Histone H3 posttranslational modified enzymes defined neutrophil plasticity and their vulnerability to IL-10 in the course of the inflammation.

BACKGROUND: Neutrophils are a heterogeneous population capable of antimicrobial functions associated with pre-activation/activation and tissue regeneration. The specific polarisation of immune cells is mediated by the modification of 'chromatin landscapes', which enables differentiated access and activity of regulatory elements that guarantee their plasticity during inflammation No specific pattern within histone posttranslational modifications (PTMs) controlling this plasticity has been identified. METHODS: Using the in vitro model of inflammation, reflecting different states of neutrophils from resting, pre-activated cells to activated and reducing tissue regeneration, we have analysed 11 different histone posttranslational modifications (PTMs), PTM enzymes associated with remodelling neutrophil chromatin, and H3K4me3 ChIP-Seq Gene Ontology analysis focusing on the processes related to histone PTMs. These findings were verified by extrapolation to adequate clinical status, using neutrophils derived from the patients with sepsis (systemic septic inflammation with LPS-stimulated neutrophils), neuromyelitis optical spectrum disorders (aseptic inflammation with pre-activated neutrophils) and periodontitis (local self-limiting septic inflammation with IL-10-positive neutrophils). RESULTS: Physiological activation of neutrophils comprises a pre-activation characterised by histone H3K27ac and H3K4me1, which position enhancers; direct LPS exposure is induced explicitly by H3K4me3 which marked Transcription Start Site (TSS) regions and low-level of H3K9me3, H3K79me2 and H3K27me3 which, in turn, marked repressed genes. Contrary to antimicrobial action, IL-10 positively induced levels of H3S10p and negatively H3K9me3, which characterised processes related to the activation of genes within heterochromatin mediated by CHD1 and H3K9me3 specific demethylase JMJD2A. IL-10 protects changes within histone PTMs induced by TNF or LPS that affected H3K4me3-specific methyltransferase SETD1A and MLL1. Neutrophils previously exposed to inflammatory factors become unvulnerable to IL-10 because previous LPS stimulation interrupts TSS regions marked by H3K4me3 of CHD1 and JMJD2A genes. Therefore, LPS-activated neutrophils are disabled to induce CHD1/JMJD2A enzymes by IL-10, making this process irreversible. Because transcription of JMJD2A and CHD1 also depends on TSS positioning by H3K4me3, neutrophils before LPS stimulation become insensitive to IL-10. CONCLUSION: Neutrophils, once pre-activated by TNF or directly stimulated by LPS, become insensitive to the anti-inflammatory effects of IL-10, and vice versa; IL-10 protects neutrophils against these proinflammatory stimuli. This phenomenon is responsible for disturbing the natural process of resolving inflammation and tissue regeneration.

Nervonic Acid Synthesis Substrates as Essential Components in Profiled Lipid Supplementation for More Effective Central Nervous System Regeneration.

Central nervous system (CNS) damage leads to severe neurological dysfunction as a result of neuronal cell death and axonal degeneration. As, in the mature CNS, neurons have little ability to regenerate their axons and reconstruct neural loss, demyelination is one of the hallmarks of neurological disorders such as multiple sclerosis (MS). Unfortunately, remyelination, as a regenerative process, is often insufficient to prevent axonal loss and improve neurological deficits after demyelination. Currently, there are still no effective therapeutic tools to restore neurological function, but interestingly, emerging studies prove the beneficial effects of lipid supplementation in a wide variety of pathological processes in the human body. In the future, available lipids with a proven beneficial effect on CNS regeneration could be included in supportive therapy, but this topic still requires further studies. Based on our and others' research, we review the role of exogenous lipids, pointing to substrates that are crucial in the remyelination process but are omitted in available studies, justifying the properly profiled supply of lipids in the human diet as a supportive therapy during CNS regeneration.

Clinical and microbiological outcomes of subgingival instrumentation supplemented with high-dose omega-3 polyunsaturated fatty acids in periodontal treatment - a randomized clinical trial.

PURPOSE: This study aimed to evaluate the impact of dietary supplementation with omega-3 polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) combined with scaling and root planing (SRP) in untreated periodontitis stage III and IV. METHODS: Forty patients were randomly assigned to the test group receiving SRP plus omega-3 PUFAs (n = 20) or control group receiving SRP alone (n = 20). Clinical changes of pocket probing depths (PD), clinical attachment level (CAL), bleeding on probing (BOP) and rates of closed pockets (PPD ≤ 4 mm without BOP) were evaluated at baseline and after 3 and 6 months. Phorphyromonas gingivalis, Tanarella forsythia, Treponema denticola and Aggregatibacter actinomycetemcomitans counts were analysed at baseline and at 6 months. Serum was subjected to lipid gas chromatography/mass spectrometry analysis at baseline and at 6 months. RESULTS: Significant improvement of all clinical parameters at 3 and 6 months was observed in both groups. For the primary outcome "change of mean PD," no significant difference was detected between the groups. Patients treated with omega-3 PUFAs demonstrated significantly lower rates of BOP, higher gain of CAL and higher number of closed pockets at 3 months in comparison to the control group. After 6 months, no clinical differences between the groups were found, with the exception of lower BOP rates. Moreover, in the test group, the number of key periodontal bacteria was significantly lower than in the control group at 6 months. Increased proportions of serum n-3 PUFAs and decreased proportions of n-6 PUFAs were detected at 6 months in the patients from the test group. CONCLUSION: High-dose omega-3 PUFA intake during non-surgical treatment of periodontitis results in short-term clinical and microbiological benefits. The study protocol was approved by the ethical committee of Medical University of Lodz (reference number RNN/251/17/KE) and registered at clinicaltrials.gov (NCT04477395) on 20/07/2020.

Cerebrospinal Fluid Biomarkers in Differential Diagnosis of Multiple Sclerosis and Systemic Inflammatory Diseases with Central Nervous System Involvement.

BACKGROUND: Diagnosis of multiple sclerosis (MS) is established on criteria according to clinical and radiological manifestation. Cerebrospinal fluid (CSF) analysis is an important part of differential diagnosis of MS and other inflammatory processes in the central nervous system (CNS). METHODS: In total, 242 CSF samples were collected from patients undergoing differential MS diagnosis because of the presence of T2-hyperintensive lesions on brain MRI. The non-MS patients were subdivided into systemic inflammatory diseases with CNS involvement (SID) or cerebrovascular diseases (CVD) or other non-inflammatory diseases (NID). All samples were analyzed for the presence of oligoclonal bands and ELISA was performed for detection of: INF gamma, IL-6, neurofilaments light chain (NF-L), GFAP, CHI3L1, CXCL13, and osteopontin. RESULTS: The level of IL-6 (p = 0.024), osteopontin (p = 0.0002), and NF-L (p = 0.002) was significantly different among groups. IL-6 (p = 0.0350) and NF-L (p = 0.0015) level was significantly higher in SID compared to NID patients. A significantly higher level of osteopontin (p = 0.00026) and NF-L (p = 0.002) in MS compared to NID population was noted. ROC analysis found weak diagnostic power for osteopontin and NFL-L. CONCLUSIONS: The classical and non-standard markers of inflammatory process and neurodegeneration do not allow for sufficient differentiation between MS and non-MS inflammatory CNS disorders. Weak diagnostic power observed for the osteopontin and NF-L needs to be further investigated.

Natural fish oil improves the differentiation and maturation of oligodendrocyte precursor cells to oligodendrocytes in vitro after interaction with the blood-brain barrier.

The blood-brain barrier (BBB) tightly controls the microenvironment of the central nervous system (CNS) to allow neurons to function properly. Additionally, emerging studies point to the beneficial effect of natural oils affecting a wide variety of physiological and pathological processes in the human body. In this study, using an in vitro model of the BBB, we tested the influence of natural fish oil mixture (FOM) vs. borage oil (BO), both rich in long-chain polyunsaturated fatty acids (LC-PUFAs) and monounsaturated fatty acids (MUFAs) such as oleic acid (C18:1n9c) or nervonic acid (NA), on human oligodendrocyte precursor cells (hOPCs) during their maturation to oligodendrocytes (OLs) regarding their ability to synthesize myelin peptides and NA. We demonstrated that FOM, opposite to BO, supplemented endothelial cells (ECs) and astrocytes forming the BBB, affecting the function of hOPCs during their maturation. This resulted in improved synthesis of myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), proteolipid protein (PLP), and NA in mature OLs. This effect is probably the result of BBB cell and hOPC stimulation via free fatty acid receptors (FFARs), which increases insulin growth factor-1 (IGF-1), ciliary neurotrophic factor (CNTF), and brain-derived neurotrophic factor (BDNF) and inhibits fibroblast growth factor 2 (FGF-2) synthesis. The unique formula of fish oil, characterized by much more varied components compared to those of BOs, also improved the enhancement of the tight junction by increasing the expression of claudin-5 and VE-cadherin on ECs. The obtained data justify consideration of naturally derived fish oil intake in human diet as affecting during remyelination.

Changes Within H3K4me3-Marked Histone Reveal Molecular Background of Neutrophil Functional Plasticity.

Neutrophils are a heterogenous population capable of both antimicrobial functions and suppressor ones, however, no specific pattern of transcription factors controlling this plasticity has been identified. We observed rapid changes in the neutrophil status after stimulation with LPS, pre-activating concentration of TNF-α, or IL-10. Chromatin immunoprecipitation sequencing (ChIP-Seq) analysis of histone H3K4me3 allowed us to identify various transcriptional start sites (TSSs) associated with plasticity and heterogeneity of human neutrophils. Gene Ontology analysis demonstrated great variation within target genes responsible for neutrophil activation, cytokine production, apoptosis, histone remodelling as well as NF-κB transcription factor pathways. These data allowed us to assign specific target genes positioned by H3K4me3-marked histone with a different pattern of gene expression related to NF-κB pathways, apoptosis, and a specific profile of cytokines/chemokines/growth factors realised by neutrophils stimulated by LPS, IL-10, or TNF-α. We discovered IL-10-induced apoptotic neutrophils being transcriptionally active cells capable of switching the profile of cytokines/chemokines/growth factors desired in resolving inflammation via non-canonical NF-κB pathway with simultaneous inhibition of canonical NF-κB pathway. As apoptotic/suppressive neutrophils induced by IL-10 via positioning genes within H3K4me3-marked histone were transcriptionally active, newly described DNA binding sites can be considered as potential targets for immunotherapy.H3K4me3 histone ChIP-Seq analysis reveals molecular drivers critical for switching neutrophils from their pro- to anti-inflammatory properties.

H3K4me3 Histone ChIP-Seq Analysis Reveals Molecular Mechanisms Responsible for Neutrophil Dysfunction in HIV-Infected Individuals.

Peripheral neutrophils in HIV-infected individuals are characterized by impairment of chemotaxis, phagocytosis, bactericidal activity, and oxidative burst ability regardless of whether patients are receiving antiretroviral therapy or not. Neutrophil dysfunction leads not only to increased susceptibility to opportunistic infections but also to tissue damage through the release of reactive oxygen species (ROS), proteases, and other potentially harmful effector molecules contributing to AIDS progression. In this study, we demonstrated high levels of histone H3 lysine K4 trimethylated (H3K4me3) and dysregulation of DNA transcription in circulating neutrophils of HIV-infected subjects. This dysregulation was accompanied by a deficient response of neutrophils to LPS, impaired cytokine/chemokine/growth factor synthesis, and increased apoptosis. Chromatin immunoprecipitation sequencing (ChIPseq) H3K4me3 histone analysis revealed that the most spectacular abnormalities were observed in the exons, introns, and promoter-TSS regions. Bioinformatic analysis of Gene Ontology, including biological processes, molecular function, and cellular components, demonstrated that the main changes were related to the genes responsible for cell activation, cytokine production, adhesive molecule expression, histone remodeling via upregulation of methyltransferase process, and downregulation of NF-κB transcription factor in canonical pathways. Abnormalities within H3K4me3 implicated LPS-mediated NF-κB canonical activation pathway that was a result of low amounts of κB DNA sites within histone H3K4me3, low NF-κB (p65 RelA) and TLR4 mRNA expression, and reduced free NF-κB (p65 RelA) accumulation in the nucleus. Genome-wide survey of H3K4me3 provided evidence that chromatin modifications lead to an impairment within the canonical NF-κB cell activation pathway causing the neutrophil dysfunction observed in HIV-infected individuals.

Omega-3 Polyunsaturated Fatty Acids EPA and DHA as an Adjunct to Non-Surgical Treatment of Periodontitis: A Randomized Clinical Trial.

Periodontitis is a chronic multifactorial inflammatory disease that leads to the loss of supportive tissues around the teeth with gradual deterioration of masticatory function and esthetics, resulting eventually in the decrease of the life quality. Host immune response triggered by bacterial biofilm is responsible for the chronic periodontal inflammation and ongoing tissue loss. Omega-3 polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have anti-inflammatory properties, thus may be used for the treatment of chronic inflammatory diseases. In this study, we aimed to evaluate the effect of dietary supplementation with omega-3 PUFA in the patients with stage III and IV periodontitis. Thirty otherwise healthy patients were treated with scaling and root planning (SRP). In the test group (n = 16), patients were additionally supplemented with 2.6 g of EPA and 1.8 g of DHA. In the control group (n = 14), patients received only SRP. Periodontal examination was performed at baseline and three months following initial therapy. Salivary samples were taken twice at baseline and at the end of the experiment. We found that there was a statistically significant reduction in the bleeding on probing (BOP) and improvement of clinical attachment loss (CAL) at three months in the test group compared to the control group. Moreover, a statistically significant higher percentage of closed pockets (probing depth ≤ 4 mm without BOP) was achieved in the test group vs. control group after three months of treatment. Accordingly, the levels of pro-inflammatory cytokines/chemokines interleukin (IL)-8 and IL-17 were markedly lower, while the level of anti-inflammatory IL-10 was significantly higher in the salivary samples of the patients supplemented with omega-3 PUFA at three months in comparison to the patients treated with SRP alone. Our findings demonstrate that dietary intervention with high-dose of omega-3 PUFA during non-surgical therapy may have potential benefits in the management of periodontitis.

Multiple Sclerosis CD49d+CD154+ As Myelin-Specific Lymphocytes Induced During Remyelination.

Multiple sclerosis (MS) is a demyelinating autoimmune disease of the central nervous system (CNS) mediated by autoreactive lymphocytes. The role of autoreactive lymphocytes in the CNS demyelination is well described, whereas very little is known about their role in remyelination during MS remission. In this study, we identified a new subpopulation of myelin-specific CD49d+CD154+ lymphocytes presented in the peripheral blood of MS patients during remission, that proliferated in vitro in response to myelin peptides. These lymphocytes possessed the unique ability to migrate towards maturing oligodendrocyte precursor cells (OPCs) and synthetize proinflammatory chemokines/cytokines. The co-culture of maturing OPCs with myelin-specific CD49d+CD154+ lymphocytes was characterized by the increase in proinflammatory chemokine/cytokine secretion that was not only a result of their cumulative effect of what OPCs and CD49d+CD154+ lymphocytes produced alone. Moreover, maturing OPCs exposed to exogenous myelin peptides managed to induce CD40-CD154-dependent CD49d+CD154+ lymphocyte proliferation. We confirmed, in vivo, the presence of CD49d+CD154+ cells close to maturating OPCs and remyelinating plaque during disease remission in the MS mouse model (C57Bl/6 mice immunized with MOG35-55) by immunohistochemistry. Three weeks after an acute phase of experimental autoimmune encephalomyelitis, CD49d+/CD154+ cells were found to be co-localized with O4+ cells (oligodendrocyte progenitors) in the areas of remyelination identified by myelin basic protein (MBP) labelling. These data suggested that myelin-specific CD49d+CD154+ lymphocytes present in the brain can interfere with remyelination mediated by oligodendrocytes probably as a result of establishing proinflammatory environment.

MS CD49d+CD154+ Lymphocytes Reprogram Oligodendrocytes into Immune Reactive Cells Affecting CNS Regeneration.

The critical aspect in multiple sclerosis (MS) progression involves insufficient regeneration of CNS resulting from deficient myelin synthesis by newly generated oligodendrocytes (OLs). Although many studies have focused on the role of autoreactive lymphocytes in the inflammatory-induced axonal loss, the problem of insufficient remyelination and disease progression is still unsolved. To determine the effect of myelin-specific lymphocytes on OL function in MS patients and in a mouse model of MS, we cultured myelin induced MS CD49d+CD154+ circulating lymphocytes as well as Experimental Autoimmune Encephalomyelitis (EAE) mouse brain-derived T and memory B cells with maturing oligodendrocyte precursor cells (OPCs). We found that myelin-specific CD49d+CD154+ lymphocytes affected OPC maturation toward formation of immune reactive OLs. Newly generated OLs were characterized by imbalanced myelin basic protein (MBP) and proteolipid protein (PLP) production as well as proinflammatory chemokine/cytokine synthesis. The analysis of cellular pathways responsible for OL reprogramming revealed that CD49d+CD154+ lymphocytes affected miRNA synthesis by dysregulation of polymerase II activity. miR-665 and ELL3 turned out to be the main targets of MS myelin-specific lymphocytes. Neutralization of high intracellular miR-665 concentration restored miRNA and MBP/PLP synthesis. Together, these data point to new targets for therapeutic intervention promoting CNS remyelination.

Naturally Occurring Nervonic Acid Ester Improves Myelin Synthesis by Human Oligodendrocytes.

The dysfunction of oligodendrocytes (OLs) is regarded as one of the major causes of inefficient remyelination in multiple sclerosis, resulting gradually in disease progression. Oligodendrocytes are derived from oligodendrocyte progenitor cells (OPCs), which populate the adult central nervous system, but their physiological capability to myelin synthesis is limited. The low intake of essential lipids for sphingomyelin synthesis in the human diet may account for increased demyelination and the reduced efficiency of the remyelination process. In our study on lipid profiling in an experimental autoimmune encephalomyelitis brain, we revealed that during acute inflammation, nervonic acid synthesis is silenced, which is the effect of shifting the lipid metabolism pathway of common substrates into proinflammatory arachidonic acid production. In the experiments on the human model of maturating oligodendrocyte precursor cells (hOPCs) in vitro, we demonstrated that fish oil mixture (FOM) affected the function of hOPCs, resulting in the improved synthesis of myelin basic protein, myelin oligodendrocyte glycoprotein, and proteolipid protein, as well as sphingomyelin. Additionally, FOM reduces proinflammatory cytokines and chemokines, and enhances fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF) synthesis by hOPCs was also demonstrated. Based on these observations, we propose that the intake of FOM rich in the nervonic acid ester may improve OL function, affecting OPC maturation and limiting inflammation.

Global exosome transcriptome profiling reveals biomarkers for multiple sclerosis.

OBJECTIVE: Accumulating evidence supports a role for exosomes in immune regulation. In this study, we investigated the total circulating exosome transcriptome in relapsing-remitting multiple sclerosis (RRMS) patients and healthy controls (HC). METHODS: Next generation sequencing (NGS) was used to define the global RNA profile of serum exosomes in 19 RRMS patients (9 in relapse, 10 in remission) and 10 HC. We analyzed 5 million reads and >50,000 transcripts per sample, including a detailed analysis of microRNAs (miRNAs) differentially expressed in RRMS. The discovery set data were validated by quantification using digital quantitative polymerase chain reaction with an independent cohort of 63 RRMS patients (33 in relapse, 30 in remission) and 32 HC. RESULTS: Exosomal RNA NGS revealed that of 15 different classes of transcripts detected, 4 circulating exosomal sequences within the miRNA category were differentially expressed in RRMS patients versus HC: hsa-miR-122-5p, hsa-miR-196b-5p, hsa-miR-301a-3p, and hsa-miR-532-5p. Serum exosomal expression of these miRNAs was significantly decreased during relapse in RRMS. These miRNAs were also decreased in patients with a gadolinium enhancement on brain magnetic resonance imaging. In vitro secretion of these miRNAs by peripheral blood mononuclear cells was also significantly impaired in RRMS. INTERPRETATION: These data show that circulating exosomes have a distinct RNA profile in RRMS. Because putative targets for these miRNAs include the signal transducer and activator of transcription 3 and the cell cycle regulator aryl hydrocarbon receptor, the data suggest a disturbed cell-to-cell communication in this disease. Thus, exosomal miRNAs might represent a useful biomarker to distinguish multiple sclerosis relapse. Ann Neurol 2017;81:703-717.

THE EFFECT OF IBUPROFEN ON bFGF, VEGF SECRETION AND CELL PROLIFERATION IN THE PRESENCE OF LPS IN HMEC-1 CELLS.

Ibuprofen belongs to the group of non-selective cyclooxygenase (COX) inhibitors, also known as traditional non-steroidal anti-inflammatory drugs (NSAIDs). Bacterial lipopolysaccharide, an inflammatory mimicking agent, is responsible for the production of prostaglandins and growth factors (VEGF and bFGF), and as inflammation and angiogenesis are closely associated with osteoarthritis, these factors play a functional role in the cardiovascular system. Therefore, the main aim of our study was to examine the effect of ibuprofen on cell viability and proliferation of HMEC-1 cells and VEGF and bFGF secretion under the inflammatory conditions. The effect of NSAID and LPS on bFGF and VEGF was analyzed by ELISA. Cell viability was measured by the MTT method and the proliferation by the [3H-thymidine test. LPS at 100 µg/mL stimulated the secretion of VEGF and bFGF by HMEC-1 cells. Ibuprofen at concentrations of 0.1 and 1 mM intensified the secretion of LPS-induced VEGF in a statistically significant manner (p < 0.05). Both concentrations of ibuprofen inhibited LPS-stimulated bFGF secretion (p < 0.05) in HMEC-1 in a concentration-dependent manner. The non-selective COX inhibitor decreased proliferation and cell viability induced by LPS in a concentration-dependent manner. The observed effects of ibuprofen on endothelial cells may further explain its effects as well as other NSAIDs on the cardiovascular system function in cardiovascular diseases.

Orexins protect neuronal cell cultures against hypoxic stress: an involvement of Akt signaling.

Orexins A and B are peptides produced mainly by hypothalamic neurons that project to numerous brain structures. We have previously demonstrated that rat cortical neurons express both types of orexin receptors, and their activation by orexins initiates different intracellular signals. The present study aimed to determine the effect of orexins on the Akt kinase activation in the rat neuronal cultures and the significance of that response in neurons subjected to hypoxic stress. We report the first evidence that orexins A and B stimulated Akt in cortical neurons in a concentration- and time-dependent manner. Orexin B more potently than orexin A increased Akt phosphorylation, but the maximal effect of both peptides on the kinase activation was very similar. Next, cultured cortical neurons were challenged with cobalt chloride, an inducer of reactive oxygen species and hypoxia-mediated signaling pathways. Under conditions of chemical hypoxia, orexins potently increased neuronal viability and protected cortical neurons against oxidative stress. Our results also indicate that Akt kinase plays an important role in the pro-survival effects of orexins in neurons, which implies a possible mechanism of the orexin-induced neuroprotection.

Orexins/hypocretins acting at Gi protein-coupled OX 2 receptors inhibit cyclic AMP synthesis in the primary neuronal cultures.

Orexins A and B are newly discovered neuropeptides with pleiotropic activity. They signal through two G protein-coupled receptors: OX(1) and OX(2). In this study, we examined the expression of orexin receptors and effects of the receptors' activation on cyclic AMP formation in the primary neuronal cell cultures from rat cerebral cortex. Both types of orexin receptors were expressed in rat cortical neurons; the level of OX(2)R was markedly higher compared to OX(1)R. Orexin A (an agonist of OX(1)R and OX(2)R) and [Ala(11)-D-Leu(15)]orexin B (a selective agonist of OX(2)R) did not affect basal cyclic AMP formation in the primary neuronal cell cultures. Both peptides (0.001-1 µM) inhibited, in a concentration-dependent manner and IC(50) values in low nanomolar range, the increase in the nucleotide production evoked by forskolin (1 µM; a direct activator of adenylyl cyclase), pituitary adenylate cyclase-activating polypeptide (PACAP27; 0.1 µM), and vasoactive intestinal peptide (VIP; 3 µM). Effects of orexin A on forskolin-, PACAP27-, and VIP-stimulated cyclic AMP synthesis were blocked by TCS OX2 29 (a selective antagonist of OX(2)R), and unaffected by SB 408124 (a selective antagonist of OX(1)R). Pretreatment of neuronal cell cultures with pertussis toxin (PTX) abolished the inhibitory action of orexin A on forskolin- and PACAP-stimulated cyclic AMP accumulation. It is suggested that in cultured rat cortical neurons orexins, acting at OX(2) receptors coupled to PTX-sensitive G(i) protein, inhibit cyclic AMP synthesis.

Orexins promote survival of rat cortical neurons.

Orexin A and B (hypocretin-1 and -2) are hypothalamic peptides that exert their biological functions by stimulation of two specific, membrane-bound receptors, OX(1)R and OX(2)R. Recently, we have demonstrated the expression of both types of orexin receptors in rat cortical neurons, with the OX(2)R level being markedly higher compared to OX(1)R. In the present study we investigated the receptor-mediated effects of orexin A, an agonist of OX(1)R and OX(2) R, orexin B and [Ala(11)-D-Leu(15)]orexin B, preferential agonists of OX(2)R, on survival of cultured neurons derived from rat cerebral cortex. The three tested peptides markedly increased neuronal viability in a concentration-dependent manner. The pro-survival properties of orexins were associated with an attenuation of caspase-3 activity. Comparable potency of orexin A, orexin B and [Ala(11)-D-Leu(15)]orexin B suggests a predominant role of OX(2)R in the studied phenomenon. Our findings provide new insights into the role of orexins in CNS as potential neuroprotective factors.

Characteristics of adrenaline-driven receptor-mediated signals in human microvessel-derived endothelial cells.

Adrenaline (0.001-1,000 muM) strongly stimulated adenosine-3',5'cyclic monophosphate (cAMP) generation in cultured human microvascular-derived endothelial cells (HMEC-1). Isoprenaline mimicked the action of adrenaline, whereas noradrenaline appeared to be decisively less potent. Experiments carried out with an array of compounds acting selectively on different types/subtypes of adrenergic receptors revealed that the adrenaline cAMP effect in HMEC-1 cells did not possess either an alpha(1) or alpha(2) component. However, the effect may have been mediated through a receptor that did not fit beta(1)-, beta(2)-, or beta(3)-receptor classification. Supporting this assertion, various double and triple beta-subtype selective drug combinations maximally inhibited the adrenaline effect by 50-60%, whereas the non-selective antagonist propranolol totally prevented the hormone-evoked cAMP effect. Based on results utilizing the phosphodiesterase (PDE)-isoform nonselective inhibitor 3-isobutyl-1-methylxanthine (IBMX) and the PDE-4-selective inhibitor rolipram, the adrenaline-driven cAMP signal appeared to be regulated by PDE-4. In addition, the present study demonstrated that phenylephrine, a presumed selective alpha(1)-adrenoceptor agonist, was capable of stimulating cAMP generation in HMEC-1 cells in a prazosin-insensitive and propranolol-sensitive manner. This result indicated that in at least this cell model system, phenylephrine may act nonspecifically. Microvessel-derived endothelial cells such as HMEC-1 exhibit functional differences when compared with macrovessel-derived endothelial cells (e.g. HUVEC sensitivity to adrenaline). Accordingly, these cell cultures represent a useful model system to study the biological effects of endogenous catecholamines, including adrenaline, as well as potential therapeutics targeting adrenergic receptors.

Human micro- and macrovessel-derived endothelial cells: a comparative study on the effects of adrenaline and a selective adenosine A2-type receptor agonist under normoxic and hypoxic conditions.

Adrenaline is a highly effective stimulator of cyclic AMP (cAMP) production in microvascular endothelial cells (ECs)--HMEC-1, showing only a moderate activity in macrovascular ECs--HUVEC. In both EC preparations, adrenaline acts via beta-type receptors. Selective stimulation of adenosine A(2)-type receptors resulted in comparable increases in cAMP formation in ECs lining micro- and macrovessels. Hypoxia largely suppressed the cAMP effects resulting from stimulation of both beta-adrenoceptors and adenosine A(2) type receptors in ECs of microvessels (HMEC-1). In contrast, hypoxia had only slight effect on these responses in ECs of macrovessels (HUVEC). The present data provide further evidence of functional differences between microvessel- and macrovessel-derived ECs.

Cyclic AMP generating system in human microvascular endothelium is highly responsive to adrenaline.

We have tested cultured human microvascular endothelial cells (HMEC-1) for their ability to synthesize cyclic adenosine 3',5'-monophosphate (cAMP) in the absence or presence of various drugs. The accumulation of cAMP was only slightly affected by the addition of a phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) to the incubation medium. A direct stimulator of adenylyl cyclase forskolin and adrenergic drugs, such as adrenaline and noradrenaline, strongly increased cAMP accumulation in IBMX-treated HMEC-1 cells, whereas some other drugs known to stimulate the nucleotide synthesis in different cell/tissues were inactive (dopamine, histamine). Adrenaline was significantly more potent than noradrenaline. The effect of adrenaline on cyclic AMP production was reproduced by a selective beta-adrenoceptor agonist isoprenaline, antagonized by beta-blocker propranolol, and was not influenced by both alpha(1) and alpha(2)-selective antagonists, prazosin and yohimbine, respectively. Adrenaline did not significantly affect the ability of HMEC-1 cells to produce vascular endothelial growth factor (VEGF) or interleukin-8 (IL-8), the major angiogenic mediators. The results indicate that under basal (non-stimulated) conditions, the cAMP generating system of HMEC-1 cells maintained in culture remains rather quiescent, yet it can strongly respond to the hormone adrenaline acting on beta-adrenergic receptors.

[VEGF as an angiogenic, neurotrophic, and neuroprotective factor]. / VEGF jako czynnik angiogenny, neurotroficzny i neuroprotekcyjny.

Vascular endothelial growth factor (VEGF, occurring in several isoforms: VEGF-A, -B, -C, -D) is a well-known endothelial cell mitogen and vascular growth and permeability factor. Recent work done over the last few years has elucidated the important role of VEGF, which participates in the regulation of normal (physiological or therapeutic) and pathological angiogenesis (VEGF-A, VEGF-B) and lymphangiogenesis (VEGF-C, VEGF-D). VEGF has also been implicated in practically every stage of angiogenesis, yet its role in the initiation of new blood vessel creation appears to be the most important. In addition to its role as a key angiogenic factor, VEGF also possesses neurotrophic and neuroprotective activity both in the peripheral and in the central nervous system, exerting a direct action on neurons, Schwann cells, astrocytes, neural stem cells, and microglia. VEGF interacts with three subtypes of VEGF receptors occurring on the cellular membrane known as VEGFR-1 (Flt-1), VEGFR-2 (Flk-1/KDR), and VEGFR-3 (Flt-4). All these receptor types possess an internal tyrosin kinase domain. Interaction of VEGF with particular subtypes of receptors activates a circuit of signaling pathways, e.g. PI3K/Akt, Ras/Raf-MEK/Erk, eNOS/NO, and IP3/Ca2+. These participate in the generation of specific biological responses connected with proliferation, migration, increasing vascular permeability, or promoting endothelial cell survival. Recent findings from experiments performed on animals with experimentally evoked focal cerebral ischemia suggest that the neuroprotective activity of VEGF runs in parallel with its ability to promote neurogenesis and angiogenesis and that these effects may operate independently through multiple mechanisms. The above-mentioned three major features characterizing the neurobiological activity of VEGF, i.e. neuroprotection, neurogenesis, and angiogenesis, together with their possible functional link(s), provide the rationale for considering VEGF-based therapy as a promising future avenue for a more effective treatment of at least some neurodegenerative disorders and stroke. Moreover, the possibility of using neutralizing factors of VEGF or VEGF receptor antagonists may reveal a way of preventing many dangerous pathologies, including post-ischemic disturbances in cardiac and neurological disorders, tumor growth, or hypervascularization in avascular structures of the eye.