BAY-9835: Discovery of the First Orally Bioavailable ADAMTS7 Inhibitor.

The matrix metalloprotease ADAMTS7 has been identified by multiple genome-wide association studies as being involved in the development of coronary artery disease. Subsequent research revealed the proteolytic function of the enzyme to be relevant for atherogenesis and restenosis after vessel injury. Based on a publicly known dual ADAMTS4/ADAMTS5 inhibitor, we have in silico designed an ADAMTS7 inhibitor of the catalytic domain, which served as a starting point for an optimization campaign. Initially our inhibitors suffered from low selectivity vs MMP12. An X-ray cocrystal structure inspired us to exploit amino acid differences in the binding site of MMP12 and ADAMTS7 to improve selectivity. Further optimization composed of employing 5-membered heteroaromatic groups as hydantoin substituents to become more potent on ADAMTS7. Finally, fine-tuning of DMPK properties yielded BAY-9835, the first orally bioavailable ADAMTS7 inhibitor. Further optimization to improve selectivity vs ADAMTS12 seems possible, and a respective starting point could be identified.

Design and Preclinical Characterization Program toward Asundexian (BAY 2433334), an Oral Factor XIa Inhibitor for the Prevention and Treatment of Thromboembolic Disorders.

Activated coagulation factor XI (FXIa) is a highly attractive antithrombotic target as it contributes to the development and progression of thrombosis but is thought to play only a minor role in hemostasis so that its inhibition may allow for decoupling of antithrombotic efficacy and bleeding time prolongation. Herein, we report our major efforts to identify an orally bioavailable, reversible FXIa inhibitor. Using a protein structure-based de novo design approach, we identified a novel micromolar hit with attractive physicochemical properties. During lead modification, a critical problem was balancing potency and absorption by focusing on the most important interactions of the lead series with FXIa while simultaneously seeking to improve metabolic stability and the cytochrome P450 interaction profile. In clinical trials, the resulting compound from our extensive research program, asundexian (BAY 2433334), proved to possess the desired DMPK properties for once-daily oral dosing, and even more importantly, the initial pharmacological hypothesis was confirmed.

New Generation of sGC Stimulators: Discovery of Imidazo[1,2-a]pyridine Carboxamide BAY 1165747 (BAY-747), a Long-Acting Soluble Guanylate Cyclase Stimulator for the Treatment of Resistant Hypertension.

Herein, we describe the identification, chemical optimization, and preclinical characterization of novel soluble guanylate cyclase (sGC) stimulators. Given the very broad therapeutic opportunities for sGC stimulators, new tailored molecules for distinct indications with specific pharmacokinetics, tissue distribution, and physicochemical properties will be required in the future. Here, we report the ultrahigh-throughput (uHTS)-based discovery of a new class of sGC stimulators from an imidazo[1,2-a]pyridine lead series. Through the extensive and staggered optimization of the initial screening hit, liabilities such as potency, metabolic stability, permeation, and solubility could be substantially improved in parallel. These efforts resulted ultimately in the discovery of the new sGC stimulators 22 and 28. It turned out that BAY 1165747 (BAY-747, 28) could be an ideal treatment alternative for patients with hypertension, especially those not responding to standard anti-hypertensive therapy (resistant hypertension). BAY-747 (28) demonstrated sustained hemodynamic effects up to 24 h in phase 1 studies.

Discovery of the Soluble Guanylate Cyclase Activator Runcaciguat (BAY 1101042).

Herein we describe the discovery, mode of action, and preclinical characterization of the soluble guanylate cyclase (sGC) activator runcaciguat. The sGC enzyme, via the formation of cyclic guanosine monophoshphate, is a key regulator of body and tissue homeostasis. sGC activators with their unique mode of action are activating the oxidized and heme-free and therefore NO-unresponsive form of sGC, which is formed under oxidative stress. The first generation of sGC activators like cinaciguat or ataciguat exhibited limitations and were discontinued. We overcame limitations of first-generation sGC activators and identified a new chemical class via high-throughput screening. The investigation of the structure-activity relationship allowed to improve potency and multiple solubility, permeability, metabolism, and drug-drug interactions parameters. This program resulted in the discovery of the oral sGC activator runcaciguat (compound 45, BAY 1101042). Runcaciguat is currently investigated in clinical phase 2 studies for the treatment of patients with chronic kidney disease and nonproliferative diabetic retinopathy.

Design, Synthesis, and Pharmacological Characterization of a Neutral, Non-Prodrug Thrombin Inhibitor with Good Oral Pharmacokinetics.

Despite extensive research on small molecule thrombin inhibitors for oral application in the past decades, only a single double prodrug with very modest oral bioavailability has reached human therapy as a marketed drug. We have undertaken major efforts to identify neutral, non-prodrug inhibitors. Using a holistic analysis of all available internal data, we were able to build computational models and apply these for the selection of a lead series with the highest possibility of achieving oral bioavailability. In our design, we relied on protein structure knowledge to address potency and identified a small window of favorable physicochemical properties to balance absorption and metabolic stability. Protein structure information on the pregnane X receptor helped in overcoming a persistent cytochrome P450 3A4 induction problem. The selected compound series was optimized to a highly potent, neutral, non-prodrug thrombin inhibitor by designing, synthesizing, and testing derivatives. The resulting optimized compound, BAY1217224, has reached first clinical trials, which have confirmed the desired pharmacokinetic properties.

Immunomodulation by an Omega-6 Fatty Acid Reduced Mixed Lipid Emulsion in Murine Acute Respiratory Distress Syndrome.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is associated with both high morbidity and mortality in intensive care units worldwide. Patients with ARDS often require parenteral nutrition with lipid emulsions as essential components. In the present study, we assessed the immunomodulatory and apoptotic effects of a modern, n-6-reduced lipid emulsion mixture in murine ARDS. METHODS: Mice received an infusion of either normal saline solution, pure long-chain triglyceride (LCT) emulsion, or SMOF (soybean oil, medium-chain triglycerides, olive oil, and fish oil) before a lipopolysaccharide (LPS) challenge. Mice were sacrificed at different time points (0, 24, or 72 h) after ARDS induction, and an analysis of inflammatory cytokines, protein concentrations, and the cellular composition of the alveolar and interstitial compartments was performed with special focus on alveolar apoptosis and necrosis. RESULTS: Mice infused with SMOF showed decreased leukocyte invasion, protein leakage, myeloperoxidase activity, and cytokine production in alveolar spaces after LPS challenge compared to animals that received LCT. There were fewer cells in the lung interstitium of the SMOF group compared to the LCT group. Both lipid emulsions exerted pro-apoptotic and pro-necrotic properties on alveolar immune cells, with significantly increased necrosis in mice infused with LCT compared to SMOF. CONCLUSION: SMOF has both anti-inflammatory and pro-resolving influences in murine ARDS. Partial replacement of n-6 fatty acids with n-3/n-9 fatty acids may therefore benefit critically ill patients at risk for ARDS who require parenteral nutrition.

Macrophage and Tumor Cell Cross-Talk Is Fundamental for Lung Tumor Progression: We Need to Talk.

Regardless of the promising results of certain immune checkpoint blockers, current immunotherapeutics have met a bottleneck concerning response rate, toxicity, and resistance in lung cancer patients. Accumulating evidence forecasts that the crosstalk between tumor and immune cells takes center stage in cancer development by modulating tumor malignancy, immune cell infiltration, and immune evasion in the tumor microenvironment (TME). Cytokines and chemokines secreted by this crosstalk play a major role in cancer development, progression, and therapeutic management. An increased infiltration of Tumor-associated macrophages (TAMs) was observed in most of the human cancers, including lung cancer. In this review, we emphasize the role of cytokines and chemokines in TAM-tumor cell crosstalk in the lung TME. Given the role of cytokines and chemokines in immunomodulation, we propose that TAM-derived cytokines and chemokines govern the cancer-promoting immune responses in the TME and offer a new immunotherapeutic option for lung cancer treatment.

Epithelial cell plasticity defines heterogeneity in lung cancer.

Lung cancer is the leading cause of cancer death for both men and women and accounts for almost 18.4% of all deaths due to cancer worldwide, with the global incidence increasing by approximately 0.5% per year. Lung cancer is regarded as a devastating type of cancer owing to its high prevalence, reduction in the health-related quality of life, frequently delayed diagnosis, low response rate, high toxicity, and resistance to available therapeutic options. The highly heterogeneous nature of this cancer with a proximal-to-distal distribution throughout the respiratory tract dramatically affects its diagnostic and therapeutic management. The diverse composition and plasticity of lung epithelial cells across the respiratory tract are regarded as significant factors underlying lung cancer heterogeneity. Therefore, definitions of the cells of origin for different types of lung cancer are urgently needed to understand lung cancer biology and to achieve early diagnosis and develop cell-targeted therapies. In the present review, we will discuss the current understanding of the cellular and molecular alterations in distinct lung epithelial cells that result in each type of lung cancer.

Allosteric Inhibition as a New Mode of Action for BAY 1213790, a Neutralizing Antibody Targeting the Activated Form of Coagulation Factor XI.

Factor XI (FXI), the zymogen of activated FXI (FXIa), is an attractive target for novel anticoagulants because FXI inhibition offers the potential to reduce thrombosis risk while minimizing the risk of bleeding. BAY 1213790, a novel anti-FXIa antibody, was generated using phage display technology. Crystal structure analysis of the FXIa-BAY 1213790 complex demonstrated that the tyrosine-rich complementarity-determining region 3 loop of the heavy chain of BAY 1213790 penetrated deepest into the FXIa binding epitope, forming a network of favorable interactions including a direct hydrogen bond from Tyr102 to the Gln451 sidechain (2.9 Å). The newly discovered binding epitope caused a structural rearrangement of the FXIa active site, revealing a novel allosteric mechanism of FXIa inhibition by BAY 1213790. BAY 1213790 specifically inhibited FXIa with a binding affinity of 2.4 nM, and in human plasma, prolonged activated partial thromboplastin time and inhibited thrombin generation in a concentration-dependent manner.

Intravenous n-3 fatty acids in the critically ill.

PURPOSE OF REVIEW: Lipid emulsions are an integral part of parenteral nutrition. Enteral nutrition is the preferred route to feed critically ill patients and parenteral nutrition is used in case of contraindications or when enteral nutrition does not reach the nutritional goals. n-3 Lipids are included into some newer lipid emulsions including fish oil or may be added by a fish oil-based lipid emulsion to lipid emulsion without fish oil. This review focuses on recent clinical trials, metaanalyses, and guidelines of parenteral nutrition with n-3 lipids in critically ill patients. RECENT FINDINGS: Two single-center studies report a mortality benefit of adding fish oil-based lipid emulsions to the parenteral nutrition. Metaanalyses performed without these two studies had demonstrated beneficial effects of n-3 lipids regarding infections, length of stay, and time of mechanical ventilation but not on mortality. However, all metaanalyses judged the database derived from the underlying studies as not sufficient for a firm recommendation. Consecutively, guidelines and expert groups issue very cautious recommendations for the use of n-3 lipids in parenteral nutrition. SUMMARY: Beneficial effects of n-3 lipids in trials and metaanalyses became available; however, high-quality multicenter randomized controlled trials are needed before more endorsing recommendation will be available.

N-3 vs. n-6 fatty acids differentially influence calcium signalling and adhesion of inflammatory activated monocytes: impact of lipid rafts.

BACKGROUND: Anti-inflammatory n-3 fatty acids (FA) like docosahexaenoic acid (DHA) opposed to the pro-inflammatory n-6 FA arachidonic acid (AA) might modulate lipid rafts within the cell membrane by differential incorporation. In inflammation, monocyte adhesion to endothelial cells is a crucial step mediated by intracellular calcium changes. We investigated whether lipid rafts mediate FA-induced modulation of adhesion and intracellular calcium. METHODS: In isolated human monocytes and monocytic U937 cells we measured adhesion to human umbilical vein endothelial cells (HUVEC) using a parallel flow chamber and a static assay, adhesion molecules by FACScan, and intracellular calcium by fluorescence. Monocyte lipid rafts were isolated by ultracentrifugation and submitted to gas chromatography for FA analysis. RESULTS: Pre-incubation with AA or DHA resulted in a predominant incorporation of the respective FA into raft compared to non-raft fraction. DHA as compared to AA significantly reduced monocyte adhesion and calcium release after stimulation with TNF-α while expression of adhesion molecules remained unchanged. Pre-treatment with a calcium chelator abolished the effect of FA on calcium and adhesion. Disruption of lipid rafts prevented FA-induced modulations. CONCLUSION: Incorporation of FA into lipid rafts seem to be crucial for modulation of adhesion under inflammatory conditions.

Discovery and SAR of Novel 2,3-Dihydroimidazo[1,2-c]quinazoline PI3K Inhibitors: Identification of Copanlisib (BAY 80-6946).

The phosphoinositide 3-kinase (PI3K) pathway is aberrantly activated in many disease states, including tumor cells, either by growth factor receptor tyrosine kinases or by the genetic mutation and amplification of key pathway components. A variety of PI3K isoforms play differential roles in cancers. As such, the development of PI3K inhibitors from novel compound classes should lead to differential pharmacological and pharmacokinetic profiles and allow exploration in various indications, combinations, and dosing regimens. A screening effort aimed at the identification of PI3Kγ inhibitors for the treatment of inflammatory diseases led to the discovery of the novel 2,3-dihydroimidazo[1,2-c]quinazoline class of PI3K inhibitors. A subsequent lead optimization program targeting cancer therapy focused on inhibition of PI3Kα and PI3Kß. Herein, initial structure-activity relationship findings for this class and the optimization that led to the identification of copanlisib (BAY 80-6946) as a clinical candidate for the treatment of solid and hematological tumors are described.

Immunomodulation by fish-oil containing lipid emulsions in murine acute respiratory distress syndrome.

INTRODUCTION: Acute respiratory distress syndrome (ARDS) is a major cause of mortality in intensive care units. Patients with ARDS often require parenteral nutrition with lipid emulsions as essential components. Besides being an energy supply, these lipid emulsions might display differential modulatory effects on lung integrity and inflammation. METHODS: In a pre-emptive strategy, we investigated the impact of three different intravenously infused lipid emulsions on lung morphology, leukocyte invasion, protein leakage and cytokines in a murine model of ARDS. Mice received an infusion of normal saline solution, a pure long-chain triglycerides (LCT) emulsion, a medium-chain triglycerides (MCT) containing mixed emulsion (LCT/MCT), or a fish oil (FO) containing mixed emulsion (LCT/MCT/FO) before lipopolysaccharide (LPS) challenge. RESULTS: Mice pre-infused with fish oil-containing lipid emulsion showed decreased leukocyte invasion, protein leakage, myeloperoxidase activity, and cytokine production in their alveolar space after LPS challenge compared to mice receiving LCT or LCT/MCT. In line with these findings, lung morphology assessed by histological staining after LPS-induced lung injury improved faster in the LCT/MCT/FO group. Concerning the above mentioned parameters, no significant difference was observed between mice infused with LCT or the combination of LCT and MCT. CONCLUSION: Fish oil-containing lipid emulsions might exert anti-inflammatory and pro-resolving effects in the murine model of acute lung injury. Partial replacement of n-6 fatty acids with n-3 fatty acids may thus be of benefit for critically ill patients at risk for ARDS which require parenteral nutrition.

Insights into BAY 60-2770 activation and S-nitrosylation-dependent desensitization of soluble guanylyl cyclase via crystal structures of homologous nostoc H-NOX domain complexes.

The soluble guanylyl cyclase (sGC) is an important receptor for nitric oxide (NO). Nitric oxide activates sGC several hundred fold to generate cGMP from GTP. Because of sGC's salutary roles in cardiovascular physiology, it has received substantial attention as a drug target. The heme domain of sGC is key to its regulation as it not only contains the NO activation site but also harbors sites for NO-independent sGC activators as well an S-nitrosylation site (ß1 C122) involved in desensitization. Here we report the crystal structure of the activator BAY 60-2770 bound to the Nostoc H-NOX domain that is homologous to sGC. The structure reveals that BAY 60-2770 has displaced the heme and acts as a heme mimetic via carboxylate-mediated interactions with the conserved YxSxR motif as well as hydrophobic interactions. Comparisons with the previously determined BAY 58-2667 bound structure reveal that BAY 60-2770 is more ordered in its hydrophobic tail region. sGC activity assays demonstrate that BAY 60-2770 has about 10% higher fold maximal stimulation compared to BAY 58-2667. S-Nitrosylation of the BAY 60-2770 substituted Nostoc H-NOX domain causes subtle changes in the vicinity of the S-nitrosylated C122 residue. These shifts could impact the adjacent YxSxR motif and αF helix and as such potentially inhibit either heme incorporation or NO-activation of sGC and thus provide a structural basis for desensitization.

cAMP phosphodiesterase inhibitors increases nitric oxide production by modulating dimethylarginine dimethylaminohydrolases.

BACKGROUND: Pulmonary arterial hypertension is characterized by a progressive increase in pulmonary vascular resistance caused by endothelial dysfunction, inward vascular remodeling, and severe loss of precapillary pulmonary vessel cross-sectional area. Asymmetrical dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, and its metabolizing enzyme dimethylarginine dimethylaminohydrolase (DDAH) play important roles in endothelial dysfunction. We investigated whether combined phosphodiesterase (PDE) 3 and 4 inhibition ameliorates endothelial function by regulating the ADMA-DDAH axis. METHODS AND RESULTS: We investigated the effects of the PDE3/4 inhibitor tolafentrine in vitro on endothelial cell survival, proliferation, and apoptosis. Effects of tolafentrine on the endothelial nitric oxide synthase/nitric oxide pathway, DDAH expression, DDAH promoter activity, and cytokine release from endothelial cells and their subsequent influence on DDAH expression were investigated. In monocrotaline-induced pulmonary arterial hypertension in rats, the effects of inhaled tolafentrine on DDAH expression and activity were investigated. Real-time-polymerase chain reaction, immunocytochemistry, and PDE activity assays suggested high expression of PDE3 and PDE4 isoforms in endothelial cells. Treatment of endothelial cells with PDE3/4 inhibitor significantly decreased ADMA-induced apoptosis via a cAMP/PKA-dependent pathway by induction of DDAH2. Chronic nebulization of PDE3/4 inhibitor significantly attenuated monocrotaline-induced hemodynamic, gas exchange abnormalities, vascular remodeling, and right heart hypertrophy. Interestingly, PDE3/4 inhibitor treatment reduced ADMA and elevated nitric oxide/cGMP levels. Mechanistically, this could be attributed to direct modulatory effects of cAMP on the promoter region of DDAH2, which was consequently found to be increased in expression and activity. Furthermore, PDE3/4 inhibitor suppressed apoptosis in endothelial cells and increased vascularization in the lung. CONCLUSION: Combined inhibition of PDE3 and 4 regresses development of pulmonary hypertension and promotes endothelial regeneration by modulating the ADMA-DDAH axis.

Structure of cinaciguat (BAY 58-2667) bound to Nostoc H-NOX domain reveals insights into heme-mimetic activation of the soluble guanylyl cyclase.

Heme is a vital molecule for all life forms with heme being capable of assisting in catalysis, binding ligands, and undergoing redox changes. Heme-related dysfunction can lead to cardiovascular diseases with the oxidation of the heme of soluble guanylyl cyclase (sGC) critically implicated in some of these cardiovascular diseases. sGC, the main nitric oxide (NO) receptor, stimulates second messenger cGMP production, whereas reactive oxygen species are known to scavenge NO and oxidize/inactivate the heme leading to sGC degradation. This vulnerability of NO-heme signaling to oxidative stress led to the discovery of an NO-independent activator of sGC, cinaciguat (BAY 58-2667), which is a candidate drug in clinical trials to treat acute decompensated heart failure. Here, we present crystallographic and mutagenesis data that reveal the mode of action of BAY 58-2667. The 2.3-A resolution structure of BAY 58-2667 bound to a heme NO and oxygen binding domain (H-NOX) from Nostoc homologous to that of sGC reveals that the trifurcated BAY 58-2667 molecule has displaced the heme and acts as a heme mimetic. Carboxylate groups of BAY 58-2667 make interactions similar to the heme-propionate groups, whereas its hydrophobic phenyl ring linker folds up within the heme cavity in a planar-like fashion. BAY 58-2667 binding causes a rotation of the alphaF helix away from the heme pocket, as this helix is normally held in place via the inhibitory His(105)-heme covalent bond. The structure provides insights into how BAY 58-2667 binds and activates sGC to rescue heme-NO dysfunction in cardiovascular diseases.

Induction of lymphocyte apoptosis in a murine model of acute lung injury--modulation by lipid emulsions.

Acute lung injury (ALI) and sepsis are the major causes of mortality in intensive care units. Lymphocytes apoptosis is a hallmark feature of late detrimental sepsis. Parenteral nutrition in critically ill patients is based on lipid emulsions, but the impact of ALI and lipid emulsions on lymphocytes has not been defined. The effects of intravenously infused conventional soybean oil (SO)-based and new olive oil (OO)-based emulsions on splenic and blood lymphocytes were investigated in a murine model of endotoxin-induced ALI. After LPS challenge and infusion of lipid emulsions, apoptosis of lymphocytes and lung injury were assessed by flow cytometry, Western blot, and histology. Induction of ALI led to a time-dependent decline in splenic and circulating lymphocyte numbers and an increase in apoptosis, with engagement of the extrinsic apoptotic pathway. Both SO- and OO-based emulsions promoted the apoptosis of splenic lymphocytes before induction of ALI. The OO-based emulsions exhibited lower proapoptotic activity than did SO-based emulsions, an observation paralleled by the induction of survival factors. Induction of ALI increased the mortality of mice receiving SO-based emulsions compared with OO-based emulsions and normal saline. Splenic lymphocyte apoptosis is apparent in murine ALI, which may be linked to detrimental outcome. Infusion of lipid emulsions per se provoked splenic lymphocyte apoptosis. Infusion of SO-based emulsions further augmented the apoptosis of splenic and circulating lymphocytes in ALI and led to increased mortality in mice. These findings may be of relevance for patients experiencing ALI that require parenteral nutrition.

Acute lung injury is reduced in fat-1 mice endogenously synthesizing n-3 fatty acids.

RATIONALE: Acute lung injury (ALI) remains an important cause of mortality in intensive care units. Inflammation is controlled by cytokines and eicosanoids derived from the n-6 fatty acid (FA) arachidonic acid (AA). The n-3 FA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and mediators derived from EPA and DHA possess reduced inflammatory potency. OBJECTIVES: To determine whether the ability of fat-1 mice to endogenously convert n-6 to n-3 FA, and thus generate an increased ratio of n-3 to n-6 FA, impacts experimental ALI. METHODS: We investigated ALI induced by intratracheal instillation of endotoxin in fat-1 and wild-type (WT) mice, assessing leukocyte numbers, protein concentration, and prostaglandin and cytokine levels in bronchoalveolar lavage fluid, as well as free FA in plasma, and lung ventilator compliance. Body temperature and motor activity of mice--markers of sickness behavior--were also recorded. MEASUREMENTS AND MAIN RESULTS: In ALI, fat-1 mice exhibited significantly reduced leukocyte invasion, protein leakage, and macrophage inflammatory protein-2 and thromboxane B(2) levels in lavage fluid compared with WT mice. Free AA levels were increased in the plasma of WT mice in response to endotoxin, whereas EPA and DHA were increased in the fat-1 group. Ventilator compliance was significantly improved in fat-1 mice. Body temperature and motor activity were decreased in ALI. fat-1 Mice recovered body temperature and motor activity faster. CONCLUSIONS: fat-1 Mice exhibited reduced features of ALI and sickness behavior. Increasing the availability of n-3 FA may thus be beneficial in critically ill patients with ALI.

Fatty acids differentially influence phosphatidylinositol 3-kinase signal transduction in endothelial cells: impact on adhesion and apoptosis.

In contrast to n-6 fatty acids like arachidonic acid (AA), the anti-inflammatory potential of n-3 fatty acids such as docosahexaenoic acid (DHA) has been demonstrated. We examined the phosphatidylinositol (PI)3-kinase dependent effects of AA versus DHA on monocyte rolling, adhesion and transmigration through inflammatory activated human umbilical venous endothelial cells (HUVEC) as well as on apoptosis, to investigate the impact on vascular inflammation. HUVEC were pre-incubated with AA, DHA or sham, and stimulated with VEGF, TNF-alpha or staurosporine. Rolling and adhesion were investigated by means of a parallel flow chamber; transmigration was performed in a static assay. Activation of PI3-kinase was measured as phosphorylation of protein kinase B (Akt). Apoptosis was determined by caspase-3 activity and annexin-V analysis. Pre-incubation of HUVEC with DHA markedly decreased TNF-alpha-induced monocyte rolling, adhesion, and transmigration, although expression of endothelial adhesion molecules was unchanged. In contrast, AA increased TNF-alpha-induced rolling. Both fatty acids did not alter TNF-alpha-mediated upregulation of the adhesion molecules ICAM-1, VCAM-1, and E-selectin. The divergent effects of AA and DHA were abrogated with PI3-kinase inhibitors. After pre-incubation with DHA, VEGF-, TNF-alpha- and staurosporine-induced phosphorylation of Akt was decreased when compared to AA. DHA pre-incubation significantly increased staurosporine-induced apoptosis. In addition, DHA in comparison to AA augmented staurosporine-mediated increase in caspase-3 activity. In conclusion, DHA-induced a reduction in rolling, adhesion and transmigration of monocytes through inflammatory activated HUVEC that is in part PI3-kinase dependent. PI3-kinase driven phosphorylation of Akt and apoptosis of HUVEC as contribution to the resolution of inflammation is differentially modulated by DHA versus AA.

Immunomodulation by n-3- versus n-6-rich lipid emulsions in murine acute lung injury--role of platelet-activating factor receptor.

OBJECTIVE: Cytokines, platelet-activating factor (PAF), and eicosanoids control local and systemic inflammation. Conventional soybean oil-based lipid emulsions used for parenteral nutrition may aggravate the leukocyte inflammatory response or adhesion to the vessel wall. Fish oil-based lipid emulsions, in contrast, may exert an anti-inflammatory effect. DESIGN: We investigated the impact of lipid emulsions on leukocyte invasion, protein leakage, and cytokines in two murine models of acute inflammation. SETTING: Research laboratory of a university hospital. SUBJECTS: Wild-type mice and PAF-receptor knockout mice. INTERVENTIONS: Mice received an infusion of normal saline, fish oil- or soybean oil-based lipid emulsions before lipopolysaccharide challenge. MEASUREMENTS AND MAIN RESULTS: Preinfusion with soybean oil resulted in increased leukocyte invasion, myeloperoxidase activity, and protein leakage and exaggerated release of tumor necrosis factor (TNF)-alpha as well as macrophage inflammatory protein (MIP)-2 into the alveolar space after intratracheal lipopolysaccharide challenge. In contrast, preinfusion with fish oil reduced leukocyte invasion, myeloperoxidase activity, protein leakage, and TNF-alpha as well as MIP-2 generation. Corresponding profiles were found in plasma following intraperitoneal lipopolysaccharide application: Soybean oil increased but fish oil decreased the TNF-alpha and MIP-2 formation. When PAF-receptor-deficient mice were challenged with lipopolysaccharide, leukocyte invasion, lung tissue myeloperoxidase, cytokine generation, and alveolar protein leakage corresponded to those observed in wild-type animals. Fish oil and soybean oil lost their diverging effects on leukocyte transmigration, myeloperoxidase activity, leakage response, and cytokine generation in these knockout mice. Similarly, the differential impact of both lipid emulsions on these lipopolysaccharide-provoked changes was suppressed after pretreating animals with a PAF-receptor antagonist. CONCLUSIONS: Fish oil- vs. soybean oil-based lipid infusions exert anti- vs. proinflammatory effects in murine models of acute inflammation. The PAF/PAF-receptor-linked signaling appears to be a prerequisite for this differential profile.