Nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) are effective substrates for NAD synthesis, which may act as vasoprotective agents. Here we characterize the effects of NMN and NR on endothelial inflammation and dysfunction and test the involvement of CD73 in these effects. The effect of NMN and NR on IL1β or TNFα -induced endothelial inflammation (ICAM-1 and vWF expression), intracellular NAD concentration and NAD-related enzyme expression (NAMPT, CD38, CD73), were studied in HAECs. The effect of NMN and NR on angiotensin II-induced impairment of endothelium-dependent vasodilation was analyzed in murine aortic rings. The involvement of CD73 in NMN and NR effects was tested using CD73 inhibitor- AOPCP, or CD73-/- mice. 24h-incubation with NMN and NR induced anti-inflammatory effects in HAEC stimulated by IL1β or TNFα, as evidenced by a reduction in ICAM-1 and vWF expression. Effects of exogenous NMN but not NR was abrogated in the presence of AOPCP, that efficiently inhibited extracellular endothelial conversion of NMN to NR, without a significant effect on the metabolism of NMN to NA. Surprisingly, intracellular NAD concentration increased in HAEC stimulated by IL1β or TNFα and this effect was associated with upregulation of NAMPT and CD73, whereas changes in CD38 expression were less pronounced. NMN and NR further increased NAD in IL1β-stimulated HAECs and AOPCP diminished NMN-induced increase in NAD, without an effect on NR-induced response. In ex vivo aortic rings stimulated with angiotensin II for 24 hours, NO-dependent vasorelaxation induced by Acetylcholine was impaired. NMN and NR, both prevented Ang II-induced endothelial dysfunction in the aorta. In aortic rings taken from CD73-/- mice NMN effect was lost, whereas NR effect was preserved. NMN and NR modulate intracellular NAD content in endothelium, inhibit endothelial inflammation and improve NO-dependent function by CD73-dependent and independent pathways, respectively. Extracellular conversion of NMN to NR by CD73 localized in the luminal surface of endothelial cells represent important vasoprotective mechanisms to maintain intracellular NAD.

A recently discovered bisubstrate inhibitor of Nicotinamide N-methyltransferase (NNMT) was found to be highly potent in biochemical assays with a single digit nanomolar IC₅₀ value but lacking in cellular activity. We, here, report a prodrug strategy designed to translate the observed potent biochemical inhibitory activity of this inhibitor into strong cellular activity. This prodrug strategy relies on the temporary protection of the amine and carboxylic acid moieties of the highly polar amino acid side chain present in the bisubstrate inhibitor. The modification of the carboxylic acid into a range of esters in the absence or presence of a trimethyl-lock (TML) amine protecting group yielded a range of candidate prodrugs. Based on the stability in an aqueous buffer, and the confirmed esterase-dependent conversion to the parent compound, the isopropyl ester was selected as the preferred acid prodrug. The isopropyl ester and isopropyl ester-TML prodrugs exhibit improved cell permeability, which also translates to significantly enhanced cellular activity as established using assays designed to measure the enzymatic activity of NNMT in live cells.

Chloroquine (CQ) is an antimalarial drug known to inhibit autophagy flux by impairing autophagosome–lysosome fusion. We hypothesized that autophagy flux altered by CQ has a considerable influence on the lipid composition of endothelial cells. Thus, we investigated endothelial responses induced by CQ on human microvascular endothelial cells (HMEC-1). HMEC-1 cells after CQ exposure were measured using a combined methodology based on label-free Raman and fluorescence imaging. Raman spectroscopy was applied to characterize subtle chemical changes in lipid contents and their distribution in the cells, while the fluorescence staining (LipidTox, LysoTracker and LC3) was used as a reference method. The results showed that CQ was not toxic to endothelial cells and did not result in the endothelial inflammation at concentrations of 1–30 µM. Notwithstanding, it yielded an increased intensity of LipidTox, LysoTracker, and LC3 staining, suggesting changes in the content of neutral lipids, lysosomotropism, and autophagy inhibition, respectively. The CQ-induced endothelial response was associated with lipid accumulation and was characterized by Raman spectroscopy. CQ-induced autophagosome accumulation in the endothelium is featured by a pronounced alteration in the lipid profile, but not in the endothelial inflammation. Raman-based assessment of CQ-induced biochemical changes offers a better understanding of the autophagy mechanism in the endothelial cells.

Platelets augment respiratory burst in neutrophils activated by selected species of gram-positive or gram-negative bacteria.

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. 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. 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. 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.

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.