3D Raman imaging of systemic endothelial dysfunction in the murine model of metastatic breast cancer

It was recently reported in the murine model of metastatic breast cancer (4T1) that tumor progression and development of metastasis is associated with systemic endothelial dysfunction characterized by impaired nitric oxide (NO) production. Using Raman 3D confocal imaging with the analysis of the individual layers of the vascular wall combined with AFM endothelial surface imaging, we demonstrated that metastasis-induced systemic endothelial dysfunction resulted in distinct chemical changes in the endothelium of the aorta. These changes, manifested as a significant increase in the protein content (18 %) and a slight decrease in the lipid content (4 %), were limited to the endothelium and did not occur in the deeper layers of the vascular wall. The altered lipid to protein ratio in the endothelium, although more pronounced in the fixed vascular wall, was also observed in the freshly isolated unfixed vascular wall samples in the aqueous environment (12 and 7 % change of protein and lipid content, respectively). Our results support the finding that the metastasis induces systemic endothelial dysfunction that may contribute to cancer progression

1-methylnicotinamide and its structural analog 1,4-dimethylpyridine for the prevention of cancer metastasis

Background: 1-methylnicotinamide (1-MNA), an endogenous metabolite of nicotinamide, has recently gained interest due to its anti-inflammatory and anti-thrombotic activities linked to the COX-2/PGI2 pathway. Given the previously reported anti-metastatic activity of prostacyclin (PGI2), we aimed to assess the effects of 1-MNA and its structurally related analog, 1,4-dimethylpyridine (1,4-DMP), in the prevention of cancer metastasis. Methods: All the studies on the anti-tumor and anti-metastatic activity of 1-MNA and 1,4-DMP were conducted using the model of murine mammary gland cancer (4T1) transplanted either orthotopically or intravenously into female BALB/c mouse. Additionally, the effect of the investigated molecules on cancer cell-induced angiogenesis was estimated using the matrigel plug assay utilizing 4T1 cells as a source of pro-angiogenic factors. Results: Neither 1-MNA nor 1,4-DMP, when given in a monotherapy of metastatic cancer, influenced the growth of 4T1 primary tumors transplanted orthotopically; however, both compounds tended to inhibit 4T1 metastases formation in lungs of mice that were orthotopically or intravenously inoculated with 4T1 or 4T1-luc2-tdTomato cells, respectively. Additionally, while 1-MNA enhanced tumor vasculature formation and markedly increased PGI2 generation, 1,4-DMP did not have such an effect. The anti-metastatic activity of 1-MNA and 1,4-DMP was further confirmed when both agents were applied with a cytostatic drug in a combined treatment of 4T1 murine mammary gland cancer what resulted in up to 80 % diminution of lung metastases formation. Conclusions: The results of the studies presented below indicate that 1-MNA and its structural analog 1,4-DMP prevent metastasis and might be beneficially implemented into the treatment of metastatic breast cancer to ensure a comprehensive strategy of metastasis control

Quantification of the Blood Platelet Reactivity in the ADP-Induced Model of Non-Lethal Pulmonary Thromboembolism in Mice with the Use of Laser Doppler Flowmetry.

The paper describes an alternative method for quantification of in vivo ADP-induced thromboembolism. The aim of the studies was to develop a method of quantification which would not require either extravasation or labelling of platelets. Our proposed approach is based on the monitoring of changes of blood flow with the use of laser Doppler flowmetry.Mice of C57Bl strain were used in the study. ADP was injected to the vena cava and blood flow was monitored with the use of a laser Doppler flowmeter in the mesentery. Measurements in platelet-depleted mice, mice pretreated with cangrelor, an ADP receptor antagonist, and eptifibatide, a blocker of fibrinogen binding to GPIIbIIIa, were conducted as the proof-of-concept in the performed experiments. Intravital microscopy and ex vivo imaging of organs was performed to identify the sites of aggregate formation resulting from ADP injection.The injection of ADP resulted in a dose-dependent reduction of the blood flow in the mesentery. These responses were fully attributable to blood platelet aggregation, as shown by the lack of the effect in platelet-depleted mice, and significantly reduced responses in mice pretreated with cangrelor and eptifibatide. No platelet aggregate formation in mesenteric vessels was revealed by intravital microscopy, while ex vivo imaging showed accumulation of fluorescent labelled platelets in the lung.Injection of ADP to the venous system results in the formation of platelet aggregates predominantly in the lung. This results in reversible blood flow cessation in peripheral blood vessels. The measurement of this blood flow cessation in the mesentery allows indirect measurement of ADP-induced pulmonary thromboembolism. We suggest that this approach can be useful for in vivo screening for antiplatelet drug candidates

Effect of Biochar on Metal Distribution and Microbiome Dynamic of a Phytostabilized Metalloid-Contaminated Soil Following Freeze–Thaw Cycles

In the present paper the effectiveness of biochar-aided phytostabilization of metal/metalloid-contaminated soil under freezing–thawing conditions and using the metal tolerating test plant Lolium perenne L. is comprehensively studied. The vegetative experiment consisted of plants cultivated for over 52 days with no exposure to freezing–thawing in a glass greenhouse, followed by 64 days under freezing–thawing in a temperature-controlled apparatus and was carried out in initial soil derived from a post-industrial urban area, characterized by the higher total content of Zn, Pb, Cu, Cr, As and Hg than the limit values included in the classification provided by the Regulation of the Polish Ministry of Environment. According to the substance priority list published by the Toxic Substances and Disease Registry Agency, As, Pb, and Hg are also indicated as being among the top three most hazardous substances. The initial soil was modified by biochar obtained from willow chips. The freeze–thaw effect on the total content of metals/metalloids (metal(-loid)s) in plant materials (roots and above-ground parts) and in phytostabilized soils (non- and biochar-amended) as well as on metal(-loid) concentration distribution/redistribution between four BCR (community bureau of reference) fractions extracted from phytostabilized soils was determined. Based on metal(-loid)s redistribution in phytostabilized soils, their stability was evaluated using the reduced partition index (Ir). Special attention was paid to investigating soil microbial composition. In both cases, before and after freezing–thawing, biochar increased plant biomass, soil pH value, and metal(-loid)s accumulation in roots, and decreased metal(-loid)s accumulation in stems and total content in the soil, respectively, as compared to the corresponding non-amended series (before and after freezing–thawing, respectively). In particular, in the phytostabilized biochar-amended series after freezing–thawing, the recorded total content of Zn, Cu, Pb, and As in roots substantially increased as well as the Hg, Cu, Cr, and Zn in the soil was significantly reduced as compared to the corresponding non-amended series after freezing–thawing. Moreover, exposure to freezing–thawing itself caused redistribution of examined metal(-loid)s from mobile and/or potentially mobile into the most stable fraction, but this transformation was favored by biochar presence, especially for Cu, Pb, Cr, and Hg. While freezing–thawing greatly affected soil microbiome composition, biochar reduced the freeze–thaw adverse effect on bacterial diversity and helped preserve bacterial groups important for efficient soil nutrient conversion. In biochar-amended soil exposed to freezing–thawing, psychrotolerant and trace element-resistant genera such as Rhodococcus sp. or Williamsia sp. were most abundant

Representative picture of a mesenteric vessel prior to (a, c) and after the reduction of blood flow upon ADP injection (10 mg/kg b.w.) (b, d).

<p>Animals were pre-injected with rhodamine (a, b) or DyLight488-labeled anti-GPIbβ antibodies (c, d).</p

Effects of cangrelor and eptifibatide on the dose-dependent changes of AUC and A_max of the LDF recordings in mice administered with the increasing doses of ADP.

<p>Data is presented as mean ± SEM; n = 7–9 mice. Animals were pre-injected with various doses of either cangrelor (A) (black circle- 0 mg/kg b.w., open circle- 0.2 mg/kg b.w., grey diamond- 1 mg/kg b.w.) or eptifibatide (B) (black circle- 0 mg/kg b.w., open circle- 0.5 mg/kg b.w., grey diamond- 4 mg/kg b.w.), and then administered with boluses of increasing doses of ADP. The curve was fitted to the one-site binding non-linear regression curve (GraphPad Prism ver. 5.0): <math><mrow><mi>y</mi><mo>=</mo><mrow><mi>a</mi><mi>*</mi><mi>x</mi></mrow><mrow><mi>b</mi><mo>+</mo><mi>x</mi></mrow></mrow></math>, where ‘a’ is the maximum achievable AUC and ‘b’ is the concentration of the agonist (ADP), at which the response reaches 50%.</p

Schematic representation of the experimental setup used in the study.

<p>The anesthetized mouse in a supine position is located on a heated surgical table. The mesenteric bed is prepared. ADP solution is injected into the <i>vena cava</i>. Blood flow is measured in the mesenteric bed with the use of a laser Doppler flow probe.</p

Changes in the four most discriminating parameters characterizing the LDF responses following the injections of increasing concentrations of ADP.

<p>Each dose of ADP was injected in two consecutive boluses to the same mouse (with intervals). The values represent means ± standard deviations for normally-distributed data (AUC, A_max, T_c) and medians with interquartile ranges for the data which significantly deviated from normal distribution (V_0r); (n = 7). Differences on raw or transformed data (V₀ –square, V_avgf—square root) were tested with repeated measures ANOVA followed by a paired Student’s t test with Bonferroni’s correction for multiple comparisons. * 0.01 < p < 0.05; ** 0.001 < p < 0.01; *** p<0.001. <b>A</b>_<b>max</b><b>—</b>the maximum decrease in blood flow; <b>AUC–</b>the area under the curve from 10% of maximum decrease (A_max) to 10% of the maximal restoration of flow (DA); <b>T</b>_<b>c</b><b>−</b>time from 90% of maximum decrease (A_max) to 90% of value of maximal restoration of flow (DA); <b>V</b>_<b>0f</b><b>–</b>the maximal rate of the restoration of blood flow.</p