PAC1 deficiency reduces chondrogenesis in atherosclerotic lesions of hypercholesterolemic ApoE-deficient mice.

BACKGROUND: Induction of chondrogenesis is associated with progressive atherosclerosis. Deficiency of the ADCYAP1 gene encoding pituitary adenylate cyclase-activating peptide (PACAP) aggravates atherosclerosis in ApoE deficient (ApoE-/-) mice. PACAP signaling regulates chondrogenesis and osteogenesis during cartilage and bone development. Therefore, this study aimed to decipher whether PACAP signaling is related to atherogenesis-related chondrogenesis in the ApoE-/- mouse model of atherosclerosis and under the influence of a high-fat diet. METHODS: For this purpose, PACAP-/-/ApoE-/-, PAC1-/-/ApoE-/-, and ApoE-/- mice, as well as wildtype (WT) mice, were studied under standard chow (SC) or cholesterol-enriched diet (CED) for 20 weeks. The amount of cartilage matrix in atherosclerotic lesions of the brachiocephalic trunk (BT) with maximal lumen stenosis was monitored by alcian blue and collagen II staining on deparaffinized cross sections. The chondrogenic RUNX family transcription factor 2 (RUNX2), macrophages [(MΦ), Iba1+], and smooth muscle cells (SMC, sm-α-actin) were immunohistochemically analyzed and quantified. RESULTS: ApoE-/- mice fed either SC or CED revealed an increase of alcian blue-positive areas within the media compared to WT mice. PAC1-/-/ApoE-/- mice under CED showed a reduction in the alcian blue-positive plaque area in the BT compared to ApoE-/- mice. In contrast, PACAP deficiency in ApoE-/- mice did not affect the chondrogenic signature under either diet. CONCLUSIONS: Our data show that PAC1 deficiency reduces chondrogenesis in atherosclerotic plaques exclusively under conditions of CED-induced hypercholesterolemia. We conclude that CED-related chondrogenesis occurs in atherosclerotic plaques via transdifferentiation of SMCs and MΦ, partly depending on PACAP signaling through PAC1. Thus, PAC1 antagonists or PACAP agonists may offer therapeutic potential against pathological chondrogenesis in atherosclerotic lesions generated under hypercholesterolemic conditions, especially in familial hypercholesterolemia. This discovery opens therapeutic perspectives to be used in the treatment against the progression of atherosclerosis.

Effect of chronic intermittent hypoxia (CIH) on neuromuscular junctions and mitochondria in slow- and fast-twitch skeletal muscles of mice-the role of iNOS.

BACKGROUND: Obstructive sleep apnea (OSA) imposes vascular and metabolic risks through chronic intermittent hypoxia (CIH) and impairs skeletal muscle performance. As studies addressing limb muscles are rare, the reasons for the lower exercise capacity are unknown. We hypothesize that CIH-related morphological alterations in neuromuscular junctions (NMJ) and mitochondrial integrity might be the cause of functional disorders in skeletal muscles. METHODS: Mice were kept under 6 weeks of CIH (alternating 7% and 21% O2 fractions every 30 s, 8 h/day, 5 days/week) compared to normoxia (NOX). Analyses included neuromuscular junctions (NMJ) postsynaptic morphology and integrity, fiber cross-sectional area (CSA) and composition (ATPase), mitochondrial ultrastructure (transmission-electron-microscopy), and relevant transcripts (RT-qPCR). Besides wildtype (WT), we included inducible nitric oxide synthase knockout mice (iNOS-/-) to evaluate whether iNOS is protective or risk-mediating. RESULTS: In WT soleus muscle, CIH vs. NOX reduced NMJ size (- 37.0%, p < 0.001) and length (- 25.0%, p < 0.05) together with fiber CSA of type IIa fibers (- 14%, p < 0.05) and increased centronucleated fiber fraction (p < 0.001). Moreover, CIH vs. NOX increased the fraction of damaged mitochondria (1.8-fold, p < 0.001). Compared to WT, iNOS-/- similarly decreased NMJ area and length with NOX (- 55%, p < 0.001 and - 33%, p < 0.05, respectively) or with CIH (- 37%, p < 0.05 and - 29%, p < 0.05), however, prompted no fiber atrophy. Moreover, increased fractions of damaged (2.1-fold, p < 0.001) or swollen (> 6-fold, p < 0.001) mitochondria were observed with iNOS-/- vs. WT under NOX and similarly under CIH. Both, CIH- and iNOS-/- massively upregulated suppressor-of-cytokine-signaling-3 (SOCS3) > 10-fold without changes in IL6 mRNA expression. Furthermore, inflammatory markers like CD68 (macrophages) and IL1ß were significantly lower in CIH vs. NOX. None of these morphological alterations with CIH- or iNOS-/- were detected in the gastrocnemius muscle. Notably, iNOS expression was undetectable in WT muscle, unlike the liver, where it was massively decreased with CIH. CONCLUSION: CIH leads to NMJ and mitochondrial damage associated with fiber atrophy/centronucleation selectively in slow-twitch muscle of WT. This effect is largely mimicked by iNOS-/- at NOX (except for atrophy). Both conditions involve massive SOCS3 upregulation likely through denervation without Il6 upregulation but accompanied by a decrease of macrophage density especially next to denervated endplates. In the absence of muscular iNOS expression in WT, this damage may arise from extramuscular, e.g., motoneuronal iNOS deficiency (through CIH or knockout) awaiting functional evaluation.

Characterization of atherosclerotic plaques in blood vessels with low oxygenated blood and blood pressure (Pulmonary trunk): role of growth differentiation factor-15 (GDF-15).

BACKGROUND: Growth differentiation factor (GDF)-15 is linked to inflammation, cancer, and atherosclerosis. GDF-15 is expressed in most tissues but is extremely induced under pathological conditions. Elevated serum levels are suggested as a risk factor and a marker for cardiovascular diseases. However, the cellular sources and the effects of GDF-15 on the cardiovascular system have not been completely elucidated including progression, and morphology of atherosclerotic plaques. Thus, this work aimed to characterize the influence of GDF-15 deficiency on the morphology of atherosclerotic plaques in blood vessels with low-oxygen blood and low blood pressure as the pulmonary trunk (PT), in hypercholesterolemic ApoE-/- mice. METHODS: GDF-15-/- ApoE-/- mice were generated by crossbreeding of ApoE-/-- and GDF-15-/- mice. After feeding a cholesterol-enriched diet (CED) for 20 weeks, samples of the brachiocephalic trunk (BT) and PT were dissected and lumen stenosis (LS) was measured. Furthermore, changes in the cellularity of the PT, amounts of apoptosis-, autophagy-, inflammation- and proliferation-relevant proteins were immunohisto-morphometrically analyzed. Additionally, we examined an atherosclerotic plaque in a human post mortem sample of the pulmonary artery. RESULTS: After CED the body weight of GDF-15-/-ApoE-/- was 22.9% higher than ApoE-/-. Double knockout mice showed also an 35.3% increase of plasma triglyceride levels, whereas plasma cholesterol was similar in both genotypes. LS in the BT and PT of GDF-15-/-ApoE-/- mice was significantly reduced by 19.0% and by 6.7% compared to ApoE-/-. Comparing LS in PT and BT of the same genotype revealed a significant 38.8% (ApoE-/-) or 26.4% (GDF-15-/-ApoE-/-) lower LS in the PT. Immunohistomorphometry of atherosclerotic lesions in PT of GDF-15-/-ApoE-/- revealed significantly increased levels (39.8% and 7.3%) of CD68 + macrophages (MΦ) and α-actin + smooth muscle cells than in ApoE-/-. The density of TUNEL + , apoptotic cells was significantly (32.9%) higher in plaques of PT of GDF-15-/-ApoE-/- than in ApoE-/-. Analysis of atherosclerotic lesion of a human pulmonary artery showed sm-α-actin, CD68+, TUNEL+, Ki67+, and APG5L/ATG+ cells as observed in PT. COX-2+ and IL-6+ immunoreactivities were predominantly located in endothelial cells and subendothelial space. In BT and PT of GDF15-/-ApoE-/- mice the necrotic area was 10% and 6.5% lower than in ApoE-/-. In BT and PT of GDF15-/-ApoE-/- we found 40% and 57% less unstable plaques than ApoE-/- mice. CONCLUSIONS: Atherosclerotic lesions occur in both, BT and PT, however, the size is smaller in PT, possibly due to the effect of the low-oxygen blood and/or lower blood pressure. GDF-15 is involved in atherosclerotic processes in BT and PT, although different mechanisms (e.g. apoptosis) in these two vessels seem to exist.

Effect of cholesterol re-supplementation and atorvastatin on plaque composition in the thoracic aorta of New Zealand white rabbits.

BACKGROUND: Effects of re-supplementation of a cholesterol-enriched diet (CEDrs) on size, cholesterol content and morphology of already existing plaques are not known to date. METHODS: A group of rabbits received standard chow (SC) for 6 weeks ("negative control"; for plasma lipid measurements only). Group I-IV received 2% CED (induction) for 6 weeks; thereafter, groups II-IV have been fed a SC (= cholesterol withdrawal) for 68 weeks. Afterwards, feeding of groups II-IV was continued as follows: Group II - 10 weeks SC, group III - 4 weeks 0.5% CED (~re-supplementation), afterwards 6 weeks SC (~withdrawal again); group IV - 4 weeks 0.5% CED (re-supplementation) + atorvastatin (2.5 mg/kg body weight/day), afterwards 6 weeks SC (~withdrawal again) + atorvastatin. Plasma lipids, but also plaque size, morphology and cholesterol contents of thoracic aortas were quantified. RESULTS: After CEDrs, plasma cholesterol levels were increased. However, after withdrawal of CEDrs, plasma cholesterol levels decreased, whereas the cholesterol content of the thoracic aorta was increased in comparison with the group without CEDrs. Plaque size remained unaffected. Atorvastatin application did not change plasma cholesterol level, cholesterol content of the thoracic aorta and plaque size in comparison with the group without drug treatment. However, atorvastatin treatment increased the density of macrophages (MΦ) compared with the group without treatment, with a significant correlation between densities of MΦ (Mac-1+) and apoptotic (TUNEL+; TP53+), antigen-presenting (HLA-DR+) or oxidatively stressed (SOD2+) cells. CONCLUSIONS: In rabbits with already existing plaques, CEDrs affects plaque morphology and cellular composition, but not plaque size. Despite missing effects on plasma cholesterol levels, cholesterol content of the thoracic aorta and size of already existing atherosclerotic plaques, atorvastatin treatment transforms the already existing lesions to a more active form, which may accelerate the remodelling to a more stable plaque.

The phenotype of the RABV glycoprotein determines cellular and global virus load in the brain and is decisive for the pace of the disease.

The Rabies lyssavirus glycoprotein (RABV-G) is largely responsible for the neuroinvasiveness of the virus and the induction of antiviral immune responses. To study the effects of RABV-G we compared the G of the attenuated RABV variant SPBN with that of the pathogenic DOG4 strain. Infection via the olfactory route caused 100% mortality in mice with both virus variants. Of note, with the attenuated SPBN, progression of the disease was accelerated, microglia response less pronounced and IL-6 expression higher than in the presence of RABV-G from the pathogenic DOG4. However, while virus spread was less extensive, viral gene expression in individual neurons was actually higher in SPBN-infected brains without causing apoptosis of infected neurons. These differences between the two variants were not observed in infected neuronal cultures indicating that the effects of RABV-G on virus spread and viral gene expression depend on factors only present in the intact brain.

Mechanisms of the anti-proliferative and anti-inflammatory effects of the herbal fixed combination STW 5 (Iberogast®) on colon adenocarcinoma (HT29) cells in vitro.

INTRODUCTION: Several conventional pharmaceuticals like non-steroidal anti-inflammatory drugs (NSAIDS) or selective cyclooxygenase-2 (COX-2) inhibitors have been demonstrated to exert anti-proliferative effects and to induce apoptosis in a variety of cell lines, e.g. colon, stomach, or prostate cancer cells. STW 5 (Iberogast(®)), a combination of nine plant extracts, is widely used in the treatment of gastrointestinal disorders, including functional dyspepsia and irritable bowel syndrome for which the involvement of an inflammatory etiology is discussed. To investigate the possible anti-proliferative effects, STW 5 and its components have been tested by using the colon-carcinoma cell line HT-29. The analyses have been performed in comparison to acetylsalicylic acid (ASA) and diclofenac (Diclo), which are well-known to reduce colon carcinoma risk. RESULTS: STW 5 showed significant anti-proliferative and pro-apoptotic effects on HT-29 cancer cells, similar to NSAIDs under test. However, using the LDH assay, STW 5 revealed significantly lower cytotoxicity than Diclo at same concentrations. In contrast to NSAIDs, STW 5 induced COX-1/COX-2, caspase-3 and Bax mRNA expressions in HT-29 and blocked LPS mediated translocation of the NF-κB p65 from the cytoplasm into the nucleus in PMA-differentiated THP-1 macrophages. These effects might be relevant, e.g. for prevention of undesirable side effects like gastric erosions. CONCLUSION: Our data suggest that the pro-apoptotic effect of STW 5 on HT-29 cells is involving multiple targets and is possibly due to an activation of the caspase cascade via mitochondrial destabilization. Active concentrations of STW 5 are, in relation to therapeutic doses, comparable to those of ASA and Diclo, suggesting a similar favorable effect on colon carcinoma risk.

Anti-inflammatory effects of the willow bark extract STW 33-I (Proaktiv(®)) in LPS-activated human monocytes and differentiated macrophages.

INTRODUCTION: Willow bark extract is frequently used in the treatment of painful rheumatological diseases, such as arthritis and back pain. Its effect has been attributed to its main component salicin, but pharmacological studies have shown that the clinical efficacy of the willow bark extract cannot be explained by its salicin content alone. Therefore different modes of action have been suggested for the anti-inflammatory effect of willow bark extract. Here, we report in vitro data revelling the effect and mode of action of the aqueous willow bark extract STW 33-I as well as a water-soluble fraction (fraction E [Fr E]) in comparison with well-known non-steroidal anti-inflammatory drugs (NSAIDs) like aspirin (ASA) and diclofenac (Diclo) on pro-inflammatorily activated human monocytes and differentiated macrophages. RESULTS: STW 33-I and the water-soluble Fr E showed concentration-dependent and significant anti-inflammatory effects in lipopolysaccharide-activated monocytes. Both inhibited the intracellular protein expression of tumour necrosis factor-alpha (TNFα) as well as the mRNA expression of TNFα and cyclooxygenase 2 (COX-2), and the release of nitric oxide (NO). In addition, apoptosis of pro-inflammatorily activated monocytes was induced. Furthermore, treatment of activated macrophages with STW 33-I inhibited the nuclear translocation of the p65 subunit of the nuclear transcription factor-kappa B (NF-κB p65). CONCLUSIONS: The present in vitro investigations suggest a significant anti-inflammatory activity of willow bark water extract STW 33-1 and of its water-soluble fraction by inhibiting pro-inflammatory cytokines (TNFα), COX-2 and nuclear translocation of the transcription factor NF-κB in pro-inflammatorily activated monocytes. Our results provide further evidence for the therapeutic use of STW 33-I in inflammation-related disorders.

Novel systemic cardiovascular disease biomarkers.

Motivated by the challenge of risk assessment in a heterogeneous population and guided by advances in our knowledge of the pathobiology of cardiovascular diseases (CVD), basic and clinical scientists have maintained substantial interest in the development and application of novel biomarkers for risk stratification of CVD. In particular, strategies to identify and combine multiple biomarkers, which may reflect diverse pathobiological contributors to the onset and complications of CVD, have been arising as an approach to improve more effectively the risk assessment and target therapy. Moreover, comparative evaluations of novel markers are necessary to estimate these candidates for integration into present and future strategies. In this review we consider the recent in-depth knowledge and advances with the use of systemic biomarkers in the area of CVD with special attention on inflammatory markers and those that can predict an individual arteriosclerotic disease stage.

The early immunological response to acute ischemic stroke: differential gene expression in subpopulations of mononuclear cells.

UNLABELLED: Peripheral blood mononuclear cells (PBMCs), i.e. lymphocytes, monocytes and macrophages are key players in the development of innate and adaptive immune responses. However, little is known about their properties in patients with acute stroke. EXPERIMENTAL PROCEDURES: We presently characterized the early time course of PBMC subpopulations in 19 patients with acute ischemic stroke and symptom onset below 6 h compared to 19 age-matched healthy subjects. Immediately after acute ischemic stroke, as well as 1 and 3 days thereafter, PBMC subpopulations (cluster of differentiation [CD]3+, CD14+, CD19+, CD68+) were isolated by magnetic bead system and the expression of proinflammatory (CD40, tumor necrosis factor-alpha [TNFalpha]), proapoptotic (caspase-3 [CPP32], poly(ADP-ribose) polymerase [PARP]) and adhesion relevant (CD38) genes was measured by quantitative polymerase chain reaction (PCR). Furthermore, besides routine parameters, plasma levels of oxidized low-density lipoproteins (oxLDL) were studied. RESULTS: In comparison to healthy subjects, patients revealed (i) twofold elevated plasma oxLDL concentrations, (ii) decreased (15%) blood cholesterol levels, and (iii) a 40% decrease in total number of lymphocytes. Furthermore, the majority of PBMC subpopulations revealed an increased expression of proinflammatory, proapoptotic or adhesion-relevant genes. Significant positive correlations were observed between expression of most of these genes in PBMCs and individual plasma oxLDL concentrations. CONCLUSION: Elevated expression of proinflammatory, proapoptotic and adhesion genes in subsets of PBMCs after ischemic stroke may contribute to an immunodepressive syndrome, possibly due to increased plasma oxLDL levels.

Expression of growth differentiation factor-15/ macrophage inhibitory cytokine-1 (GDF-15/MIC-1) in the perinatal, adult, and injured rat brain.

We and others have recently cloned a new member of the transforming growth factor-beta superfamily, growth differentiation factor-15/ macrophage inhibitory cytokine-1 (GDF-15/MIC-1). Using in situ hybridization and immunohistochemistry, we determined the distribution of GDF-15/MIC-1 mRNA and protein in the perinatal and cryolesioned adult rat brain. The choroid plexus epithelium of all ventricles represents the site of strongest and almost exclusive mRNA expression in the normal perinatal and adult brain. The newborn rat brain reveals GDF-15/MIC-1 immunoreactivity (ir) in ependymal cells lining the ventricles, in the striatal subventricular zone, and in populations of nonneural cells of the thalamic/hippocampal lamina affixa, in addition to that in the choroid plexus. Unilateral cryogenic cortical lesioning induced a significant increase of GDF-15/MIC-1 mRNA expression and ir at the lesion site and expression in presumed neurons within the dorsal thalamic area. At the lesion site, GDF-15/MIC-1-producing cells showed immuncytochemical features of neurons, macrophages, and activated microglial cells. Fluorescent microscopy revealed both intra- and extracellular GDF-15/MIC-1 ir. Up-regulation of GDF-15/MIC-1 in activated macrophages (Mstraight phi) is also supported by RT-PCR, ICC, and Western blot experiments showing pronounced induction of GDF-15/MIC-1 expression (mRNA and protein) in retinoic acid/phorbol ester-stimulated human M phi. Our data suggest that 1) GDF-15/MIC-1 is secreted into the cerebrospinal fluid and 2) in the newborn brain may penetrate through the ependymal lining and act on developing neurons and/or glial cells. As a constituent of cells in the lamina affixa, the protein might be involved in the regulation of mesenchyme-epithelial interactions. Finally, GDF-15/MIC-1 may also act within the antiinflammatory cytokine network activated in CNS lesions.

Ceramide induces aSMase expression: implications for oxLDL-induced apoptosis.

Sphingomyelinase (SMase) stimulation and subsequent ceramide generation are suggested to be involved in signal transduction of stress-induced apoptosis. We now show that apoptosis of human macrophages (MPhi) and fibroblasts initiated by oxidized low density lipoproteins (minimally modified LDL, mmLDL) is associated with an increase in acid SMase (aSMase, E.C. 3.1.4.12) expression and ceramide concentration. Application of a novel, potent, and specific inhibitor of aSMase expression (NB6) diminished the effects of mmLDL and C6-ceramide treatment by inhibiting transcription via Sp1 and AP-2. Moreover, apoptosis was abolished after mmLDL and C6-ceramide treatment of hereditary aSMase-deficient fibroblasts (from Niemann-Pick patients). We suggest that in mmLDL-initiated apoptosis 1) enhanced ceramide generation via aSMase appears to be required as well as 2) a positive feedback control of aSMase expression by the increase in intracellular ceramide concentration.

Characterization of apoptotic macrophages in atheromatous tissue of humans and heritable hyperlipidemic rabbits.

Apoptotic macrophages are regularly found in atherosclerotic plaques indicating programmed cell death as one of their regulatory controls. The objective of this study was to characterize in more detail apoptotic macrophages in atherosclerotic lesions of humans and heritable hyperlipidemic (HHL) rabbits. Macrophages were immunohistochemically analyzed using antibodies directed against alphaMbeta2-integrins (CD11b/CD18), CD44, major histocompatibility complex (MHC) class I and II, inducible nitric oxide synthase (iNOS), manganese superoxide dismutase (MnSOD), tumor necrosis factor alpha (TNFalpha), p53, c-jun/AP-1 and rabbit macrophages (RAM-11) and the TUNEL (TdT-mediated dUTP nick end labeling) technique. Colocalization studies of human atherosclerotic carotid and aortic tissue showed apoptotic plaque macrophages also being MnSOD-, alphaMbeta2-integrin-, CD44-, MHC class I- and II-, iNOS-, TNFalpha- and p53-immunoreactive. Similar results occurred in atherosclerotic aortas of HHL rabbits. Computer-assisted morphometric analyses revealed a positive correlation of the area density of MnSOD-immunoreactive macrophages with those of alphaMbeta2-integrin- and CD44-immunoreactive ones, but not with those of MHC class I- and II- as well as of RAM-11-immunoreactive macrophages. We conclude that apoptotic macrophages located in atherosclerotic vessel wall are activated, antigen-presenting, integrin-expressing and oxidatively stressed cells. Since all these processes have been demonstrated to cause apoptosis of macrophages in vitro, we propose their potency accelerates the susceptibility of the macrophages to programmed cell death in atherosclerotic lesions.

Isolation of a frog lectin using polylysine to reduce non-specific interactions with heparin binding ligand

Polylysine is a polycation used in histology to glue tissue sections to glass slides. It was successfully used to eliminate positively charged proteins from heparin-agarose beds applied for affinity chromatography facilitating the preparative steps in the isolation and purification of heparin binding lectins from the ovary of the frog Leptodactylus occellatus. The addition pretreatment of agarose-heparin beds with polylysine increased more than 3-fold the hemagglutinating capacity of eluted lectin

Isolation of a frog lectin using polylysine to reduce non-specific interactions with heparin binding ligand.

Polylysine is a polycation used in histology to glue tissue sections to glass slides. It was successfully used to eliminate positively charged proteins from heparin-agarose beds applied for affinity chromatography facilitating the preparative steps in the isolation and purification of heparin binding lectins from the ovary of the frog Leptodactylus occellatus. The addition pretreatment of agarose-heparin beds with polylysine increased more than 3-fold the hemagglutinating capacity of eluted lectin.