Background: Platelets facilitate hematogenous metastasis in part by promoting cancer cell immunoevasion, although our understanding of platelet function in modulating the adaptive immune system in cancer is limited. A major negative regulator of the adaptive response is the immune checkpoint protein Programmed Death Ligand 1 (PD-L1).Objectives: As platelets secrete factors that may increase PD-L1 expression, we investigated whether they up-regulate cancer cell PD-L1, thus promoting immunoevasion, and whether common anti-platelet drugs inhibit this process.Methods: Platelets were isolated from human volunteers. A549 lung, PD-L1 null A549, and 786-O renal cancer cells were incubated with and without platelets, and cancer cell PD-L1 expression was measured by qPCR and flow cytometry. Additionally, platelet-cancer cell incubations were performed in the presence of common antiplatelet drugs, and with growth factor neutralizing antibodies. Following incubation with platelets, A549 were co-cultured with T-cells and interleukin-2 (IL-2) levels were measured by flow cytometry as a marker of T-cell activation.Results: Platelets increased PD-L1 mRNA and surface protein expression by A549 and 786-0 cells. Combined neutralization of VEGF and PDGF prevented the plateletinduced up-regulation of PD-L1 by A549, as did the anti-platelet drug eptifibatide.A549 incubated with platelets demonstrated a reduced ability to activate human T-cells, an effect reversed by eptifibatide. Conclusions:As platelets promote immunoevasion of the adaptive immune response by increasing cancer cell PD-L1 expression and as anti-platelet drugs prevent this immunoevasive response, the investigation of anti-platelet drugs as adjuvant therapy to immune checkpoint inhibitors may be warranted in the treatment of cancer.

: Nitric oxide (NO), an important endogenous signalling molecule released from vascular endothelial cells and nerves, activates the enzyme soluble guanylate cyclase to catalyze production of cyclic guanosine monophosphate (cGMP) from guanosine triphosphate. cGMP, in turn, activates protein kinase G to phosphorylate a range of effector proteins in smooth muscle cells that reduce intracellular Ca2+ levels to inhibit both contractility and proliferation. The enzyme phosphodiesterase type 5 (PDE5) curtails the actions of cGMP by hydrolyzing it into inactive 5’-GMP. Small molecule PDE5 inhibitors (PDE5is) such as sildenafil prolong the availability of cGMP and so enhance NO-mediated signalling. PDE5is are the first line treatment for erectile dysfunction but are also now approved for treatment of pulmonary arterial hypertension (PAH) in adults. Persistent pulmonary hypertension in neonates (PPHN) is currently treated with inhaled NO but this is an expensive option and around 1/3 of newborns are unresponsive resulting in the need for alternative approaches. Here we summarize the development, chemistry and pharmacology of PDE5is, the use of sildenafil for erectile dysfunction and PAH, and then critically review current evidence for the utility of further repurposing of sildenafil as a treatment for PPHN.

BackgroundCancer cells have the ability to activate platelets, and platelets play an important role facilitating hematogenous metastasis. One mechanism by which platelets promote metastasis is their ability to shield cancer cells from natural killer cell‐mediated death. Although much is known of how platelets affect the innate immune response, our understanding of platelet function in modulating the adaptive immune system in cancer is limited. A major negative regulator of the adaptive immune response in cancer is the immune checkpoint transmembrane protein Programmed Death Ligand 1 (PD‐L1). PD‐L1 interacts with its receptor PD on T‐cells resulting in T‐cell anergy and/or apoptosis. This cancer cell survival mechanism is exploited in various types of cancer, including lung and kidney. Interestingly, platelets upon activation secrete a number of factors that have the potential to increase cancer cell PD‐L1 expression. Therefore, we hypothesized that cancer cell‐activated platelets increase cancer cell PD‐L1 expression and that common anti‐platelet drugs inhibit this platelet‐induced up‐regulation of PD‐L1.MethodsPlatelets were isolated from the blood of healthy human volunteers. Human A549 lung and 786‐O renal cancer cells were incubated with and without platelets for 24 hours and cancer cell PD‐L1 surface expression was measured by flow cytometry and mRNA by qPCR. In other experiments, platelet‐cancer cell incubations were performed in the presence of anti‐platelet drugs acetylsalicylic acid (ASA – 100 mM), Prasugrel active metabolite (PAM – 10 mM) or Integrelin (10 mM).ResultsPlatelets caused a significant increase in PD‐L1 surface expression by A549 (6.7±3.1% of A549 vs. 16.3±3.9% of A549 + platelets, P < 0.05) and 786‐0 cells (11.2±3.1% of 786‐O vs. 21.8±4,8% of 786‐O + platelets, P < 0.05). This increase in surface expression occurred as a result of a 3‐fold increase in PD‐L1 mRNA in A549 incubated with platelets. Platelets did not express PD‐L1 mRNA. Importantly, anti‐platelet drugs Prasugrel active metabolite (PAM) and Integrelin, but not acetylsalicylic acid, prevented the platelet‐dependent increase in PD‐L1 expression by A549.ConclusionsPlatelets up‐regulate the transcriptional expression of PD‐L1 by cancer cells, and Prasugrel and Integrelin can inhibit this up‐regulation. Further experiments are needed to determine whether platelet‐induced expression of PD‐L1 on cancer cells protects them from T‐cell induced death, and whether investigation of anti‐platelet drugs as adjuvant therapy in addition to immune checkpoint inhibitors is warranted.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fiber rich diets have been shown to prevent the onset and reduce severity of hypertension1, a condition affecting 20‐30% of adults in North America. High fiber diets facilitate the production of short chain fatty acids (SCFAs) by gut bacteria of which, in humans, acetate is the most abundant, followed by propionate and butyrate. It has been proposed that these three SCFAs may have direct effects on blood vessels and so may contribute to regulation of blood pressure2. Further research is needed to determine the mechanisms underlying the vascular actions of SCFAs. We tested the hypothesis that SCFAs can induce vasodilation and limit nerve‐evoked vasoconstriction in rat mesenteric vasculature. Direct effects on vascular tone were assessed by constructing cumulative concentration‐response curves to SCFAs in isolated third order rat mesenteric arteries mounted in a wire myograph and pre‐stimulated with phenylephrine (PE) to increase tone. The effects of SCFAs on nerve‐evoked vasoconstriction were assessed by constructing frequency‐responses curves to perivascular nerve stimulation in a perfused rat mesenteric bed in the presence and absence of SCFAs added to the perfusate. In both experimental approaches, pharmacological tools were used to investigate the mechanism of action of SCFAs. Propionate (3 μM ‐ 1 mM) and acetate (0.3 μM ‐ 1 mM) caused reductions in PE‐evoked tone with maximum relaxation of 32.7 ± 7% and 31.6 ± 8.2%, respectively, and attenuated nerve‐evoked vasoconstriction in mesenteric arteries at 10 mM ‐ 60 mM propionate and 30 mM ‐ 50 mM acetate. These effects were inhibited by blockers of nitric oxide (NO) synthase (L‐NG‐nitroarginine methyl ester, L‐NAME, 100 μM) and endothelial Ca2+‐activated K+ (KCa) channels (apamin, 50 nM; TRAM‐34, 1 μM). L‐NAME alone significantly inhibited relaxations elicited by acetate indicating a predominant role for NO, whereas the actions of propionate were significantly inhibited by apamin and TRAM‐34, indicating a predominant role for endothelial KCa channels in this response. These data indicate that SCFAs may cause vasodilation in mesenteric resistance arteries via activation of endothelial pathways and that there may be a differential contribution of NO and KCa channels to responses of individual SCFAs. 1. Appel LJ, Moore TJ, Obarzanek E, et al. A clinical trial of the effects of dietary patterns on blood pressure. DASH Collaborative Research Group. N Engl J Med. 1997;336(16):1117‐1124. doi:10.1056/NEJM199704173361601 2. Pluznick J. A novel SCFA receptor, the microbiota, and blood pressure regulation. Gut Microbes. 2014;5(2):202‐207. doi:10.4161/gmic.27492