Search

Background For patients treated with dual antiplatelet therapy, standardized drug-specific 3-to-7 day cessation is recommended prior to major surgery to reach sufficient platelet function recovery. Here we investigated the hypothesis that supplemental fibrinogen might mitigate the inhibitory effects of antiplatelet therapy.Methods and Results To this end blood from healthy donors was treated in vitro with platelet inhibitors, and in vitro thrombus formation and platelet activation were assessed. Ticagrelor, acetylsalicylic acid, the combination of both, and tirofiban all markedly attenuated the formation of adherent thrombi, when whole blood was perfused through collagen-coated microchannels at physiological shear rates. Addition of fibrinogen restored in vitro thrombus formation in the presence of antiplatelet drugs and heparin. However, platelet activation, as investigated in assays of P-selectin expression and calcium flux, was not altered by fibrinogen supplementation. Most importantly, fibrinogen was able to restore in vitro thrombogenesis in patients on maintenance dual antiplatelet therapy after percutaneous coronary intervention.Conclusion Thus, our in vitro data support the notion that supplementation of fibrinogen influences the perioperative hemostasis in patients undergoing surgery during antiplatelet therapy by promoting thrombogenesis without significantly interfering with platelet activation.

Placental hypervascularization has been reported in pregnancy-related pathologies such as gestational diabetes mellitus (GDM). Nevertheless, the underlying causes behind this abnormality are not well understood. In this study, we addressed the expression of SUCNR1 (cognate succinate receptor) in human placental endothelial cells and hypothesized that succinate-SUCNR1 axis might play a role in the placental hypervascularization reported in GDM. We measured significantly higher succinate levels in placental tissue lysates from women with GDM relative to matched controls. In parallel, SUCNR1 protein expression was upregulated in GDM tissue lysates as well as in isolated diabetic fetoplacental arterial endothelial cells (FpECAds). A positive correlation of SUCNR1 and vascular endothelial growth factor (VEGF) protein levels in tissue lysates indicated a potential link between succinate-SUCNR1 axis and placental angiogenesis. In our in-vitro experiments, succinate prompted hallmarks of angiogenesis in human umbilical vein endothelial cells (HUVECs) such as proliferation, migration and spheroid sprouting. These results were further validated in fetoplacental arterial endothelial cells (FpECAs), where succinate induced endothelial tube formation. VEGF gene expression was increased in response to succinate in both HUVECs and FpECAs. Yet, knockdown of SUCNR1 in HUVECs led to suppression of VEGF gene expression and abrogated the migratory ability and wound healing in response to succinate. In conclusion, our data underline SUCNR1 as a promising metabolic target in human placenta and as a potential driver of enhanced placental angiogenesis in GDM.

Life-threatening inflammatory conditions such as acute respiratory distress syndrome or sepsis often go hand in hand with severe vascular leakage. During inflammation, endothelial cell integrity and intact barrier function are crucial to limit leukocyte and plasma extravasation. Prostaglandin D

Prostaglandin E2 (PGE2) signaling is known to modulate inflammation and vascular resistance. Receptors of PGE2 [E-type prostanoid receptors (EP)] might be an attractive pharmacological target in immune-mediated diseases such as glomerulonephritis. We hypothesized that selective EP4 antagonism improves nephrotoxic serum nephritis (NTS) by its anti-inflammatory properties. Mice were subjected to NTS and treated with the EP4 antagonist ONO AE3-208 (10 mg·kg body wt−1·day−1] or vehicle starting from disease initiation. In one set of experiments, treatment was started 4 days after NTS induction. Tubular epithelial cells were evaluated in vitro under starving conditions. EP4 antagonist treatment significantly improved the NTS phenotype without affecting blood pressure levels. Remarkably, the improved NTS phenotype was also observed when treatment was started 4 days after NTS induction. EP4 antagonism decreased tubular chemokine (C-X-C motif) ligand ( Cxcl) 1 and Cxcl-5 expression and thereby subsequently reduced interstitial neutrophil infiltration into the kidney. In vitro, tubular epithelial cells increasingly expressed Cxcl-5 mRNA and Cxcl-5 protein when treated with PGE2 or an EP4 agonist under starving conditions, which was blunted by EP4 antagonist treatment. Together, EP4 antagonism improves the NTS phenotype, probably by decreasing mainly Cxcl-5 production in tubular cells, thereby reducing renal neutrophil infiltration.

Pulmonary arterial hypertension (PAH) is a rare disease characterized by increased pulmonary pressure and vascular remodelling as a consequence of smooth muscle cell proliferation, endothelial cell dysfunction and inflammatory infiltrates. Meprin α is a metalloproteinase whose substrates include adhesion and cell–cell contact molecules involved in the process of immune cell extravasation. In this study, we aimed to unravel the role of meprin α in PAH-induced vascular remodelling. Our results showed that meprin α was present in the apical membrane of endothelial cells in the lungs and pulmonary arteries of donors and idiopathic PAH (IPAH) patients. Elevated circulating meprin α levels were detected in the plasma of IPAH patients. In vitro binding assays and electron microscopy confirmed binding of meprin α to the glycocalyx of human pulmonary artery endothelial cells (hPAECs). Enzymatic and genetic approaches identified heparan sulphate (HS) as an important determinant of the meprin α binding capacity to hPAEC. Meprin α treatment protected from excessive neutrophil infiltration and the protective effect observed in the presence of neutrophils was partially reversed by removal of HS from hPAEC. Importantly, HS levels in pulmonary arteries were decreased in IPAH patients and binding of meprin α to HS was impaired in IPAH hPAEC. In summary, our results suggest a role of HS in docking meprin α to the endothelium and thus in the modulation of inflammatory cell extravasation. In IPAH, the decreased endothelial HS results in the reduction of meprin α binding which might contribute to enhanced inflammatory cell extravasation and potentially to pathological vascular remodelling.

The putative cannabinoid receptor GPR55 has been shown to play a tumor-promoting role in various cancers, and is involved in many physiological and pathological processes of the gastrointestinal (GI) tract. While the cannabinoid receptor 1 (CB1 ) has been reported to suppress intestinal tumor growth, the role of GPR55 in the development of GI cancers is unclear. We, therefore, aimed at elucidating the role of GPR55 in colorectal cancer (CRC), the third most common cancer worldwide. Using azoxymethane (AOM)- and dextran sulfate sodium (DSS)-driven CRC mouse models, we found that GPR55 plays a tumor-promoting role that involves alterations of leukocyte populations, i.e. myeloid-derived suppressor cells and T lymphocytes, within the tumor tissues. Concomitantly, expression levels of COX-2 and STAT3 were reduced in tumor tissue of GPR55 knockout mice, indicating reduced presence of tumor-promoting factors. By employing the experimental CRC models to CB1 knockout and CB1 /GPR55 double knockout mice, we can further show that GPR55 plays an opposing role to CB1 . We report that GPR55 and CB1 mRNA expression are differentially regulated in the experimental models and in a cohort of 86 CRC patients. Epigenetic methylation of CNR1 and GPR55 was also differentially regulated in human CRC tissue compared to control samples. Collectively, our data suggest that GPR55 and CB1 play differential roles in colon carcinogenesis where the former seems to act as oncogene and the latter as tumor suppressor.

Background Idiopathic pulmonary fibrosis (IPF) is a disease with high 5-year mortality and few therapeutic options. Prostaglandin (PG) E2 exhibits antifibrotic properties and is reduced in bronchoalveolar lavage from patients with IPF. 15-Prostaglandin dehydrogenase (15-PGDH) is the key enzyme in PGE2 metabolism under the control of TGF-β and microRNA 218. Objective We sought to investigate the expression of 15-PGDH in IPF and the therapeutic potential of a specific inhibitor of this enzyme in a mouse model and human tissue. Methods In vitro studies, including fibrocyte differentiation, regulation of 15-PGDH, RT-PCR, and Western blot, were performed using peripheral blood from healthy donors and patients with IPF and A549 cells. Immunohistochemistry, immunofluorescence, 15-PGDH activity assays, and in situ hybridization as well as ex vivo IPF tissue culture experiments were done using healthy donor and IPF lungs. Therapeutic effects of 15-PGDH inhibition were studied in the bleomycin mouse model of pulmonary fibrosis. Results We demonstrate that 15-PGDH shows areas of increased expression in patients with IPF. Inhibition of this enzyme increases PGE2 levels and reduces collagen production in IPF precision cut lung slices and in the bleomycin model. Inhibitor-treated mice show amelioration of lung function, decreased alveolar epithelial cell apoptosis, and fibroblast proliferation. Pulmonary fibrocyte accumulation is also decreased by inhibitor treatment in mice, similar to PGE2 that inhibits fibrocyte differentiation from blood of healthy donors and patients with IPF. Finally, microRNA 218-5p, which is downregulated in patients with IPF, suppressed 15-PGDH expression in vivo and in vitro. Conclusions These findings highlight the role of 15-PGDH in IPF and suggest 15-PGDH inhibition as a promising therapeutic approach.

Life-threatening inflammatory conditions such as acute respiratory distress syndrome or sepsis often go hand in hand with severe vascular leakage. During inflammation, endothelial cell integrity and intact barrier function are crucial to limit leukocyte and plasma extravasation. Prostaglandin D2 (PGD2) is a potent inflammatory lipid mediator with vasoactive properties. Previous studies suggest that PGD2 is involved in the regulation of endothelial barrier function; however, it is unclear whether this is also true for primary human pulmonary microvascular endothelial cells. Furthermore, as PGD2 is a highly promiscuous ligand, we set out to determine which receptors are important in human pulmonary endothelial cells. In the current study, we found that PGD2 and the DP1 agonist BW245c potently strengthened pulmonary and dermal microvascular endothelial cell barrier function and protected against thrombin-induced barrier disruption. Yet surprisingly, these effects were mediated only to a negligible extent via DP1 receptor activation. In contrast, we observed that the EP4 receptor was most important and mediated the barrier enhancement by PGD2 and BW245c. Stimulation with PGE2 or PGD2 reduced AKT phosphorylation which could be reversed by prior blockade of EP4 receptors. These data demonstrate a novel mechanism by which PGD2 may modulate inflammation and emphasizes the role of EP4 receptors in human endothelial cell function.

Eosinophils are important multifaceted effector cells involved in allergic inflammation. Following allergen challenge, eosinophils and other immune cells release secreted phospholipases, generating lysophosphatidylcholines (LPCs). LPCs are potent lipid mediators, and serum levels of LPCs associate with asthma severity, suggesting a regulatory activity of LPCs in asthma development. As of yet, the direct effects of LPCs on eosinophils remain unclear. In the present study, we tested the effects of the major LPC species (16:0, 18:0 and 18:1) on eosinophils isolated from healthy human donors. Addition of saturated LPCs in the presence of albumin rapidly disrupted cholesterol-rich nanodomains on eosinophil cell membranes and suppressed multiple eosinophil effector responses, such as CD11b upregulation, degranulation, chemotaxis, and downstream signaling. Furthermore, we demonstrate in a mouse model of allergic cell recruitment, that LPC treatment markedly reduces immune cell infiltration into the lungs. Our observations suggest a strong modulatory activity of LPCs in the regulation of eosinophilic inflammation in vitro and in vivo.

Background Lung eosinophilia is a hallmark of asthma, and eosinophils are believed to play a crucial role in the pathogenesis of allergic inflammatory diseases. Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are produced in high amounts in the gastrointestinal tract by commensal bacteria and can be absorbed into the bloodstream. Although there is recent evidence that SCFAs are beneficial in allergic asthma models, the effect on eosinophils has remained elusive. Objective The role of SCFAs was investigated in human eosinophil function and a mouse model of allergic asthma. Methods Eosinophils were purified from self-reported allergic or healthy donors. Migration, adhesion to the endothelium, and eosinophil survival were studied in vitro. Ca2+ flux, apoptosis, mitochondrial membrane potential, and expression of surface markers were determined by using flow cytometry and in part by using real-time PCR. Allergic airway inflammation was assessed in vivo in an ovalbumin-induced asthma model by using invasive spirometry. Results For the first time, we observed that SCFAs were able to attenuate human eosinophils at several functional levels, including (1) adhesion to the endothelium, (2) migration, and (3) survival. These effects were independent from GPR41 and GPR43 but were accompanied by histone acetylation and mimicked by trichostatin A, a pan–histone deacetylase inhibitor. In vivo butyrate ameliorated allergen-induced airway and lung eosinophilia, reduced type 2 cytokine levels in bronchial fluid, and improved airway hyperresponsiveness in mice. Conclusion These in vitro and in vivo findings highlight the importance of SCFAs, especially butyrate as a promising therapeutic agent in allergic inflammatory diseases.

Prostaglandin E

Background and Purpose Pulmonary vascular dysfunction is a key event in acute lung injury. We recently demonstrated that PGE2, via activation of E-prostanoid (EP)4 receptors, strongly enhances microvascular barrier function in vitro. The aim of this study was to investigate the beneficial effects of concomitant EP4 receptor activation in murine models of acute pulmonary inflammation. Experimental Approach Pulmonary inflammation in male BALB/c mice was induced by LPS (20 μg per mouse intranasally) or oleic acid (0.15 μL·g-1, i.v.). In-vitro, endothelial barrier function was determined by measuring electrical impedance. Key Results PGE2 activation of EP4 receptors reduced neutrophil infiltration, pulmonary vascular leakage and TNF-α concentration in bronchoalveolar lavage fluid from LPS-induced pulmonary inflammation. Similarly, pulmonary vascular hyperpermeability induced by oleic acid was counteracted by EP4 receptor activation. In lung function assays, the EP4 agonist ONO AE1-329 restored the increased resistance and reduced compliance upon methacholine challenge in mice treated with LPS or oleic acid. In agreement with these findings, EP4 receptor activation increased the in vitro vascular barrier function of human and mouse pulmonary microvascular endothelial cells and diminished the barrier disruption induced by LPS. The EP2 agonist ONO AE1-259 likewise reversed LPS-induced lung dysfunction without enhancing vascular barrier function. Conclusion and Implications Our results show that activation of the EP4 receptor strengthens the microvascular barrier function and thereby ameliorates the pathology of acute lung inflammation, including neutrophil infiltration, vascular oedema formation and airway dysfunction. This suggests a potential benefit for EP4 agonists in acute pulmonary inflammation.

Endothelial dysfunction is a hallmark of inflammatory conditions. We recently demonstrated that prostaglandin (PG)E(2) enhances the resistance of pulmonary endothelium in vitro and counteracts lipopolysaccharide (LPS)-induced pulmonary inflammation in vivo via EP4 receptors. The aim of this study was to investigate the role of the EP1/EP3 receptor agonist 17-phenyl-trinor-(pt)-PGE(2) on acute lung inflammation in a mouse model. In LPS-induced pulmonary inflammation in mice, 17-pt-PGE(2) reduced neutrophil infiltration and inhibited vascular leakage. These effects were unaltered by an EP1 antagonist, but reversed by EP4 receptor antagonists. 17-pt-PGE(2) increased the resistance of pulmonary microvascular endothelial cells and prevented thrombin-induced disruption of endothelial junctions. Again, these effects were not mediated via EP1 or EP3 but through activation of the EP4 receptor, as demonstrated by the lack of effect of more selective EP1 and EP3 receptor agonists, prevention of these effects by EP4 antagonists and EP4 receptor knock-down by siRNA. In contrast, the aggregation enhancing effect of 17-pt-PGE(2) in human platelets was mediated via EP3 receptors. Our results demonstrate that 17-pt-PGE(2) enhances the endothelial barrier in vitro on pulmonary microvascular endothelial cells, and accordingly ameliorates the recruitment of neutrophils, via EP4 receptors in vivo. This suggests a beneficial effect of 17-pt-PGE(2) on pulmonary inflammatory diseases.