Role of the Stress- and Inflammation-Induced Cytokine GDF-15 in Cardiovascular Diseases: From Basic Research to Clinical Relevance.

Stress- and inflammation-induced growth differentiation factor-15 (GDF-15) is proposed as a biomarker for mortality and disease progression in patients with atherosclerosis and/or cardiovascular disease (CVD). The development of atherosclerotic lesions depends, among other factors, on inflammatory processes, oxidative stress, and impaired lipid homeostasis. As a consequence, activation and dysfunction of endothelial cells, release of chemokines, growth factors and lipid mediators occur. GDF-15 is suggested as an acute-phase modifier of transforming growth factor (TGF)-ßRII-dependent pro-inflammatory responses leading to rupture of atherosclerotic plaques, although the exact biological function is poorly understood to date. GDF-15 is upregulated in many disease processes, and its effects may be highly context-dependent. To date, it is unclear whether the upregulation of GDF-15 leads to disease progression or provides protection against disease. Concerning CVD, cardiomyocytes are already known to produce and release GDF-15 in response to angiotensin II stimulation, ischemia, and mechanical stretch. Cardiomyocytes, macrophages, vascular smooth muscle cells, endothelial cells, and adipocytes also release GDF-15 in response to oxidative as well as metabolic stress or stimulation with pro-inflammatory cytokines. Given the critically discussed pathophysiological and cellular functions and the important clinical significance of GDF-15 as a biomarker in CVD, we have summarized here the basic research findings on different cell types. In the context of cellular stress and inflammation, we further elucidated the signaling pathway of GDF-15 in coronary artery disease (CAD), the most common CVD in developing and industrial nations.

Bioresorbable zinc stent with ultra-thin center struts attenuates stent jail in porcine femoral artery bifurcations.

INTRODUCTION: An ultra-thin, fracture-resistant and bioresorbable stent may be advantageous for provisional stenting in vessel bifurcations, if catheter passage and side-branch post-dilatation is facilitated to prevent a 'stent jail' by struts obstructing the orifice of a major side branch. MATERIAL AND METHODS: We studied a highly radiopaque, slowly bioresorbable zinc alloy stent characterized by a novel design of a radiopaque-marked region of ultra-thin struts in the center of the stent. The stent is characterized by an extended range flexibility and high fracture resistance. Zn-stents and Zn-drug eluting stents (DES) were implanted opposite to rigid Nitinol stents into both femoral artery bifurcations of 21 juvenile pigs, followed for one and three months and studied by angiography and histomorphometry.Results and conclusion: Bare Zn-stents with thinner stent struts showed less neointimal hyperplasia compared to Zn-stents with thicker struts. Neointimal formation was further reduced by 12% in Zn-alloy DES. Both, bare Zn-stents and Zn-DES, can be precisely positioned into the femoral artery bifurcation, allowing easy balloon catheter passage through the very thin strut mesh. Side branch orifices remained open after Zn-stent deployment without stent jailing. No stent fractures or particles emboli occurred after the deployment. A Zn-stent with ultra-thin center struts may be useful for provisional stenting in vessel bifurcations.

Systemic LPS induces toll-like receptor 3 (TLR3) expression and apoptosis in testicular mouse tissue.

It is well known that sepsis and inflammation reduce male fertility. Within the testis, toll-like receptor 3 (TLR3) is constitutively expressed and recognizes double-stranded RNA (dsRNA) from viruses, degraded bacteria, damaged tissues and necrotic cells. To characterize the potential role of TLR3 in response to testicular infections, its expression and downstream signaling were investigated upon challenge with lipopolysaccharides (LPS) in two mouse strains that differ in their immuno-competence regarding T cell-regulated immunity. Thereto, Balb/c and Foxn1nu mice were randomized into six interventional groups treated with either i.v. application of saline or LPS followed by 20 min, 5 h 30 min and 18 h of observation and two sham-treated control groups. LPS administration induced a significant stress response; the amplification was manifested for TLR3 and interleukin 6 (IL6) mRNA in the impaired testis 5 h 30 min after LPS injection. TLR3 immunostaining revealed that TLR3 was primarily localized in spermatocytes. The TLR3 expression displayed different temporal dynamics between both mouse strains. However, immunofluorescence staining indicated only punctual interferon regulatory factor 3 (IRF3) expression upon LPS treatment along with minor alterations in interferon ß (IFNß) mRNA expression. Induction of acute inflammation was closely followed by a significant shift of the Bax/Bcl2 ratio to pro-apoptotic signaling accompanied by augmented TUNEL-positive cells 18 h after LPS injection with again differing patterns in both mouse strains. In conclusion, this study shows the involvement of TLR3 in response to LPS-induced testicular inflammation in immuno-competent and -incompetent mice, yet lacking transmission into its signaling pathway.

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.

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.

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.

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.

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.

Lower hypoxic ventilatory response in smokers compared to non-smokers during abstinence from cigarettes.

BACKGROUND: Carotid body O2-chemosensitivity determines the hypoxic ventilatory response (HVR) as part of crucial regulatory reflex within oxygen homeostasis. Nicotine has been suggested to attenuate HVR in neonates of smoking mothers. However, whether smoking affects HVR in adulthood has remained unclear and probably blurred by acute ventilatory stimulation through cigarette smoke. We hypothesized that HVR is substantially reduced in smokers when studied after an overnight abstinence from cigarettes i.e. after nicotine elimination. METHODS: We therefore determined the isocapnic HVR of 23 healthy male smokers (age 33.9 ± 2.0 years, BMI 24.2 ± 0.5 kg m-2, mean ± SEM) with a smoking history of >8 years after 12 h of abstinence and compared it to that of 23 healthy male non-smokers matched for age and BMI. RESULTS: Smokers and non-smokers were comparable with regard to factors known to affect isocapnic HVR such as plasma levels of glucose and thiols as well as intracellular levels of glutathione in blood mononuclear cells. As a new finding, abstinent smokers had a significantly lower isocapnic HVR (0.024 ± 0.002 vs. 0.037 ± 0.003 l min-1 %-1BMI-1, P = 0.002) compared to non-smokers. However, upon re-exposure to cigarettes the smokers' HVR increased immediately to the non-smokers' level. CONCLUSIONS: This is the first report of a substantial HVR reduction in abstinent adult smokers which appears to be masked by daily smoking routine and may therefore have been previously overlooked. A low HVR may be suggested as a novel link between smoking and aggravated hypoxemia during sleep especially in relevant clinical conditions such as COPD.

Novel Nucleolipids of Pyrimidine ß-D-Ribonucleosides: Combinatorial Synthesis, Spectroscopic Characterization, and Cytostatic/Cytotoxic Activities.

Four series of nucleolipids with either uridine, 5-methyluridine, 5-fluorouridine, and 6-azauridine as ß-D-ribonucleoside component have been prepared in a combinatorial (not parallel!) manner (see Formulae). All compounds have been characterized by elemental analyses, ESI mass spectrometry as well as by (1) H-, and (13) C-NMR, and UV spectroscopy. A selection of eight nucleolipids with different lipophilizing moieties, based on earlier findings, as well as of 5-fluorouridine as control were first tested on their cytotoxic effect towards PMA-differentiated human THP-1 macrophages. Those compounds which did not exhibit a significant inhibitory effect on the survival of the macrophages were next tested on their cytostatic/cytotoxic effect towards the human astrocytoma/oligodendroglioma GOS-3 cells as well as against the rat malignant neuroectodermal BT4Ca cell line. Additionally, induction of apoptosis of the cell lines was evaluated. It turned out that particularly a combined lipophilization of the nucleosides by an 2',3'-O-ethyl levulinate residue plus a farnesyl moiety at N(3) of the pyrimidine moiety of the corresponding nucleolipids leads to an active compound with the highest probability.

Improved survival in osteosarcoma patients with atypical low vascularization.

BACKGROUND: Osteosarcoma is considered a highly vascularized bone tumor with early metastatic dissemination through intratumoral blood vessels mostly into the lung. Novel targets for therapy such as tumor vascularization are highly warranted since little progress has been achieved in the last 30 years. However, proof of relevance for vascularization as a major prognostic parameter has been hampered by tumor heterogeneity, difficulty in detecting microvessels by immunohistochemistry, and small study cohorts. Most recently, we demonstrated that highly standardized whole-slide imaging could overcome these limitations (Kunz et al., PloS One 9(3):e90727, 2014). In this study, we applied this method to a multicenter cohort of 131 osteosarcoma patients to test osteosarcoma vascularization as a prognostic determinant. METHODS: Computer-assisted whole-slide analysis, together with enzymatic epitope retrieval, was used for CD31-based microvessel quantification in 131 pretreatment formalin-fixed and paraffin-embedded biopsies from three bone tumor centers. Kaplan-Meier-estimated survival and chemoresponse were determined and multivariate analysis was performed. Conventional hot-spot-based microvessel density (MVD) determination was compared with whole-slide imaging. RESULTS: We detected high estimated overall (p ≤ 0.008) and relapse-free (p ≤ 0.004) survival in 25 % of osteosarcoma patients with low osteosarcoma vascularization in contrast to other patient groups. Furthermore, all patients with low osteosarcoma vascularization showed a good response to neoadjuvant chemotherapy. Comparison of conventional MVD determination with whole-slide imaging suggests false high quantification or even exclusion of samples with low osteosarcoma vascularization due to difficult CD31 detection in previous studies. CONCLUSION: Low intratumoral vascularization at the time of diagnosis is a strong predictor for prolonged survival and good response to neoadjuvant chemotherapy in osteosarcoma.

Ameliorated or Acquired Cytostatic/Cytotoxic Properties of Nucleosides by Lipophilization.

A series of nucleolipids, containing one of the ß-D-ribonucleosides 5-fluorouridine, 6-azauridine, uridine, or 5-methyluridine were lipophilized, either at the O-2',3'-position and/or at N(3) of the nucleobase with a large variety of hydrophobic residues. The resulting nucleolipids as well as the parent nucleosides and the lipid precursors were investigated in vitro with respect to their antitumor activity towards i) ten human tumor cell lines from the NCI 60 panel and ii) partly against three further tumor cell lines, namely a) human astrocytoma/oligodendro glioma GOs-3, b) rat malignantneuroectodermal BT4Ca, and c) differentiated human THP-1 macrophages. Inspection of the doseresponse curves allows two main conclusions concerning lipid determinants lending the corresponding nucleoside an ameliorated or an acquired antitumor activity: i) introduction of either a symmetrical O-2',3'-nonadecylidene ketal group or introduction of an O-2',3'-ethyl levulinate moiety plus an N(3)-farnesyl group leads often to nucleolipids with significant cytostatic/cytotoxic properties; ii) for the two canonical and non-toxic nucleosides uridine and 5-methyluridine, the condensation with also non-toxic lipids gives nucleolipids with a pronounced antitumor activity.

Effects of pre-competitional rapid weight loss on nutrition, vitamin status and oxidative stress in elite boxers.

Dietary intake, vitamin status and oxidative stress were evaluated in 17 elite male boxers. Ten of them frequently reduced body weight rapidly before competitions (Weight Loss Group) and 7 did not practice rapid weight loss (Control Group). Food record checklists, blood samples for determination of vitamin status and plasma glutathione levels were obtained during a week of weight maintenance, a pre-competition week and a post-competition week. The average dietary intakes in both groups were 33 ± 8 kcal·kg(-1), 3.7 ± 1.1 g·kg(-1) carbohydrates, 1.5 ± 0.4 g·kg(-1) protein, 1.2 ± 0.4 g·kg(-1) fat and 2.2 ± 1.0 L water per day (excluding pre-competition week in Weight Loss Group). Energy (18 ± 7 kcal·kg(-1)), carbohydrate (2.2 ± 0.8 g·kg(-1)), protein (0.8 ± 0.4 g·kg(-1)), fat (0.6 ± 0.3 g·kg(-1)) and water (1.6 ± 0.6 L) consumption (P-values <0.001) and intakes of most vitamins (P-values < 0.05) were significantly reduced during the pre-competition week in the Weight Loss Group. In both groups, the intakes of vitamins A, E and folate were below recommended values throughout the three periods; however, blood vitamin and plasma glutathione levels did not change significantly. Our findings indicate a low-caloric and low-carbohydrate diet in elite boxers, regardless of participating in rapid weight loss or not. Apparently, the pre-competitional malnutitrition in the Weight Loss Group did not induce alterations in the vitamin and glutathione status.

Cytostatic/cytotoxic effects of 5-fluorouridine nucleolipids on colon, hepatocellular, and renal carcinoma cells: in vitro identification of a potential cytotoxic multi-anticancer drug.

The insufficient penetration through the cell membranes is one of the major drawbacks of chemotherapeutics such as 5-fluorouracil (5-FU; 1). To improve the penetration, a useful strategy is the attachment of lipophilic moieties. Thus, we have synthesized a series of nucleolipid derivatives of 5-fluorouridine (5-FUrd; 2a), carrying lipophilic moieties at N(3) and/or at the 2',3'-O position, i.e., 3a, 3b, 4-7, and tested their cytostatic/cytotoxic activities towards three carcinoma cell lines (colon (HT-29), hepatocellular (HepG2), and renal (RENCA)) in comparison with 5-FU (1) and 5-FUrd (2a). After 48 h of incubation, four derivatives, 3a, 3b, 5, and 7, showed inhibitory effects on the survival of HT-29, HepG2, and RENCA cells. Additionally, to differentiate between anticancer and side-effects, we tested the cytotoxicity of the derivatives in human macrophages. Interestingly, the derivatives 4, 5, and 6 did not exhibit any effects on survival of THP-1 macrophages. Furthermore, we investigated the apoptosis induction of compound 1 and 2a, and the above-mentioned derivatives in HT-29 cells. Derivative 5 showed the highest significant (p<0.05; p<0.01) increase of the apoptosis at 80 µM after 2-h or 4-h treatment, as well as after 6-h incubation at 40 µM (p<0.05). Real-time PCR revealed that 40-µM derivative 5 showed a 1.8-fold increase of the pro-apoptotic caspase-3 gene and a twofold significant increase (p<0.01 and p<0.05 vs. control and 1, resp.) of the tumor suppressor TP53 gene, whereas the other compounds did not show any effect. We demonstrated that some 5-FUrd derivatives such as compound 5 are more effective than 5-FU or 5-FUrd concerning a cytotoxic (vs. cytostatic (5-FU, 5-FUrd)) effect on different cancer cell lines, but without cytotoxic side-effects on differentiated macrophages. Thus, compound 5 is suggested as a novel potent cytotoxic multi-anti-cancer drug.

Increased Myocardial MAO-A, Atrogin-1, and IL-1ß Expression in Transgenic Mice with Pancreatic Carcinoma-Benefit of MAO-A Inhibition for Cardiac Cachexia.

Cancer cachexia (CC) continues to challenge clinicians by massively impairing patients' prognosis, mobility, and quality of life through skeletal muscle wasting. CC also includes cardiac cachexia as characterized by atrophy, compromised metabolism, innervation and function of the myocardium through factors awaiting clarification for therapeutic targeting. Because monoamine oxidase-A (MAO-A) is a myocardial source of H2O2 and implicated in myofibrillar protein catabolism and heart failure, we presently studied myocardial MAO-A expression, inflammatory cells, and capillarization together with transcripts of pro-inflammatory, -angiogenic, -apoptotic, and -proteolytic signals (by qRT-PCR) in a 3x-transgenic (LSL-KrasG12D/+; LSL-TrP53R172H/+; Pdx1-Cre) mouse model of orthotopic pancreatic ductal adenoarcinoma (PDAC) compared to wild-type (WT) mice. Moreover, we evaluated the effect of MAO-A inhibition by application of harmine hydrochloride (HH, 8 weeks, i.p., no sham control) on PDAC-related myocardial alterations. Myocardial MAO-A protein content was significantly increased (1.69-fold) in PDAC compared to WT mice. PDAC was associated with an increased percentage of atrogin-1+ (p < 0.001), IL-1ß+ (p < 0.01), COX2+ (p < 0.001), and CD68+ (p > 0.05) cells and enhanced transcripts of pro-inflammatory IL-1ß (2.47-fold), COX2 (1.53-fold), TNF (1.87-fold), and SOCS3 (1.64-fold). Moreover, PDAC was associated with a reduction in capillary density (-17%, p < 0.05) and transcripts of KDR (0.46-fold) but not of VEGFA, Notch1, or Notch3. Importantly, HH treatment largely reversed the PDAC-related increases in atrogin-1+, IL-1ß+, and TNF+ cell fraction as well as in COX2, IL-1ß, TNF, and SOCS3 transcripts, whereas capillary density and KDR transcripts failed to improve. In mice with PDAC, increased myocardial pro-atrophic/-inflammatory signals are attributable to increased expression of MAO-A, because they are significantly improved with MAO-A inhibition as a potential novel therapeutic option. The PDAC-related loss in myocardial capillary density may be due to other mechanisms awaiting evaluation with consideration of cardiomyocyte size, cardiac function and physical activity.

Neuropilin-1 modulates vascular endothelial growth factor-induced poly(ADP-ribose)-polymerase leading to reduced cerebrovascular apoptosis.

Cerebral ischemia is encompassed by cerebrovascular apoptosis, yet the mechanisms behind apoptosis regulation are not fully understood. We previously demonstrated inhibition of endothelial apoptosis by vascular endothelial growth factor (VEGF) through upregulation of poly(ADP-ribose)-polymerase (PARP) expression. However, PARP overactivation through oxidative stress can lead to necrosis. This study tested the hypothesis that neuropilin-1 (NP-1), an alternative VEGF receptor, regulates the response to cerebral ischemia by modulating PARP expression and, in turn, apoptosis inhibition by VEGF. In endothelial cell culture, NP-1 colocalized with VEGF receptor-2 (VEGFR-2) and acted as its coreceptor. This significantly enhanced VEGF-induced PARP mRNA and protein expression demonstrated by receptor-specific inhibitors and VEGF-A isoforms. NP-1 augmented the inhibitory effect of VEGF/VEGFR-2 interaction on apoptosis induced by adhesion inhibition through the αV-integrin inhibitor cRGDfV. NP-1/VEGFR-2 signal transduction involved JNK and Akt. In rat models of permanent and temporary middle cerebral artery occlusion, the ischemic cerebral hemispheres displayed endothelial and neuronal apoptosis next to increased endothelial NP-1 and VEGFR-2 expression compared to non-ischemic cerebral hemispheres, sham-operated or untreated controls. Increased vascular superoxide dismutase-1 and catalase expression as well as decreased glycogen reserves indicated oxidative stress in the ischemic brain. Of note, protein levels of intact PARP remained stable despite pro-apoptotic conditions through increased PARP mRNA production during cerebral ischemia. In conclusion, NP-1 is upregulated in conditions of imminent cerebrovascular apoptosis to reinforce apoptosis inhibition and modulate VEGF-dependent PARP expression and activation. We propose that NP-1 is a key modulator of VEGF maintaining cerebrovascular integrity during ischemia. Modulating the function of NP-1 to target PARP could help to prevent cellular damage in cerebrovascular disease.

Synthesis of 5-fluorouridine nucleolipid derivatives and their cytostatic/cytotoxic activities on human HT-29 colon carcinoma cells.

One of the major drawbacks of chemotherapeutics is their insufficient penetration through cell membranes due to a high hydrophobicity. Thus, we have synthesized a series of selected nucleolipid derivatives of 5-fluorouridine (5-FUrd; 2a), carrying lipophilic moieties at N(3) and/or in the 2',3'-O-position (i.e., 3a-7a and 3c), and tested their cytostatic/cytotoxic activities using HT-29 human colon carcinoma cells, in comparison with, e.g., 5-FU (1) and 5-FUrd (2a). Incorporation and intracellular localization of the substances under test were performed after conjugation with the fluorochrome Atto 425. We showed that all 5'-O-labelled Atto 425 derivatives were incorporated by the human HT-29 cells and accumulated in their cytoplasm. Moreover, after 24-h treatment of HT-29 human colon carcinoma cells, 1 or 2a (10, 20, 40, or 80 µM) revealed a significant (14-23 or 33-45%, resp.) decrease of the viability in comparison with the (negative) control. Interestingly, derivatives 3a and 3c (40 and 80 µM) led to a significant (77-95 or 89-96%, resp.) inhibition of survival of human HT29 cells, i.e., these two substances were ca. 63-72% or ca. 75%, respectively more effective than 5-FU (1; positive control). Furthermore, derivative 5a showed a significant, i.e., 30 and 86%, inhibition of the survival at 40 and 80 µM, respectively in comparison with the (negative) control. Some synthesized 5-FUrd derivatives turned out to be more effective than 5-FU (1) or 5-FUrd (2a).

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.

Comparative analysis of full-length 16s ribosomal RNA genome sequencing in human fecal samples using primer sets with different degrees of degeneracy.

Next-generation sequencing has revolutionized the field of microbiology research and greatly expanded our knowledge of complex bacterial communities. Nanopore sequencing provides distinct advantages, combining cost-effectiveness, ease of use, high throughput, and high taxonomic resolution through its ability to process long amplicons, such as the entire 16s rRNA genome. We examine the performance of the conventional 27F primer (27F-I) included in the 16S Barcoding Kit distributed by Oxford Nanopore Technologies (ONT) and that of a more degenerate 27F primer (27F-II) in the context of highly complex bacterial communities in 73 human fecal samples. The results show striking differences in both taxonomic diversity and relative abundance of a substantial number of taxa between the two primer sets. Primer 27F-I reveals a significantly lower biodiversity and, for example, at the taxonomic level of the phyla, a dominance of Firmicutes and Proteobacteria as determined by relative abundances, as well as an unusually high ratio of Firmicutes/Bacteriodetes when compared to the more degenerate primer set (27F-II). Considering the findings in the context of the gut microbiomes common in Western industrial societies, as reported in the American Gut Project, the more degenerate primer set (27F-II) reflects the composition and diversity of the fecal microbiome significantly better than the 27F-I primer. This study provides a fundamentally relevant comparative analysis of the in situ performance of two primer sets designed for sequencing of the entire 16s rRNA genome and suggests that the more degenerate primer set (27F-II) should be preferred for nanopore sequencing-based analyses of the human fecal microbiome.