PACAP regulates VPAC1 expression, inflammatory processes and lipid homeostasis in M1- and M2-macrophages.

Background: Pituitary adenylate cyclase-activating polypeptide (PACAP) acts as an anti-atherogenic neuropeptide and plays an important role in cytoprotective, as well as inflammatory processes, and cardiovascular regulation. Therefore, the aim of this study is to investigate the regulatory effects of PACAP and its receptor VPAC1 in relation to inflammatory processes and lipid homeostasis in different macrophage (MΦ) subtypes. Methods: To investigate the role of PACAP deficiency in the pathogenesis of atherosclerosis under standard chow (SC) or cholesterol-enriched diet (CED) in vivo, PACAP-/- mice were crossbred with ApoE-/- to generate PACAP-/-/ApoE-/- mice. Lumen stenosis in the aortic arch and different MΦ-subtypes were analyzed in atherosclerotic plaques by quantitative immunohistochemistry. Undifferentiated bone marrow-derived cells (BMDC) from 30-weeks-old ApoE-/- and PACAP-/-/ApoE-/- mice were isolated, differentiated into BMDM1- and BMDM2-MΦ, and incubated with oxidized low-density lipoprotein (oxLDL). In addition, PMA-differentiated human THP-1 MΦ were further differentiated into M1-/M2-MΦ and subsequently treated with PACAP38, the VPAC1 agonist [(Ala11,22,28)VIP], the antagonist (PG 97-269), and/or oxLDL. Uptake/accumulation of oxLDL was analyzed by oxLDL-DyLight™488 and Bodipy™ 493/503. The mRNA expression was analyzed by qRT-PCR, protein levels by Western blot, and cytokine release by ELISA. Results: In vivo, after 30 weeks of SC, PACAP-/-/ApoE-/- mice showed increased lumen stenosis compared with ApoE-/- mice. In atherosclerotic plaques of PACAP-/-/ApoE-/- mice under CED, immunoreactive areas of VPAC1, CD86, and CD163 were increased compared with ApoE-/- mice. In vitro, VPAC1 protein levels were increased in PACAP-/-/ApoE-/- BMDM compared with ApoE-/- BMDM, resulting in increased TNF-α mRNA expression in BMDM1-MΦ and decreased TNF-α release in BMDM2-MΦ. Concerning lipid homeostasis, PACAP deficiency decreased the area of lipid droplets in BMDM1-/M2-MΦ with concomitant increasing adipose differentiation-related protein level. In THP-1 M1-/M2-MΦ, the VPAC1 antagonist increased the uptake of oxLDL, whereas the VPAC1 agonist decreased the oxLDL-induced intracellular triglyceride content. Conclusion: Our data suggest that PACAP via VPAC1 signaling plays an important regulatory role in inflammatory processes in atherosclerotic plaques and in lipid homeostasis in different MΦ-subtypes, thereby affecting foam cell formation. Therefore, VPAC1 agonists or PACAP may represent a new class of anti-atherogenic therapeutics.

Effects of Monoamino-Oxidase-A (MAO-A) Inhibition on Skeletal Muscle Inflammation and Wasting through Pancreatic Ductal Adenocarcinoma in Triple Transgenic Mice.

Cancer cachexia describes a syndrome of muscle wasting and lipolysis that is still largely untreatable and negatively impacts prognosis, mobility, and healthcare costs. Since upregulation of skeletal muscle monoamine-oxidase-A (MAO-A), a source of reactive oxygen species, may contribute to cachexia, we investigated the effects of the MAO-inhibitor harmine-hydrochloride (HH, intraperitoneal, 8 weeks) on muscle wasting in a triple-transgenic mouse model of pancreatic ductal adenocarcinoma (PDAC) and wild type (WT) mice. Gastrocnemius and soleus muscle cryo-cross-sections were analyzed for fiber type-specific cross-sectional area (CSA), fraction and capillarization using ATPase- and lectin-stainings. Transcripts of pro-apoptotic, -atrophic, and -inflammatory signals were determined by RT-qPCR. Furthermore, we evaluated the integrity of neuromuscular junction (NMJ, pre-/post-synaptic co-staining) and mitochondrial ultrastructure (transmission electron microscopy). MAO-A expression in gastrocnemius muscle was increased with PDAC vs. WT (immunohistochemistry: p < 0.05; Western blot: by trend). PDAC expectedly reduced fiber CSA and upregulated IL-1ß in both calf muscles, while MuRF1 expression increased in soleus muscle only. Although IL-1ß decreased, HH caused an additional 38.65% (p < 0.001) decrease in gastrocnemius muscle (IIBX) fiber CSA. Moreover, soleus muscle CSA remained unchanged despite the downregulation of E3-ligases FBXO32 (p < 0.05) and MuRF1 (p < 0.01) through HH. Notably, HH significantly decreased the post-synaptic NMJ area (quadriceps muscle) and glutathione levels (gastrocnemius muscle), thereby increasing mitochondrial damage and centronucleation in soleus and gastrocnemius type IIBX fibers. Moreover, although pro-atrophic/-inflammatory signals are reversed, HH unfortunately fails to stop and rather promotes PDAC-related muscle wasting, possibly via denervation or mitochondrial damage. These differential adverse vs. therapeutic effects warrant studies regarding dose-dependent benefits and risks with consideration of other targets of HH, such as the dual-specificity tyrosine phosphorylation regulated kinases 1A and B (DYRK1A/B).

Phytohustil® and root extract of Althaea officinalis L. exert anti-inflammatory and anti-oxidative properties and improve the migratory capacity of endothelial cells in vitro.

Introduction: Althaea officinalis L.'s root extract (REA) has been used as a medicinal plant since ancient times to treat a cough. Applying REA leads to a protective film that induces a faster regeneration of the lesioned laryngopharyngeal mucosa caused by dry coughs. The buccopharyngeal mucosa is a highly vascularized tissue. In this regard, anti-inflammatory/-oxidant phytochemicals that improve the repair of the lesion site, e.g., neovascularization in the wound, are critical for promoting healing. For this reason, it is essential to investigate the effects of Phytohustil® and REA on different cellular components of the mucosa under conditions similar to those found in the injured mucosa. Thus, this in vitro study investigated the anti-inflammatory/oxidative and pro-migratory properties of Phytohustil® cough syrup on vascular endothelial cells. Methods: Human umbilical vein endothelial cells (HUVEC) were pretreated (24 h) with Phytohustil®, its excipients, or REA, followed by incubation with hydrogen peroxide (H2O2; 1 h; pro-oxidative) or with lipopolysaccharides (LPS; 3 h; pro-inflammatory). Viability and cytotoxicity were measured by PrestoBlue® assay. Intracellular reactive oxygen species (ROS) were quantified with 20-70-dichlorofluorescein diacetate (DCFDA). The release of interleukin 6 (IL6) was determined by enzyme-linked immunosorbent assay (ELISA). The migratory capacity of HUVEC was measured using a scratch assay. Results: Our results show that Phytohustil®, its excipients and REA were not cytotoxic. Pretreatment of HUVEC (24 h) with Phytohustil® or REA inhibited the LPS-activated IL6 release. Phytohustil® or REA inhibited the H2O2-induced cytotoxicity and intracellular ROS production. Phytohustil® and REA significantly stimulated wound closure compared to the control. Conclusion: Our data show that Phytohustil® and REA have anti-inflammatory/-oxidant properties and improve the migratory capacity of vascular endothelial cells. These properties may contribute to the healing characteristics of Phytohustil® and support the benefit of Phytohustil® in patient's treatment of irritated oral mucosa.

Increased Density of Growth Differentiation Factor-15+ Immunoreactive M1/M2 Macrophages in Prostate Cancer of Different Gleason Scores Compared with Benign Prostate Hyperplasia.

Although growth differentiation factor-15 (GDF-15) is highly expressed in PCa, its role in the development and progression of PCa is unclear. The present study aims to determine the density of GDF-15+ cells and immune cells (M1-/M2 macrophages [MΦ], lymphocytes) in PCa of different Gleason scores (GS) compared to BPH. Immunohistochemistry and double immunofluorescence were performed on paraffin-embedded human PCa and BPH biopsies with antibodies directed against GDF-15, CD68 (M1 MΦ), CD163 (M2 MΦ), CD4, CD8, CD19 (T /B lymphocytes), or PD-L1. PGP9.5 served as a marker for innervation and neuroendocrine cells. GDF-15+ cell density was higher in all GS than in BPH. CD68+ MΦ density in GS9 and CD163+ MΦ exceeded that in BPH. GDF-15+ cell density correlated significantly positively with CD68+ or CD163+ MΦ density in extratumoral areas. Double immunoreactive GDF-15+/CD68+ cells were found as transepithelial migrating MΦ. Stromal CD68+ MΦ lacked GDF-15+. The area of PGP9.5+ innervation was higher in GS9 than in BPH. PGP9.5+ cells, occasionally copositive for GDF-15+, also occurred in the glandular epithelium. In GS6, but not in BPH, GDF-15+, PD-L1+, and CD68+ cells were found in epithelium within luminal excrescences. The degree of extra-/intra-tumoral GDF-15 increases in M1/M2Φ is proposed to be useful to stratify progredient malignancy of PCa. GDF-15 is a potential target for anti-tumor therapy.

Inflammation and Wasting of Skeletal Muscles in Kras-p53-Mutant Mice with Intraepithelial Neoplasia and Pancreatic Cancer-When Does Cachexia Start?

Skeletal muscle wasting critically impairs the survival and quality of life in patients with pancreatic ductal adenocarcinoma (PDAC). To identify the local factors initiating muscle wasting, we studied inflammation, fiber cross-sectional area (CSA), composition, amino acid metabolism and capillarization, as well as the integrity of neuromuscular junctions (NMJ, pre-/postsynaptic co-staining) and mitochondria (electron microscopy) in the hindlimb muscle of LSL-KrasG12D/+; LSL-TrP53R172H/+; Pdx1-Cre mice with intraepithelial-neoplasia (PanIN) 1-3 and PDAC, compared to wild-type mice (WT). Significant decreases in fiber CSA occurred with PDAC but not with PanIN 1-3, compared to WT: These were found in the gastrocnemius (type 2x: −20.0%) and soleus (type 2a: −21.0%, type 1: −14.2%) muscle with accentuation in the male soleus (type 2a: −24.8%, type 1: −17.4%) and female gastrocnemius muscle (−29.6%). Significantly higher densities of endomysial CD68+ and cyclooxygenase-2+ (COX2+) cells were detected in mice with PDAC, compared to WT mice. Surprisingly, CD68+ and COX2+ cell densities were also higher in mice with PanIN 1-3 in both muscles. Significant positive correlations existed between muscular and hepatic CD68+ or COX2+ cell densities. Moreover, in the gastrocnemius muscle, suppressor-of-cytokine-3 (SOCS3) expressions was upregulated >2.7-fold with PanIN 1A-3 and PDAC. The intracellular pools of proteinogenic amino acids and glutathione significantly increased with PanIN 1A-3 compared to WT. Capillarization, NMJ, and mitochondrial ultrastructure remained unchanged with PanIN or PDAC. In conclusion, the onset of fiber atrophy coincides with the manifestation of PDAC and high-grade local (and hepatic) inflammatory infiltration without compromised microcirculation, innervation or mitochondria. Surprisingly, muscular and hepatic inflammation, SOCS3 upregulation and (proteolytic) increases in free amino acids and glutathione were already detectable in mice with precancerous PanINs. Studies of initial local triggers and defense mechanisms regarding cachexia are warranted for targeted anti-inflammatory prevention.

Anti-inflammatory and Anti-oxidative Effects of Phytohustil® and Root Extract of Althaea officinalis L. on Macrophages in vitro.

INTRODUCTION: The medicinal plant marshmallow Althaea officinalis L. (A. officinalis), is used for the treatment of cough since centuries. Application of medicinal extracts of marshmallow roots shows immediate effects like a protective film on the inflamed mucosa. Because the soothing layer reduce irritation of the mucous system, a faster regeneration is supported by defense mechanisms required to protect the respiratory tract from environmental injury. Macrophages (MΦ), which belong to a group of multipurpose defensive cells, provide the first line of defense against mucosal invasive pathogens. The present study was performed to investigate, whether the herbal medicinal product has anti-inflammatory or anti-oxidative effects on pro-inflammatorily activated MΦ or after oxidative stress induction. Special attention should be payed to elucidate the effects of A. officinalis on the mechanism of intracellular defense as well as on migratory capacity of the MΦ. RESULTS: Treatment of PMA-differentiated human THP-1 MΦ with Phytohustil® increased their viability without affecting the cell number. Phytohustil® or root extracts of A. officinalis (REAo) - an active component of Phytohustil® - were able to protect human MΦ against H2O2-induced cytotoxicity and H2O2-induced ROS production. Phytohustil®, REAo or diclofenac used as anti-inflammatory reference substance, inhibited the LPS-induced release of tumor necrosis factor-alpha (TNF-α) as well as of IL6 in MΦ. Treatment with Phytohustil®, its excipients or REAo did not impair the mitochondrial membrane potential (MMP). Finally, Phytohustil® and REAo activated the migratory capacity of MΦ. CONCLUSION: The present in vitro investigations indicate protective, i.e., anti-oxidative and anti-inflammatory effects of REAo and Phytohustil®, additionally improving the migratory capacity of MΦ. These antiinflammatory effects were similar or even better than diclofenac. Thus, our data support and may explain the positive effect of Phytohustil® observed in patients during the therapy of inflamed buccal mucosal membranes or treatment of cough.

PAC1 deficiency attenuates progression of atherosclerosis in ApoE deficient mice under cholesterol-enriched diet.

The neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP) is vasoactive and cytoprotective and exerts immunoregulatory functions throughout the nervous, neuroendocrine cardiovascular and immune systems in health and disease. PACAP mainly acts through PAC1 receptor signaling in neuronal communication, but the role of PAC1 in immune regulation of atherosclerosis is not known. Here, we generated PAC1-/-/ApoE-/- mice to test, whether PAC1-/- influences plasma cholesterol-/triglyceride levels and/or atherogenesis in the brachiocephalic trunk (BT) seen in ApoE-/- mice, under standard chow (SC) or cholesterol-enriched diet (CED). Furthermore, the effect of PAC1-/-, on inflammatory, autophagy-, apoptosis- and necroptosis-relevant proteins in atherosclerotic plaques was determined. In plaques of PAC1-/-/ApoE-/- mice fed a SC, the immunoreactivity for apoptotic, autophagic, necroptotic and proinflammatory proteins was increased, however, proliferation was unaffected. Interestingly, without affecting hyperlipidemia, PAC1-/- in ApoE-/- mice remarkably reduced CED-induced lumen stenosis seen in ApoE-/- mice. Thus, PAC1-/- allows unchecked inflammation, necroptosis and decreased proliferation during SC, apparently priming the BT to develop reduced atheroma under subsequent CED. Remarkably, no differences in inflammation/necroptosis signatures in the atheroma under CED between PAC1-/-/ApoE-/- and ApoE-/- mice were observed. These data indicate that selective PAC1 antagonists should offer potential as a novel class of atheroprotective therapeutics, especially during hypercholesterolemia.

In Vitro Effects of St. John's Wort Extract Against Inflammatory and Oxidative Stress and in the Phagocytic and Migratory Activity of Mouse SIM-A9 Microglia.

Introduction: Herbal medicinal plants as Hypericum perforatum L., known as St. John's wort (SJW) have been in use for a long time. SJW that is specifically used for the treatment of depressive disorders. Inflammatory cytokines derived from microglia play an important role in the regulation of the synthesis and reuptake of glutamate and influence synaptic function, morphology and neuronal plasticity. The present study was performed to investigate, whether STW3-VI, a special SJW extract has protective effects on mouse SIM-A9 microglia against cytotoxic and proinflammatory effects of ROS, glutamate, NMDA or cortisol. Additionally, we investigated the effects of SJW on migratory and phagocytic properties of microglia. Results: Pre-treatment (48 h) of microglia with STW3-VI (5 or 10 µg/ml)-in contrast to desipramine-inhibited the H2O2-induced TNF-α release by 20-40%. Pre-treatment (48 h) of microglia with STW3-VI (5 or 10 µg/ml) delayed the 3 or 4 mM H2O2-induced intracellular ROS level by 26.9 and 44.4%, respectively. Furthermore, pre-treatment (48 h) of microglia with STW3-VI (5 µg/ml) - in contrast to desipramine - lowered the glutamate-induced cytotoxicity by 13.2%. Besides, pre-treatment (48 h) of microglia with STW3-VI (5 or 10 µg/ml) or desipramine (5 µM) inhibited the NMDA-induced decrease of the viability by 16.5-28.8% or 12%, respectively. Finally, pre-treatment (48 h) of microglia with STW3-VI (5 or 10 µg/ml)-in contrast to desipramine - reduced the cortisol-induced cytotoxicity by 15.5 and 12.9%. Treatment of microglia with STW3-VI (10 or 100 µg/ml) increased the migratory and the phagocytic capacities by 100 and 40%. Conclusion: Our data provide evidence that STW3-VI-in contrast to desipramine - protects microglia from oxidative stress, NMDA- or glutamate-induced cytotoxicity, and has anti-inflammatory properties that are accompanied by improvement of their migratory and phagocytic capacity. These protective (particularly the anti-inflammatory) properties may be beneficial in the treatment of depressive disorders.

Data on autophagy markers in human macrophages exposed to oxLDL and growth differentiation factor-15.

Growth differentiation factor-15 (GDF-15) is a member of the TGF-ß superfamily, identical to MΦ-inhibitory cytokine-1 (MIC-1). GDF-15 is associated with e.g. cardiovascular disease, inflammation and development of atherosclerosis and is highly expressed in macrophages (MΦ) of atherosclerotic lesions. Moreover, there exists an indication for the involvement of oxidized-low density lipoprotein (oxLDL) uptake and autophagic processes by MΦ regarding arteriosclerotic progression. Thus, we were interested to investigate a potential regulatory effect of GDF-15 on autophagy signaling pathway in human MΦ during foam cell formation. Here, we present western blot data of ATG5, ATG12/ATG5-complex and p62 regarding the GDF-15 concentration. For further interpretation of the data presented in this article, please see the research article "Growth differentiation factor-15 regulates oxLDL-induced lipid homeostasis and autophagy in human macrophages" [1].

Anti-inflammatory effects of Phytodolor® (STW 1) and components (poplar, ash and goldenrod) on human monocytes/macrophages.

BACKGROUND: Populus tremula L. (Poplar), Fraxinus excelsior L. (ash) and Solidago virgaurea L. (goldenrod) have been used for medicinal purposes through centuries, to treat pain, fever and inflammation, but their mechanisms of action are still not fully understood. The present study was performed to investigate, whether the herbal medicinal product Phytodolor® (STW 1) and its components have anti-inflammatory effects on activated human monocytes and differentiated human macrophages to elucidate their modes of action in comparison with well-known analgesic, non-steroidal anti-inflammatory drug (NSAIDs) as diclofenac. METHODS: Adherent human monocytes obtained from peripheral blood mononuclear cells (PBMCs) were cultured in serum-free medium and pre-treated with 50-100 µg/ml of diclofenac, STW 1, their components, poplar, ash or goldenrod or its combination (0.05% to 2%). Thereafter, monocytes were activated with 0.1 or 1 µg/ml LPS for 24 h. The intracellular expressions of TNF-α or PTGS2 were determined by cell-based ELISA. Apoptotic cells were identified by YO-PRO-1 staining. Protein or total RNA were isolated to perform SDS-PAGE/Western blot and qRT-PCR analyses. PMA-differentiated human THP-1 macrophages were pre-treated with diclofenac (50 µg/ml) or STW1 (0.1%) and afterwards with LPS (1 µg/ml) and the translocation of the intracellular p62 NF-κB subunit was detected by immunofluorescence. RESULTS: STW 1 inhibited the intracellular content of TNF-α and PTGS2 protein, as well as of TNF-α and PTGS2 gene expression and induced apoptosis in LPS-activated human monocytes under serum free conditions. Furthermore, STW 1 inhibited the translocation of the p65 subunit of the redox-regulated NF-κB into the nucleus in LPS-activated human macrophages. CONCLUSION: The present in vitro investigations suggest a significant anti-inflammatory activity of STW 1 and its components by inhibiting pro-inflammatory cytokine as TNF-α and the key enzyme PTGS2 in LPS-activated human monocytes, which is, at least partly mediated through the suppression of NF-κB activation. Our results provide evidence for distinctive anti-inflammatory effects of STW 1 and its components on LPS-activated human monocytes/macrophages and, thus, for the therapeutic use of STW 1 in inflammation and pain related disorders.

Nucleolipids of the Nucleoside Antibiotics Formycins A and B: Synthesis and Biomedical Characterization Particularly Using Glioblastoma Cells.

Two lipophilic derivatives of formycin A (1) and formycin B (5) carrying an O-2',3'-(ethyl levulinate) ketal group have been prepared. These were base-alkylated at N(1) (for 1) and N(1) and N(6) (for 5) with both isopentenyl and all-trans-farnesyl residues. Upon the prenylation, side reactions were observed, resulting in the formation of nucleolipids with a novel tricyclic nucleobase (→4a, 4b). In the case of formycin B, O-2',3'-(ethyl levulinate) (6) farnesylation gave the double prenylated nucleolipid 7. All new compounds were characterized by 1 H-, 13 C-, UV/VIS and fluorescence spectroscopy, by ESI-MS spectrometry and/or by elemental analysis. Log P determinations between water and octanol as well as water and cyclohexane of a selection of compounds allowed qualitative conclusions concerning their potential blood-brain barrier passage efficiency. All compounds were investigated in vitro with respect to their cytotoxic activity toward rat malignant neuroectodermal BT4Ca as well as against a series of human glioblastoma cell lines (GOS 3, U-87 MG and GBM 2014/42). In order to differentiate between anticancer and side effects of the novel nucleolipids, we also studied their activity on PMA-differentiated human THP-1 macrophages. Here, we show that particularly the formycin A derivative 3b possesses promising antitumor properties in several cancer cell lines with profound cytotoxic effects partly on human glioblastoma cells, with a higher efficacy than the chemotherapeutic drug 5-fluorouridine.

Guanosine Nucleolipids: Synthesis, Characterization, Aggregation and X-Ray Crystallographic Identification of Electricity-Conducting G-Ribbons.

The lipophilization of ß-d-riboguanosine (1) with various symmetric as well as asymmetric ketones is described (→3a-3f). The formation of the corresponding O-2',3'-ketals is accompanied by the appearance of various fluorescent by-products which were isolated chromatographically as mixtures and tentatively analyzed by ESI-MS spectrometry. The mainly formed guanosine nucleolipids were isolated and characterized by elemental analyses, 1 H-, 13 C-NMR and UV spectroscopy. For a drug profiling, static topological polar surface areas as well as 10 logPOW values were calculated by an increment-based method as well as experimentally for the systems 1-octanol-H2 O and cyclohexane-H2 O. The guanosine-O-2',3'-ketal derivatives 3b and 3a could be crystallized in (D6 )DMSO - the latter after one year of standing at ambient temperature. X-ray analysis revealed the formation of self-assembled ribbons consisting of two structurally similar 3b nucleolipid conformers as well as integrated (D6 )DMSO molecules. In the case of 3a ⋅ DMSO, the ribbon is formed by a single type of guanosine nucleolipid molecules. The crystalline material 3b ⋅ DMSO was further analyzed by differential scanning calorimetry (DSC) and temperature-dependent polarization microscopy. Crystallization was also performed on interdigitated electrodes (Au, distance, 5 µm) and visualized by scanning electron microscopy. Resistance and amperage measurements clearly demonstrate that the electrode-bridging 3b crystals are electrically conducting. All O-2',3'-guanosine ketals were tested on their cytostatic/cytotoxic activity towards phorbol 12-myristate 13-acetate (PMA)-differentiated human THP-1 macrophages as well as against human astrocytoma/oligodendroglioma GOS-3 cells and against rat malignant neuroectodermal BT4Ca cells.

Growth differentiation factor-15 regulates oxLDL-induced lipid homeostasis and autophagy in human macrophages.

BACKGROUND AND AIMS: Growth differentiation factor-15 (GDF-15)/macrophage inhibitory cytokine-1 (MIC-1/GDF15) is associated with cardiovascular disease, inflammation and development of atherosclerosis and is highly expressed in macrophages (MΦ) of atherosclerotic lesions. Thus, we were interested in investigating the influence of GDF-15 in lipid homeostasis and autophagy in human MΦ during foam cell formation. METHODS AND RESULTS: Oxidized-low density lipoprotein (50 µg/ml oxLDL), recombinant (r)GDF-15, transiently silenced GDF-15 (siGDF-15 MΦ), as well as with negative siRNA transfected (nsiGDF-15 MΦ) PMA-differentiated human THP-1 MΦ, were used to investigate the effects of GDF-15 on autophagic processes and lipid accumulation. Oil Red O staining revealed that rGDF-15 alone, but also in combination with oxLDL, significantly increased the lipid accumulation in THP-1 MΦ; a reverse effect was detected in siGDF-15 MΦ. Western-blot analyses and confocal laser scanning microscopy showed an increase of Atg5, Atg12/Atg5 protein complex and p62 protein in THP-1 MΦ co-incubated with rGDF-15 and oxLDL, as well as an increase of p62 accumulation compared to rGDF-15-treated MΦ. Vice versa, siGDF-15 MΦ showed a reduced p62 accumulation compared to nsiGDF-15 MΦ. The present study indicates that GDF-15, especially in combination with oxLDL, regulates the expression of autophagy-relevant proteins (p62, Atg5 and Atg12/Atg5 protein complex) and p62 accumulation in human MΦ. CONCLUSIONS: GDF-15, in combination with oxLDL, impairs autophagic processes with consequences for lipid homeostasis in human MΦ, indicating its novel important pathophysiological role in atherosclerotic plaque development and progression.

Synthesis of New Potential Lipophilic Co-Drugs of 2-Chloro-2'-deoxyadenosine (Cladribine, 2-CdA, Mavenclad®, Leustatin®) and 6-Azauridine (z6 U) with Valproic Acid.

2-Chloro-2'-deoxyadenosine (cladribine, 1) was acylated with valproic acid (2) under various reaction conditions yielding 2-chloro-2'-deoxy-3',5'-O-divalproyladenosine (3) as well as the 3'-O- and 5'-O-monovalproylated derivatives, 2-chloro-2'-deoxy-3'-O-valproyladenosine (4) and 2-chloro-2'-deoxy-5'-O-valproyladenosine (5), as new co-drugs. In addition, 6-azauridine-2',3'-O-(ethyl levulinate) (8) was valproylated at the 5'-OH group (→9). All products were characterized by 1 H- and 13 C-NMR spectroscopy and ESI mass spectrometry. The structure of the by-product 6 (N-cyclohexyl-N-(cyclohexylcarbamoyl)-2-propylpentanamide), formed upon valproylation of cladribine in the presence of N,N-dimethylaminopyridine and dicyclohexylcarbodiimide, was analyzed by X-ray crystallography. Cladribine as well as its valproylated co-drugs were tested upon their cancerostatic/cancerotoxic activity in human astrocytoma/oligodendroglioma GOS-3 cells, in rat malignant neuro ectodermal BT4Ca cells, as well as in phorbol-12-myristate 13-acetate (PMA)-differentiated human THP-1 macrophages. The most important result of these experiments is the finding that only the 3'-O-valproylated derivative 4 exhibits a significant antitumor activity while the 5'-O- as well as the 3',5'-O-divalproylated cladribine derivatives 3 and 5 proved to be inactive.

PACAP deficiency aggravates atherosclerosis in ApoE deficient mice.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) plays an important role in cytoprotection, inflammation and cardiovascular regulation. Thus, we studied the involvement of PACAP in atherogenesis. Differentiated human THP-1 macrophages (MΦ) were stimulated with oxidized low-density lipoproteins (oxLDL) and the influence of PACAP38 treatment on lipid content and TNF release was determined. To test the effect of PACAP deficiency (PACAP-/-) on the development of atherosclerosis under standard chow (SC) or cholesterol-enriched diet (CED) in vivo, PACAP-/- mice were crossbred with ApoE-/- to generate PACAP-/-/ApoE-/- mice. Blood cholesterol and triglyceride levels were quantified. Lumen stenosis in the brachiocephalic trunk, cellularity and amounts of pro-inflammatory as well as autophagy-, apoptosis- and necroptosis-relevant proteins were analysed in atherosclerotic plaques by quantitative immunohistochemistry. In vitro, PACAP38 inhibited oxLDL-induced intracellular lipid storage as well as TNF release in MФ. In vivo, after SC, but not under CED, PACAP-/-/ApoE-/- mice showed an increased lumen stenosis compared to ApoE-/- mice. In atherosclerotic plaques of PACAP-/-/ApoE-/- mice, the immunoreactive areas of TNF+, IL-1ß+, autophagic, apoptotic and necroptotic cells were increased. In contrast, the overall cell density was decreased compared to ApoE-/- under SC, while no differences were seen under CED. Similar plasma cholesterol levels were observed in PACAP-/-/ApoE-/- and ApoE-/- mice under the respective feeding regime. Thus, PACAP-/-/ApoE-/- mice represent a novel mouse model of accelerated atherosclerosis where CED is not required. Our data indicate that PACAP acts as an endogenous atheroprotective neuropeptide. Thus, stable PACAP agonists may have potential as anti-atherosclerotic therapeutics. The specific PACAP receptor(s) mediating atheroprotection remain(s) to be identified.

Combinatorial Synthesis of New Pyrimidine- and Purine-ß-d-Ribonucleoside Nucleolipids: Their Distribution Between Aqueous and Organic Phases and Their In Vitro Activity Against Human- and Rat Glioblastoma Cells In Vitro.

Two series of nucleolipids, O-2',3'-heptanylidene- as well as O-2',3'-undecanylidene ketals of six ß-d-ribonucleosides (type A) and partly N-farnesyl derivatives thereof (type B) were prepared in a combinatorial manner. All novel compounds were characterized by elemental analysis and/or ESI mass spectrometry and by UV-, 1 H-, and 13 C-NMR spectroscopy. Conformational parameters of the nucleosides and nucleolipids were calculated from various 3 J(H,H), 3 J(1 H,13 C), and 5 J(F,H) coupling constants. For a drug profiling, the parent nucleosides and their lipophilic derivatives were studied with respect to their distribution (log P) between water and n-octanol as well as water and cyclohexane. From these data, qualitative conclusions were drawn concerning their possible blood-brain barrier passage efficiency. Moreover, nucleolipids were characterized by their molecular descriptor amphiphilic ratio (a.r.), which describes the balance between the hydrophilicity of the nucleoside headgroup and the lipophilicity of the lipid tail. All compounds were investigated in vitro with respect to their cytostatic/cytotoxic activity toward human glioblastoma (GOS 3) as well as rat malignant neuroectodermal BT4Ca cell lines in vitro. In order to differentiate between anticancer and side-effects of the novel nucleolipids, they were also studied on their activity on differentiated human THP-1 macrophages.

NB 06: From a simple lysosomotropic aSMase inhibitor to tools for elucidating the role of lysosomes in signaling apoptosis and LPS-induced inflammation.

Ceramide generation is involved in signal transduction of cellular stress response, in particular during stress-induced apoptosis in response to stimuli such as minimally modified Low-density lipoproteins, TNFalpha and exogenous C6-ceramide. In this paper we describe 48 diverse synthetic products and evaluate their lysosomotropic and acid sphingomyelinase inhibiting activities in macrophages. A stimuli-induced increase of C16-ceramide in macrophages can be almost completely suppressed by representative compound NB 06 providing an effective protection of macrophages against apoptosis. Compounds like NB 06 thus offer highly interesting fields of application besides prevention of apoptosis of macrophages in atherosclerotic plaques in vessel walls. Most importantly, they can be used for blocking pH-dependent lysosomal processes and enzymes in general as well as for analyzing lysosomal dependent cellular signaling. Modulation of gene expression of several prominent inflammatory messengers IL1B, IL6, IL23A, CCL4 and CCL20 further indicate potentially beneficial effects in the field of (systemic) infections involving bacterial endotoxins like LPS or infections with influenza A virus.

Indication for differential sorting of the rat v-SNARE splice isoforms VAMP-1a and -1b.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are essential constituents of the intracellular trafficking machinery. The variable C-terminus in the 2 rat VAMP-1 splice isoforms VAMP-1a and -1b potentially acts as a sorting signal, because similar changes at the C-terminal end of a human VAMP-1 splice isoform resulted in its sorting to mitochondria. To evaluate the differences in the subcellular localization of these two v-SNARE proteins, VAMP-1a and -1b proteins tagged with green fluorescent protein (GFP) and red fluorescent protein (RFP) were expressed in HeLa, COS-7, and MDCK cells and evaluated by conventional confocal as well as total internal reflection fluorescence microscopy. Regions consistent with the endoplasmic reticulum and Golgi apparatus demonstrated a major overlap of both signals. In the periphery, vesicular structures were observed that mainly expressed one of the 2 isoforms. Within our experimental settings, we could not observe sorting of any of the 2 isoforms to mitochondria or peroxisomes, whereas both isoforms were found expressed in a minor subset of singular vesicles, which sporadically appeared to co-localize with the exocyst marker EXOC3/Sec6. Because vesicular structures were seen that expressed only one of the two splice variants, it is possible that VAMP-1a and VAMP-1b are sorted to distinct cellular compartments that require further characterization.

Krill Oil-In-Water Emulsion Protects against Lipopolysaccharide-Induced Proinflammatory Activation of Macrophages In Vitro.

BACKGROUND: Parenteral nutrition is often a mandatory therapeutic strategy for cases of septicemia. Likewise, therapeutic application of anti-oxidants, anti-inflammatory therapy, and endotoxin lowering, by removal or inactivation, might be beneficial to ameliorate the systemic inflammatory response during the acute phases of critical illness. Concerning anti-inflammatory properties in this setting, omega-3 fatty acids of marine origin have been frequently described. This study investigated the anti-inflammatory and LPS-inactivating properties of krill oil (KO)-in-water emulsion in human macrophages in vitro. MATERIALS AND METHODS: Differentiated THP-1 macrophages were activated using specific ultrapure-LPS that binds only on the toll-like receptor 4 (TLR4) in order to determine the inhibitory properties of the KO emulsion on the LPS-binding capacity, and the subsequent release of TNF-α. RESULTS: KO emulsion inhibited the macrophage binding of LPS to the TLR4 by 50% (at 12.5 µg/mL) and 75% (at 25 µg/mL), whereas, at 50 µg/mL, completely abolished the LPS binding. Moreover, KO (12.5 µg/mL, 25 µg/mL, or 50 µg/mL) also inhibited (30%, 40%, or 75%, respectively) the TNF-α release after activation with 0.01 µg/mL LPS in comparison with LPS treatment alone. CONCLUSION: KO emulsion influences the LPS-induced pro-inflammatory activation of macrophages, possibly due to inactivation of the LPS binding capacity.

Oxidized LDL-induced JAB1 influences NF-κB independent inflammatory signaling in human macrophages during foam cell formation.

BACKGROUND: Oxidized low-density lipoprotein (oxLDL) mediates the transformation of macrophages (MΦ) to cholesterol-rich foam cells and the release of pro-inflammatory cytokines during atherogenesis. JAB1 (Jun activation domain binding protein-1) is present in all stages of human plaques, involved in the Toll-like receptor-mediated activation of p38 mitogen-activated protein kinase (MAPK) and controls nuclear factor-kappa B (NF-κB) activation. Thus, we were interested in the role of JAB1 during foam cell formation of MΦ after oxLDL exposition. METHODS AND RESULTS: We found that JAB1 was present in CD68-immunoreactive (-ir) MΦ in atherosclerotic plaques of apolipoprotein E knockout (ApoE-/-) mice after a high cholesterol/fat diet. Furthermore, differentiated human U937 MΦ - incubated with oxLDL (4 h) to induce foam cell formation - showed a significant increase of JAB1 (50 µg/ml: 1.39 + 0.15-fold; 100 µg/ml: 1.80 + 0.26-fold; 200 µg/ml: 2.05 + 0.30-fold; p < 0.05) on the protein level compared to the control. Independent from JAB1 silencing, we found an increase of total cholesterol (TC), free cholesterol (FC) and cholesteryl ester (CE) after oxLDL exposition. However, siJAB1-MФ showed a reduction of tumor necrosis factor-alpha (TNF-α) (36%; p < 0.05 vs. non-transfected MФ) and interleukin (IL)-6 (30%; p < 0.05 vs. non-transfected MФ) mRNA expression, as well as TNF-α (46%; p < 0.05 vs. non-transfected MФ) and IL-6 (32%; p < 0.05 vs. non-transfected MФ) protein secretion after oxLDL exposition. In parallel with an upregulation of inflammatory cytokines (TNF-α, IL-6) after oxLDL exposition, we found a significant (p < 0.05) increase of 37% in p38 MAPK activation after 4 h oxLDL-treatment, independent from NF-kB signaling. In this context, we showed regional co-localization of JAB1 with p38 MAPK in atherosclerotic plaques of ApoE-/- mice. Moreover, we detected interaction of JAB1 with p38 MAPK in U937 cells. CONCLUSION: We demonstrate that oxLDL induces JAB1 expression and influences its cellular localization, whereby the p38 MAPK signaling pathway is modified with consequences for inflammation of human MΦ in foam cells and atherosclerotic lesions.