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.

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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-αENaCKO 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-αENaCKO vs. control mice. In LPS-treated mice, both Ach- and flow-induced vasodilation was more severely impaired in endo-αENaCKO 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-αENaCKO vs. control mice. The impaired endothelial function in endotoxemia in endo-αENaCKO 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.

Over the past decade rapid advances have occurred in the understanding of RNA expression and its regulation. Quantitative polymerase chain reactions (qPCR) have become the gold standard for quantifying gene expression. Microfluidic next generation, high throughput qPCR now permits the detection of transcript copy number in thousands of reactions simultaneously, dramatically increasing the sensitivity over standard qPCR. Here we present a gene expression analysis method applicable to both standard polymerase chain reactions (qPCR) and high throughput qPCR. This technique is adjusted to the input sample quantity (e.g., the number of cells) and is independent of control gene expression. It is efficiency-corrected and with the use of a universal reference sample (commercial complementary DNA (cDNA)) permits the normalization of results between different batches and between different instruments – regardless of potential differences in transcript amplification efficiency. Modifications of the input quantity method include (1) the achievement of absolute quantification and (2) a non-efficiency corrected analysis. When compared to other commonly used algorithms the input quantity method proved to be valid. This method is of particular value for clinical studies of whole blood and circulating leukocytes where cell counts are readily available.

In microarray studies alterations in gene expression in circulating leukocytes have shown utility for ischemic stroke diagnosis. We studied forty candidate markers identified in three gene expression profiles to (1) quantitate individual transcript expression, (2) identify transcript clusters and (3) assess the clinical diagnostic utility of the clusters identified for ischemic stroke detection. Using high throughput next generation qPCR 16 of the 40 transcripts were significantly up-regulated in stroke patients relative to control subjects (p<0.05). Six clusters of between 5 and 7 transcripts discriminated between stroke and control (p values between 1.01e-9 and 0.03). A 7 transcript cluster containing PLBD1, PYGL, BST1, DUSP1, FOS, VCAN and FCGR1A showed high accuracy for stroke classification (AUC=0.854). These results validate and improve upon the diagnostic value of transcripts identified in microarray studies for ischemic stroke. The clusters identified show promise for acute ischemic stroke detection.

The significance and utility of innovative imaging techniques in arterial clot analysis, which enable far more detailed and automated analysis compared to standard methods, are presented. The examination of two types of human thrombi is shown, representing the main ischemic stroke etiologies: fibrin–predominant clot of large vessel origin and red blood cells–rich clot of cardioembolic origin. The synergy effect of Fourier–transform infrared spectroscopy (FTIR), Raman spectroscopy (RS) and atomic force microscopy (AFM) techniques supported by chemometrics in comparison with reference histological staining was presented. The main advantage of such approach refers to free–label and non–destructive quantitative imaging of clinically valid, biochemical parameters in whole sample (FTIR–low resolution) and selected regions (RS–ultra–high resolution). We may include here analysis of lipid content, its distribution and total degree of unsaturation as well as analysis of protein content (mainly fibrin and hemoproteins). The AFM studies enhanced the vibrational data, showed clearly shape and thickness of clot features as well as visualized the fibrin framework. The extraordinary sensitivity of FTIR and RS imaging toward detection and discrimination of clinically valid parameters in clot confirms its applicability in assessment of thrombi origin.

BackgroundAlthough advanced heart failure (HF) is a clinically documented risk factor for vascular cognitive impairment, the occurrence and pathomechanisms of vascular cognitive impairment in early stages of HF are equivocal. Here, we characterize vascular cognitive impairment in the early stages of HF development and assess whether cerebral hypoperfusion or prothrombotic conditions are involved.Methods and ResultsTgαq*44 mice with slowly developing isolated HF triggered by cardiomyocyte‐specific overexpression of G‐αq*44 protein were studied before the end‐stage HF, at the ages of 3, 6, and 10 months: before left ventricle dysfunction; at the stage of early left ventricle diastolic dysfunction (with preserved ejection fraction); and left ventricle diastolic/systolic dysfunction, respectively. In 6‐ to 10‐month‐old but not in 3‐month‐old Tgαq*44 mice, behavioral and cognitive impairment was identified with compromised blood‐brain barrier permeability, most significantly in brain cortex, that was associated with myelin sheet loss and changes in astrocytes and microglia. Brain endothelial cells displayed increased E‐selectin immunoreactivity, which was accompanied by increased amyloid‐β1‐42 accumulation in piriform cortex and increased cortical oxidative stress (8‐OHdG immunoreactivity). Resting cerebral blood flow measured by magnetic resonance imaging in vivo was preserved, but ex vivo NO‐dependent cortical arteriole flow regulation was impaired. Platelet hyperreactivity was present in 3‐ to 10‐month‐old Tgαq*44 mice, but it was not associated with increased platelet‐dependent thrombogenicity.ConclusionsWe report for the first time that vascular cognitive impairment is already present in the early stage of HF development, even before left ventricle systolic dysfunction. The underlying pathomechanism, independent of brain hypoperfusion, involves preceding platelet hyperreactivity and brain endothelium inflammatory activation.

Clinical studies of gene expression are increasingly using the whole blood, peripheral blood mononuclear cells, and leukocyte subsets involved in the innate and adaptive immune responses. However, the small amount of RNA available in the clinical setting is a limitation for commonly used methods such as quantitative polymerase chain reactions (qPCR) and microarrays. Our aim was to design 96 gene assays to simultaneously measure gene expression in the whole blood and seven leukocyte subsets using a new-generation qPCR method-high-throughput nanofluidic reverse transcription qPCR (HT RT-qPCR). The leukocyte subset purity was 94% to 98% for seven subsets and was less for the γδ T-cell receptor subset (80%). The HT RT-qPCR replicate sample measurements were highly reproducible (r = 0.997, p < 2.2 × 10 −16 ), and the ΔΔCt values from HT RT-qPCR correlated significantly with those from qPCR. The control genes were differentially expressed across the eight leukocyte subsets in the control subjects (p = 1.3 × 10 −5 , analysis of variance). Two analytical methods, absolute and relative, gave concordant results and were significantly correlated (p = 1.9 × 10 −9). HT RT-qPCR permits the rapid, reproducible, and quantitative measurement of multiple transcripts using minimal sample amounts. The protocol described yielded leukocyte subsets of high purity and identified two analytic methods for use.