Vascular protein disulfide isomerase A1 mediates endothelial dysfunction induced by angiotensin II in mice.

AIM: Protein disulfide isomerases (PDIs) are involved in platelet aggregation and intravascular thrombosis, but their role in regulating endothelial function is unclear. Here, we characterized the involvement of vascular PDIA1 in angiotensin II (Ang II)-induced endothelial dysfunction in mice. METHODS: Endothelial dysfunction was induced in C57BL/6JCmd male mice via Ang II subcutaneous infusion, and PDIA1 was inhibited with bepristat. Endothelial function was assessed in vivo with magnetic resonance imaging and ex vivo with a myography, while arterial stiffness was measured as pulse wave velocity. Nitric oxide (NO) bioavailability was measured in the aorta (spin-trapping electron paramagnetic resonance) and plasma (NO2 - and NO3 - levels). Oxidative stress, eNOS uncoupling (DHE-based aorta staining), and thrombin activity (thrombin-antithrombin complex; calibrated automated thrombography) were evaluated. RESULTS: The inhibition of PDIA1 by bepristat in Ang II-treated mice prevented the impairment of NO-dependent vasodilation in the aorta as evidenced by the response to acetylcholine in vivo, increased systemic NO bioavailability and the aortic NO production, and decreased vascular stiffness. Bepristat's effect on NO-dependent function was recapitulated ex vivo in Ang II-induced endothelial dysfunction in isolated aorta. Furthermore, bepristat diminished the Ang II-induced eNOS uncoupling and overproduction of ROS without affecting thrombin activity. CONCLUSION: In Ang II-treated mice, the inhibition of PDIA1 normalized the NO-ROS balance, prevented endothelial eNOS uncoupling, and, thereby, improved vascular function. These results indicate the importance of vascular PDIA1 in regulating endothelial function, but further studies are needed to elucidate the details of the mechanisms involved.

Optimal values of the Electron Monte Carlo dose engine parameters.

Background: The aim of this study was to indicate the most favorable - in terms of to the time of calculation and the uncertainty of determining the dose distribution - values of the parameters for the Electron Monte Carlo (eMC) algorithm in the Eclipse treatment planning system. Materials and methods: Using the eMC algorithm and the variability of the values of its individual parameters, calculations of the electron dose distribution in the full-scattering virtual water phantom were performed, obtaining percentage depth doses, beam profiles, absolute dose values in points and calculation times. The reference data included water tank measurements such as relative dose distributions and absolute point doses. Results: For 63 sets of calculation data created from selected values of the parameters for the eMC algorithm, calculation times were analyzed and the absolute calculated and measured doses were compared. Performing a statistical analysis made it possible to determine whether the differences in the values of deviations between the actual dose and the calculated dose in individual regions of the percentage depth dose curve and the beam profile are statistically significant between the analyzed sets of parameters. Conclusions: Taking into account obtained results from the analysis of the discrepancy between the distribution of the calculated and measured dose, the correspondence of the absolute value of the calculated and measured dose and the duration of the calculation of the dose distribution, the optimal set of parameters was indicated for the eMC algorithm which allows obtaining the dose distribution and the number of monitor units in an acceptable time.

COVID-19-related social isolation and symptoms of depression and anxiety in young men in Poland: Does insomnia mediate the relationship?

The need for physical distancing due to COVID-19 mitigation efforts forced prolonged social isolation, which may affect sleep and lead to mental health problems. Previous research has shown that young adults are particularly vulnerable to psychological stress caused by social isolation, the negative psychological impact of the pandemic, and greater frequency and severity of sleep problems. Therefore, the main goal of the present study was to examine whether insomnia could constitute a mediation mechanism that explains the relationship between social isolation experienced during the COVID-19 pandemic and mental health outcomes (depression and anxiety) reported up to 1.5 years later. The study was conducted among young (M±SD; 24.08±3.75) men (N = 1025) in Poland. Data were collected by means of self-report questionnaires, including The Social Isolation Index, The Athens Insomnia Scale, The State-Trait Anxiety Inventory (STAI-S) and Beck's Depression Inventory (BDI-II). The results show that insomnia mediates the relationships between social isolation and both anxiety and depression. The current findings emphasize the role of insomnia in the relationships between social isolation experienced during COVID-19 and negative emotional states. From a clinical perspective, the results suggest that implementing therapeutic components that address social isolation in insomnia treatment programs may prevent the development of depression and anxiety symptoms among young men.

Endothelial-mesenchymal transition induced by metastatic 4T1 breast cancer cells in pulmonary endothelium in aged mice.

Ageing is a major risk factor for cancer metastasis but the underlying mechanisms remain unclear. Here, we characterised ageing effects on cancer-induced endothelial-mesenchymal transition (EndMT) in the pulmonary circulation of female BALB/c mice in a metastatic 4T1 breast cancer model. The effect of intravenously injected 4T1 cells on pulmonary endothelium, pulmonary metastasis, lung tissue architecture, and systemic endothelium was compared between 40-week-old and 20-week-old mice. The 40-week-old mice showed features of ongoing EndMT in their lungs before 4T1 breast cancer cell injection. Moreover, they had preexisting endothelial dysfunction in the aorta detected by in vivo magnetic resonance imaging (MRI) compared to 20-week-old mice. The injection of 4T1 breast cancer cells into 40-week-old mice resulted in rapid EndMT progression in their lungs. In contrast, injection of 4T1 breast cancer cells into 20-week-old mice resulted in initiation and less pronounced EndMT progression. Although the number of metastases did not differ significantly between 20-week-old and 40-week-old mice, the lungs of older mice displayed altered lung tissue architecture and biochemical content, reflected in higher Amide II/Amide I ratio, higher fibronectin levels, and hypoxia-inducible factor 1 subunit alpha (HIF1α) levels as well as lower nitric oxide (NO) production. Our results indicate that age-dependent pre-existing endothelial dysfunction in the pulmonary endothelium of 40-week-old mice predisposed them to rapid EndMT progression in the presence of circulating 4T1 breast cancer cells what might contribute to a more severe metastatic breast cancer phenotype in these ageing mice compared to younger mice.

Influence of optional measurement parameters in the Eclipse treatment planning system on the quality of the dosimetric model of the biomedical accelerator using the Acuros XB algorithm.

Background: To properly configure a treatment planning system, a measurement data set is needed, which consists of the values required for its configuration. The aim is to obtain a dosimetric model of the beam that is as compatible as possible with the measured values. The set of required data can be supplemented with optional values. The aim of the study was to assess the influence of optional measurement data on the compliance of the calculations with the measurements. Materials and methods: Dosimetric measurements, model configuration and dose distribution calculations were performed for the photon radiation beams generated by the VMS TrueBeam® linear accelerator. Beams were configured on an Eclipse™ v. 15.6 system using the Acuros v. 15.6 algorithm. The measured and calculated data were entered into the Alfard™ software for comparison with the calculated dose distributions. In the last stage, the absolute dose values at the designated points were also compared. The obtained data were statistically analysed with Statistica™ v. 13.3. Results: The work showed that the differences in the shape of the beam profile, depth dose and the dose value in points were not related to the use of optional data. Differences in dose distributions are within the tolerance. It cannot be determined under which conditions the use of optional data has a more favourable effect on the reflection of the actual dose values. Conclusions: The use of optional data in modelling photon radiation beams does not significantly improve the compliance of the calculated and measured dose values.

Comparison of anti-cancer effects of novel protein disulphide isomerase (PDI) inhibitors in breast cancer cells characterized by high and low PDIA17 expression.

BACKGROUND: Protein disulphide isomerases (PDIs) play an important role in cancer progression. However, the relative contribution of the various isoforms of PDI in tumorigenesis is not clear. METHODS: The content of PDI isoforms in 22 cancer cells lines was investigated using LC-MS/MS-based proteomic analysis. The effects of PDIA1, PDIA3 and PDIA17 inhibition on the proliferation, migration and adhesion of MCF-7 and MDA-MB-231 cells, identified as high and low PDIA17 expressing cells, respectively, were assessed using novel aromatic N-sulphonamides of aziridine-2-carboxylic acid derivatives as PDI inhibitors. RESULTS: PDIA1 and PDIA3 were the most abundant in cancer cell lysates and were also detected extracellularly in breast cancer cells (MDA-MB-231 and MCF-7). Some cancer cell lines (e.g., MCF-7, HT-29) showed upregulated expression of PDIA17, whereas in others (e.g., MDA-MB-231, 67NR), PDIA17 was not detected. The simultaneous inhibition of PDIA1 and PDIA3 showed similar anti-proliferative effects in MCF-7 and MDA-MB-231 breast cancer cells. However, the inhibition of PDIA1 and PDIA17 in the MCF-7 cell line resulted in more effective anti-adhesive and anti-proliferative effects. CONCLUSIONS: PDIA1 and PDIA3 represent major isoforms of multiple cancer cells, and their non-selective inhibition displays significant anti-proliferative effects irrespective of whether or not PDIA17 is present. The more pronounced anti-adhesive effects of PDI inhibition in hormone-sensitive MCF-7 cells featured by higher levels of PDIs when compared to triple-negative MDA-MB-231 cells suggests that targeting extracellular PDIA1 and PDIA3 with or without additional PDIA17 inhibition may represent a strategy for personalized anti-adhesive, anti-metastatic therapy in cancers with high PDI expression.

Direct Thrombin Inhibitor Dabigatran Compromises Pulmonary Endothelial Integrity in a Murine Model of Breast Cancer Metastasis to the Lungs; the Role of Platelets and Inflammation-Associated Haemostasis.

Activation of the coagulation cascade favours metastatic spread, but antithrombotic therapy might also have detrimental effects on cancer progression. In this study, we characterized the effects of dabigatran, a direct reversible thrombin inhibitor, on the pulmonary endothelial barrier and metastatic spread in a murine model of breast cancer metastasis. Dabigatran etexilate (100 mg kg-1) was administered to mice twice daily by oral gavage. Pulmonary metastasis, pulmonary endothelium permeability in vivo, and platelet reactivity were evaluated after intravenous injection of 4T1 breast cancer cells into BALB/c mice. The effect of dabigatran on platelet-dependent protection of pulmonary endothelial barrier in the presence of an inflammatory stimulus was also verified in vitro using human lung microvascular endothelial cell (HLMVEC) cultures. Dabigatran-treated mice harbored more metastases in their lungs and displayed increased pulmonary endothelium permeability after cancer cell injection. It was not associated with altered lung fibrin deposition, changes in INFγ, or complement activation. In the in vitro model of the pulmonary endothelial barrier, dabigatran inhibited platelet-mediated protection of pulmonary endothelium. In a murine model of breast cancer metastasis, dabigatran treatment promoted pulmonary metastasis by the inhibition of platelet-dependent protection of pulmonary endothelial barrier integrity.

Protein disulfide isomerase-A1 regulates intraplatelet reactive oxygen species-thromboxane A2 -dependent pathway in human platelets.

BACKGROUND: Platelet-derived protein disulfide isomerase 1 (PDIA1) regulates thrombus formation, but its role in the regulation of platelet function is not fully understood. AIMS: The aim of this study was to characterize the role of PDIA1 in human platelets. METHODS: Proteomic analysis of PDI isoforms in platelets was performed using liquid chromatography tandem mass spectometry, and the expression of PDIs on platelets in response to collagen, TRAP-14, or ADP was measured with flow cytometry. The effects of bepristat, a selective PDIA1 inhibitor, on platelet aggregation, expression of platelet surface activation markers, thromboxane A2 (TxA2 ), and reactive oxygen species (ROS) generation were evaluated by optical aggregometry, flow cytometry, ELISA, and dihydrodichlorofluorescein diacetate-based fluorescent assay, respectively. RESULTS: PDIA1 was less abundant compared with PDIA3 in resting platelets and platelets stimulated with TRAP-14, collagen, or ADP. Collagen, but not ADP, induced a significant increase in PDIA1 expression. Bepristat potently inhibited the aggregation of washed platelets induced by collagen or convulxin, but only weakly inhibited platelet aggregation induced by TRAP-14 or thrombin, and had the negligible effect on platelet aggregation induced by arachidonic acid. Inhibition of PDIA1 by bepristat resulted in the reduction of TxA2 and ROS production in collagen- or thrombin-stimulated platelets. Furthermore, bepristat reduced the activation of αIIbß3 integrin and expression of P-selectin. CONCLUSIONS: PDIA1 acts as an intraplatelet regulator of the ROS-TxA2 pathway in collagen-GP VI receptor-mediated platelet activation that is a mechanistically distinct pathway from extracellular regulation of αIIbß3 integrin by PDIA3.

Systemic Administration of Insulin Receptor Antagonist Results in Endothelial and Perivascular Adipose Tissue Dysfunction in Mice.

Hyperglycemia linked to diabetes results in endothelial dysfunction. In the present work, we comprehensively characterized effects of short-term hyperglycemia induced by administration of an insulin receptor antagonist, the S961 peptide, on endothelium and perivascular adipose tissue (PVAT) in mice. Endothelial function of the thoracic and abdominal aorta in 12-week-old male C57Bl/6Jrj mice treated for two weeks with S961 infusion via osmotic pumps was assessed in vivo using magnetic resonance imaging and ex vivo by detection of nitric oxide (NO) production using electron paramagnetic resonance spectroscopy. Additional methods were used to analyze PVAT, aortic segments and endothelial-specific plasma biomarkers. Systemic disruption of insulin signaling resulted in severe impairment of NO-dependent endothelial function and a loss of vasoprotective function of PVAT affecting the thoracic as well as abdominal parts of the aorta, however a fall in adiponectin expression and decreased uncoupling protein 1-positive area were more pronounced in the thoracic aorta. Results suggest that dysfunctional PVAT contributes to vascular pathology induced by altered insulin signaling in diabetes, in the absence of fat overload and obesity.

A preliminary metabolites identification of a novel compound with ß-adrenolytic activity.

BACKGROUND: The identification of main metabolites and assessment of renal excretion of a novel compound with ß-adrenolytic activity (2RS)-1-(1H-indol-4-yloxy)-3-((2-(2-methoxyphenoxy)ethyl)amino)propan-2-ol, briefly called (RS)-9 or 2F109, were studied in vivo in rat serum, urine, faeces, liver, intestine, lungs and kidneys, and in vitro in rat liver microsomes. METHODS: Structures of the metabolites have been developed by comparing the high-resolution product ion mass spectra of metabolites and the parent compound based on the differences in mass values of main fragments. Quantitative analysis of (RS)-9 was done using a system of liquid chromatography coupled with a triple quadrupole mass spectrometer API 2000. Identification studies of predicted metabolites were made by a high-resolution mass spectrometer LTQ XL Orbitrap Discovery and using a Roxy™ system, for online electrochemical mimicry of oxidative metabolism by cytochrome P450s connected to QTRAP 5500. RESULTS: For (RS)-9 (m/z 357.2084) phase I metabolites derived from oxidation process: hydroxyl derivatives (m/z 373.2470) and dihydroxyl derivatives (m/z 389.4318), and phase II metabolites: N-methylated compound (m/z 371.1612), O-glucuronide (m/z 533.5118), and sulfate (m/z 437.2350) were identified. CONCLUSION: (RS)-9 was extensively metabolised to several phase I and II metabolites, and renal excretion was a minor route in its elimination.

Protein Disulphide Isomerase A1 Is Involved in the Regulation of Breast Cancer Cell Adhesion and Transmigration via Lung Microvascular Endothelial Cells.

Cancer cell cross-talk with the host endothelium plays a crucial role in metastasis, but the underlying mechanisms are still not fully understood. We studied the involvement of protein disulphide isomerase A1 (PDIA1) in human breast cancer cell (MCF-7 and MDA-MB-231) adhesion and transendothelial migration. For comparison, the role of PDIA1 in proliferation, migration, cell cycle and apoptosis was also assessed. Pharmacological inhibitor, bepristat 2a and PDIA1 silencing were used to inhibit PDIA1. Inhibition of PDIA1 by bepristat 2a markedly decreased the adhesion of breast cancer cells to collagen type I, fibronectin and human lung microvascular endothelial cells. Transendothelial migration of breast cancer cells across the endothelial monolayer was also inhibited by bepristat 2a, an effect not associated with changes in ICAM-1 expression or changes in cellular bioenergetics. The silencing of PDIA1 produced less pronounced anti-adhesive effects. However, inhibiting extracellular free thiols by non-penetrating blocker p-chloromercuribenzene sulphonate substantially inhibited adhesion. Using a proteomic approach, we identified that ß1 and α2 integrins were the most abundant among all integrins in breast cancer cells as well as in lung microvascular endothelial cells, suggesting that integrins could represent a target for PDIA1. In conclusion, extracellular PDIA1 plays a major role in regulating the adhesion of cancer cells and their transendothelial migration, in addition to regulating cell cycle and caspase 3/7 activation by intracellular PDIA1. PDIA1-dependent regulation of cancer-endothelial cell interactions involves disulphide exchange and most likely integrin activation but is not mediated by the regulation of ICAM-1 expression or changes in cellular bioenergetics in breast cancer or endothelial cells.

In Vivo Magnetic Resonance Imaging-Based Detection of Heterogeneous Endothelial Response in Thoracic and Abdominal Aorta to Short-Term High-Fat Diet Ascribed to Differences in Perivascular Adipose Tissue in Mice.

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.

Nanostructuring of Si substrates by a metal-assisted chemical etching and dewetting process.

In this work, we reported on the development of lithography-free technology for the fabrication of nanopatterned Si substrates. The combination of two phenomena, the solid-state dewetting process and metal-assisted wet chemical etching, allowed for fabrication of Si nanocolumns on large areas in a relatively simple way. The process of dewetting the thin metal layer enabled formation of nickel nanoislands, which were used as a shadow mask in the deposition of a catalytic metal pattern. Application of the two-stage dewetting process with the repetition of the metal deposition and annealing step enabled us to obtain a significant increase in the surface coverage ratio and the surface density of the nanoislands. As a catalytic metal, a gold layer was applied in the metal-assisted wet chemical etching process. The obtained columnar nanostructures showed a great verticality and had a high aspect ratio. In the conducted studies, the maximum etching rate (at RT) was higher than 1.2 µm min-1. The etching rate increased with increasing concentration of oxidizing (H2O2) and etching (HF) agent, with a tendency to saturate for more concentrated solutions. The etching rate was significantly higher for Si substrates with a crystallographic orientation (115) than for (111), but there was no privileged direction of etching except for the direction vertical to the substrate. With increasing layer thickness of the catalytic metal a decrease in the metal-assisted wet chemical etching process efficiency was observed. The developed technology allows for fabrication of patterned substrates with a wide range of lateral dimension of nanocolumns and their density.