation models in vitro and in vivo. Conversely, pathologically elevated cyanide production in nonketotic hyperglycinaemia is detrimental to cells. Our findings indicate that cyanide should be considered part of the same group of endogenous mammalian regulatory gasotransmitters as nitric oxide, carbon monoxide and hydrogen sulfide.

Porphyromonas gingivalis, an anaerobic Gram-negative bacterium critically involved in the development of human periodontitis, belongs to the late colonizers of the oral cavity. The success of this pathogen in the host colonization and infection results from the presence of several virulence factors, including extracellular peptidylarginine deiminase (PPAD), an enzyme that converts protein arginine residues to citrullines. A common opportunistic fungal pathogen of humans, Candida albicans, is also frequently identified among microorganisms that reside at subgingival sites. The aim of the current work was to verify if protein citrullination can influence the formation of mixed biofilms by both microorganisms under hypoxic and normoxic conditions. Quantitative estimations of the bacterial adhesion to fungal cells demonstrated the importance of PPAD activity in this process, since the level of binding of P. gingivalis mutant strain deprived of PPAD was significantly lower than that observed for the wild-type strain. These results were consistent with mass spectrometric detection of the citrullination of selected surface-exposed C. albicans proteins. Furthermore, a viability of P. gingivalis cells under normoxia increased in the presence of fungal biofilm compared with the bacteria that formed single-species biofilm. These findings suggest a possible protection of these strict anaerobes under unfavorable aerobic conditions by C. albicans during mixed biofilm formation.

The role of epithelial sodium channel (ENaC) activity in the regulation of endothelial function is not clear. Here, we analyze the role of ENaC in the regulation of endothelium-dependent vasodilation and endothelial permeability in vivo in mice with conditional αENaC subunit gene inactivation in the endothelium (endo-αENaC^KO mice) using unique MRI-based analysis of acetylcholine-, flow-mediated dilation and vascular permeability. Mice were challenged or not with lipopolysaccharide (LPS, from Salmonella typhosa, 10 mg/kg, i.p.). In addition, changes in vascular permeability in ex vivo organs were analyzed by Evans Blue assay, while changes in vascular permeability in perfused mesenteric artery were determined by a FITC-dextran-based assay. In basal conditions, Ach-induced response was completely lost, flow-induced vasodilation was inhibited approximately by half but endothelial permeability was not changed in endo-αENaC^KO vs. control mice. In LPS-treated mice, both Ach- and flow-induced vasodilation was more severely impaired in endo-αENaC^KO vs. control mice. There was also a dramatic increase in permeability in lungs, brain and isolated vessels as evidenced by in vivo and ex vivo analysis in endotoxemic endo-αENaC^KO vs. control mice. The impaired endothelial function in endotoxemia in endo-αENaC^KO was associated with a decrease of lectin and CD31 endothelial staining in the lung as compared with control mice. In conclusion, the activity of endothelial ENaC in vivo contributes to endothelial-dependent vasodilation in the physiological conditions and the preservation of endothelial barrier integrity in endotoxemia.

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.