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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.

// Marta Smeda 1 , Anna Kieronska 1 , Bartosz Proniewski 1 , Agnieszka Jasztal 1 , Anna Selmi 1 , Krystyna Wandzel 1 , Agnieszka Zakrzewska 1 , Tomasz Wojcik 1 , Kamil Przyborowski 1 , Katarzyna Derszniak 1 , Marta Stojak 1 , Dawid Kaczor 1 , Elzbieta Buczek 1 , Cezary Watala 2 , Joanna Wietrzyk 3 and Stefan Chlopicki 1, 4 1 Jagiellonian Centre for Experimental Therapeutics, Jagiellonian University, Bobrzynskiego 14, Krakow 30-348, Poland 2 Department of Haemostasis and Haemostatic Disorders, Medical University of Lodz, Kosciuszki 4, Lodz 90-419, Poland 3 Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Department of Experimental Oncology, Rudolfa Weigla 4, Wroclaw 53-114, Poland 4 Chair of Pharmacology, Jagiellonian University, Medical College, Grzegorzecka 16, Krakow 31-531, Poland Correspondence to: Stefan Chlopicki, email: stefan.chlopicki@jcet.eu Keywords: mouse breast cancer; vascular mimicry; platelet inhibition; aspirin; clopidogrel Received: November 01, 2017 Accepted: February 25, 2018 Published: April 03, 2018 ABSTRACT Platelet inhibition has been considered an effective strategy for combating cancer metastasis and compromising disease malignancy although recent clinical data provided evidence that long-term platelet inhibition might increase incidence of cancer deaths in initially cancer-free patients. In the present study we demonstrated that dual anti-platelet therapy based on aspirin and clopidogrel (ASA+Cl), a routine regiment in cardiovascular patients, when given to cancer-bearing mice injected orthotopically with 4T1 breast cancer cells, promoted progression of the disease and reduced mice survival in association with induction of vascular mimicry (VM) in primary tumour. In contrast, treatment with ASA+Cl or platelet depletion did reduce pulmonary metastasis in mice, if 4T1 cells were injected intravenously. In conclusion, distinct platelet-dependent mechanisms inhibited by ASA+Cl treatment promoted cancer malignancy and VM in the presence of primary tumour and afforded protection against pulmonary metastasis in the absence of primary tumour. In view of our data, long-term inhibition of platelet function by dual anti-platelet therapy (ASA+Cl) might pose a hazard when applied to a patient with undiagnosed and untreated malignant cancer prone to undergo VM.

Raman spectroscopy is a label free, versatile, simple and fast method that is increasingly used to detect pathological changes in the cells and tissues that could be useful in medical diagnostics. In this work, we tested the hypothesis that Raman spectroscopy may serve to detect endothelial dysfunction in murine models of lifestyle diseases associated with endothelial dysfunction. For that purpose, we analysed spectra from ex vivo vessels taken from mice with diabetes, hypertension and cancer metastasis. We extracted 50–70 random, single spectra, recorded in 0.2 s, from endothelium of mice with diseases and respective control animals and subjected them to hierarchical cluster analysis. Independently on the sample preparation protocol, very good discrimination was obtained for three-tested murine models, i.e. diabetes, hypertension and cancer metastasis. Obtained sensitivity and specificity parameters were between 93% and 96% (with the exception of sensitivity in the diabetes model equalled to 88%). Our results show that single, random spectra of endothelium, recorded in less than a second, contains enough information on biochemical content of the endothelium to detect endothelial dysfunction. Furthermore, we demonstrated that biochemical profile of the endothelial dysfunction in diabetes, hypertension or cancer metastasis differs with a very high specificity and sensitivity. This conclusion can be a good starting point for the development of in vivo fast diagnostic methodology of endothelium in the future. Copyright © 2016 John Wiley & Sons, Ltd.

Diabetes increases the risk of pulmonary hypertension and is associated with alterations in pulmonary vascular function. Still, it is not clear whether alterations in the phenotype of pulmonary endothelium induced by diabetes are distinct, as compared to peripheral endothelium. In the present work, we characterized differences between diabetic complications in the lung and aorta in db/db mice with advanced diabetes. Male, 20-week-old db/db mice displayed increased HbA1c and glucose concentration compatible with advanced diabetes. Diabetic lungs had signs of mild fibrosis, and pulmonary endothelium displayed significantly ultrastructural changes. In the isolated, perfused lung from db/db mice, filtration coefficient (Kf,c) and contractile response to TXA2 analogue were enhanced, while endothelial NO-dependent modulation of pulmonary response to hypoxic ventilation and cumulative production of NO2− were impaired, with no changes in immunostaining for eNOS expression. In turn, 6-keto-PGF1α release from the isolated lung from db/db mice was increased, as well as immunostaining of thrombomodulin (CD141). In contrast to the lung, NO-dependent, acetylcholine-induced vasodilation, ionophore-stimulated NO2− generation, and production of 6-keto-PGF1α were all impaired in aortic rings from db/db mice. Although eNOS immunostaining was not changed, that of CD141 was clearly lowered. Interestingly, diabetes-induced nitration of proteins in aorta was higher than that in the lungs. In summary, diabetes induced marked ultrastructural changes in pulmonary endothelium that were associated with the increased permeability of pulmonary microcirculation, impaired NO-dependent vascular function, with compensatory increase in PGI2 production, and increased CD141 expression. In contrast, endothelial dysfunction in the aorta was featured by impaired NO-, PGI2-dependent function and diminished CD141 expression.

Background Patients with cancer develop endothelial dysfunction and subsequently display a higher risk of cardiovascular events. The aim of the present work was to examine changes in nitric oxide (NO)- and prostacyclin (PGI2)-dependent endothelial function in the systemic conduit artery (aorta), in relation to the formation of lung metastases and to local and systemic inflammation in a murine orthotopic model of metastatic breast cancer. Methods BALB/c female mice were orthotopically inoculated with 4T1 breast cancer cells. Development of lung metastases, lung inflammation, changes in blood count, systemic inflammatory response (e.g. SAA, SAP and IL-6), as well as changes in NO- and PGI2-dependent endothelial function in the aorta, were examined 2, 4, 5 and 6 weeks following cancer cell transplantation. Results As early as 2 weeks following transplantation of breast cancer cells, in the early metastatic stage, lungs displayed histopathological signs of inflammation, NO production was impaired and nitrosylhemoglobin concentration in plasma was decreased. After 4 to 6 weeks, along with metastatic development, progressive leukocytosis and systemic inflammation (as seen through increased SAA, SAP, haptoglobin and IL-6 plasma concentrations) were observed. Six weeks following cancer cell inoculation, but not earlier, endothelial dysfunction in aorta was detected; this involved a decrease in basal NO production and a decrease in NO-dependent vasodilatation, that was associated with a compensatory increase in cyclooxygenase-2 (COX-2)- derived PGI2 production. Conclusions In 4 T1 metastatic breast cancer in mice early pulmonary metastasis was correlated with lung inflammation, with an early decrease in pulmonary as well as systemic NO availability. Late metastasis was associated with robust, cancer-related, systemic inflammation and impairment of NO-dependent endothelial function in the aorta that was associated with compensatory upregulation of the COX-2-derived PGI2 pathway.

The role of nitric oxide (•NO) in metastatic cancer progression has not been fully explained. In the present work we characterized the development of •NO deficiency in the lung and examined changes in •NO and prostacyclin (PGI2)-dependent endothelial function in the systemic conduit artery (aorta), in relation to the progression of 4T1 metastatic breast cancer injected orthotopically in Balb/c mice. Furthermore, orthotopic and intravenous models of 4T1 metastatic cancer in mice were used to study the differential effects of L-NAME-induced •NO deficiency on the primary tumor and the metastatic burden. •NO was assessed in isolated aorta and lungs using EPR spin-trapping and chemiluminescence, while NOx in blood were measured using either EPR, chemiluminescence or Griess reaction. Early pulmonary metastasis was correlated with lung inflammation. Pulmonary endothelial function and systemic •NO availability in 4T1 breast cancer-bearing mice were compromised prior to the development of pulmonary metastasis,preceding the onset of its phenotypic switch toward a mesenchymal phenotype (EndMT). Late metastasis was associated with robust, cancer-related, systemic inflammation and impairment of NO-dependent endothelial function in the aorta that was associated with compensatory upregulation of the COX-2-derived PGI2 pathway. L-NAME-induced •NO-deficiency showed that •NO plays a major role in the primary tumor development, but it is not the key mediator responsible for the regulation of cancer cell extravasation to the lungs in our model, despite clear-cut •NO-deficiency in the lung, reflected by a fall in systemic •NO bioavailability, both occurring early in the pre-metastatic phase.

Aim: Increased risk of pulmonary hypertension and not clear response of pulmonary endothelium to diabetes were reasons to compare nitric oxide (NO) and PGI-dependent function in pulmonary and systemic circulation in diabetes type II. Methods: In fasted db mice (male, 20 weeks) we compared the function of pulmonary (isolated perfused lung-IPL) and aortic endothelium (wire myograph). It was supported by determination of nitrite(NO 2 - )/nitrate(NO 3 - ), PGI production, immunostaining (IMS) and western blot. Results: In diabetic IPL NO-dependent modulation of hypoxic pulmonary vasoconstriction was impaired but filtration coefficient increased; response to U46619 was enhanced; cumulative generation of NO 2 - was decreased (NO 3 - , basals not changed) but 6-keto-PGF 1α increased. In diabetic lungs CD141 but not eNOS IMS was increased, fibrosis of parenchyma was weak and ultrastructural endothelial changes pronounced. In diabetic aortic rings impairment of acetylcholine-induced vasodilation, ionophore-stimulated NO 2 - (not basal) and 6-keto-PGF 1α production were observed. CD141 but not eNOS IMS was lower. In diabetes general protein nitration and nitration of PGI synthase (PGIS) were substantially increased in aorta but not in the lungs. Conclusions: Advanced diabetes affects pulmonary circulation resulting in (1) increased permeability of microcirculation and vasoreactivity; (2) impairment of NO-dependent vascular function paralleled with aorta; (3) increased PGI production contrasted with aorta; (4) weak nitration of pulmonary PGIS as oppose to systemic. In the diabetic lungs impairment of NO-dependent function was not alongside production of PGI; it may be due to the relative resistance to non-enzymatic nitration of PGIS.