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.

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.

Objectives: Carbon monoxide (CO) produced by haem oxygenases or released by CO-releasing molecules (CORM) affords antiplatelet effects, but the mechanism involved has not been defined. Here, we tested the hypothesis that CO–induced inhibition of human platelet aggregation is mediated by modulation of platelet bioenergetics. Approach and Results: To analyze the effects of CORM-A1 on human platelet aggregation and bioenergetics, a light transmission aggregometry, Seahorse XFe technique and liquid chromatography tandem-mass spectrometry–based metabolomics were used. CORM-A1–induced inhibition of platelet aggregation was accompanied by the inhibition of mitochondrial respiration and glycolysis. Interestingly, specific inhibitors of these processes applied individually, in contrast to combined treatment, did not inhibit platelet aggregation considerably. A CORM-A1–induced delay of tricarboxylic acid cycle was associated with oxidized nicotinamide adenine dinucleotide (NAD ⁺ ) depletion, compatible with the inhibition of oxidative phosphorylation. CORM-A1 provoked an increase in concentrations of proximal (before GAPDH [glyceraldehyde 3-phosphate dehydrogenase]), but not distal glycolysis metabolites, suggesting that CO delayed glycolysis at the level of NAD ⁺ –dependent GAPDH; however, GAPDH activity was directly not inhibited. In the presence of exogenous pyruvate, CORM-A1–induced inhibition of platelet aggregation and glycolysis were lost, but were restored by the inhibition of lactate dehydrogenase, involved in cytosolic NAD ⁺ regeneration, pointing out to the key role of NAD ⁺ depletion in the inhibition of platelet bioenergetics by CORM-A1. Conclusions: The antiplatelet effect of CO is mediated by inhibition of mitochondrial respiration—attributed to the inhibition of cytochrome c oxidase, and inhibition of glycolysis—ascribed to cytosolic NAD ⁺ depletion.

Glufosfamide (β-D-glucose-isophosphoramide mustard, D-19575) belongs to the oxazaphosphorine class. Glufosfamide is a novel glucose conjugate of ifosfamide in which isophosphoramide mustard, the alkylating metabolite of ifosfamide, is glycosidically linked to the β-D-glucose molecule. Glufosfamide represents an attractive new agent for cancer therapy. Its mode of action on normal and pathological cells is still under experimental and clinical investigations. An assessment of the anticancer potential of glufosfamide is of key importance in therapy. The researchers reviewed the current knowledge available on glufosfamide tested in the preclinical studies/clinical trials, based on a collection of the original papers and conference abstracts published and relevant articles searched in the SCOPUS and MEDLINE database and websites.