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1.
Vascul Pharmacol ; : 106808, 2020 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-33130016

RESUMO

BACKGROUND: The G-protein-coupled apelin receptor and its apelin ligand are an emerging regulatory system of the vascular homeostasis. To date, the implications of the apelin/apelin receptor system in athero-thrombosis are not completely clarified yet. This study determines the expression of the apelin receptor on human platelets, the effect of different apelin isoforms on platelet aggregation and the potential role of the apelin/apelin receptor system in acute myocardial infarction. METHODS: We applied immunofluorescence staining, Western Blot analysis, aggregometry, and flow cytometry to elucidate the role of the apelin receptor in activated platelets. Furthermore, in an observational pilot study, we assessed platelet apelin recpetor expression and apelin-17 plasma levels in patients with acute myocardial infarction (AMI, n = 27). RESULTS: Immunofluorescence staining indicates that the apelin receptor is located at the cell membrane in resting platelets and diminishes upon activation with a selective thrombin receptor-activating peptide (AP1, 3 to 100 µM). Western Blot analyses of AP1-activated platelets and their supernatants suggest that the apelin receptor is not predominantly internalized but is released from activated platelets. The isoform apelin-17 attenuated AP-1-induced platelet activation in-vitro, presumably via a NO-dependent mechanism. Furthermore, platelet apelin receptor expression was significantly reduced in patients with AMI (n = 27) compared to age-matched controls (n = 14; p < 0.05) and inversely correlated with troponin I plasma levels (r = -0.46; p = 0.03). Besides that, circulating apelin-17 was significantly reduced in MI patients compared to the control group. CONCLUSION: Taken together, our data support a crucial role of the platelet apelinergic system assuming an antithrombotic effect and therefore holding a potential diagnostic and therapeutic impact.

2.
J Neurooncol ; 147(3): 577-585, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32246395

RESUMO

PURPOSE: Disialoganglioside GD2 is expressed by glioblastoma multiforme (GBM) cells representing a promising target for anti-GD2 immunotherapeutic approaches. The aim of the present study was to investigate anti-tumor efficacy of the chimeric anti-GD2 antibody (Ab) dinutuximab beta against GBM. METHODS: Expression levels of GD2 and complement regulatory proteins (CRP; CD46, CD55 and CD59) on well-known and newly established primary tumor originated GBM cell lines were analyzed by flow cytometry. Ab-dependent cellular (ADCC) and complement-dependent cytotoxicity (CDC) mediated by dinutuximab beta against GBM cells were determined by a non-radioactive calcein-AM-based assay. RESULTS: Analysis of primary GBM cells revealed a heterogeneous GD2 expression that varied between the cell lines analyzed with higher expression levels in the tumor surface compared to the core originated cells. Both GD2-positive and -negative tumor cells were detected in every cell line analyzed. In contrast to CDC, ADCC mediated by dinutuximab beta was observed against the majority of GBM cells. Importantly, CDC-resistant cells showed high expression of the CRP CD46, CD55 and CD59. CONCLUSION: Our present data show anti-tumor effects mediated by dinutuximab beta against GBM cells providing a rationale for a GD2-directed immunotherapy against GBM. Due to high CRP expression, a combining of GD2-targeting with CRP blockade might be a further treatment option for GBM.

3.
Parasitol Res ; 118(7): 2223-2233, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31187225

RESUMO

Blood coagulation in vertebrates is a complex mechanism that involves the precisely coordinated and regulated action of a cascade of factors in order to prevent excessive blood loss upon wounding. Any blood sucking ectoparasite, however, has to circumvent this mechanism to ensure the uptake of an adequate blood meal. Inhibitors of blood coagulation in the saliva are hence widespread among these animals. Thrombin as a key factor of blood coagulation is a prominent target of such inhibitors, and hirudin is probably the best known among the thrombin inhibitors. Hirudin was originally described in the genus Hirudo, but occurs in other leech genera like Hirudinaria and Macrobdella as well. Besides several isoforms of hirudin, a new class of putative leech saliva components, the hirudin-like factors (HLFs), was identified in both genera Hirudo and Hirudinaria. Here, we describe the expression, purification, and functional characterization of three HLFs (HLF5, 6, and 8, respectively) and two additional hirudins (HM3 and HM4) of Hirudinaria manillensis. While HLF6 lacked any inhibitory activity on thrombin, HLF5 as well as HLF8 clearly exhibited anticoagulatory properties. The inhibitory activity of HLF5 and HLF8, however, was much lower compared with both HM3 and HM4 of Hirudinaria manillensis as well as the hirudin variants 1 (HV1) and 2 (HV2) of Hirudo medicinalis. Neither an inhibition of trypsin nor a platelet aggregation was caused by HLF8. Our data indicates the presence of two classes (rather than isoforms) of hirudins in Hirudinaria manillensis with markedly different inhibitory activity on human thrombin.


Assuntos
Antitrombinas/metabolismo , Coagulação Sanguínea/efeitos dos fármacos , Hirudinas/metabolismo , Hirudo medicinalis/metabolismo , Trombina/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Humanos , Proteínas Recombinantes/metabolismo , Tripsina/metabolismo
4.
Eur Heart J ; 40(40): 3318-3332, 2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31004144

RESUMO

AIMS: Heart failure with preserved ejection fraction (HFpEF) and pathological cardiac aging share a complex pathophysiology, including extracellular matrix remodelling (EMR). Protease-activated receptor 2 (PAR2) deficiency is associated with EMR. The roles of PAR1 and PAR2 have not been studied in HFpEF, age-dependent cardiac fibrosis, or diastolic dysfunction (DD). METHODS AND RESULTS: Evaluation of endomyocardial biopsies from patients with HFpEF (n = 14) revealed that a reduced cardiac PAR2 expression was associated with aggravated DD and increased myocardial fibrosis (r = -0.7336, P = 0.0028). In line, 1-year-old PAR2-knockout (PAR2ko) mice suffered from DD with preserved systolic function, associated with an increased age-dependent α-smooth muscle actin expression, collagen deposition (1.7-fold increase, P = 0.0003), lysyl oxidase activity, collagen cross-linking (2.2-fold increase, P = 0.0008), endothelial activation, and inflammation. In the absence of PAR2, the receptor-regulating protein caveolin-1 was down-regulated, contributing to an augmented profibrotic PAR1 and transforming growth factor beta (TGF-ß)-dependent signalling. This enhanced TGF-ß/PAR1 signalling caused N-proteinase (ADAMTS3) and C-proteinase (BMP1)-related increased collagen I production from cardiac fibroblasts (CFs). PAR2 overexpression in PAR2ko CFs reversed these effects. The treatment with the PAR1 antagonist, vorapaxar, reduced cardiac fibrosis by 44% (P = 0.03) and reduced inflammation in a metabolic disease model (apolipoprotein E-ko mice). Patients with HFpEF with upstream PAR inhibition via FXa inhibitors (n = 40) also exhibited reduced circulating markers of fibrosis and DD compared with patients treated with vitamin K antagonists (n = 20). CONCLUSIONS: Protease-activated receptor 2 is an important regulator of profibrotic PAR1 and TGF-ß signalling in the heart. Modulation of the FXa/FIIa-PAR1/PAR2/TGF-ß-axis might be a promising therapeutic approach to reduce HFpEF.

5.
Cancers (Basel) ; 11(4)2019 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-31013620

RESUMO

The link between thrombocytosis and malignancy has been well known for many years and its associations with worse outcomes have been reported mainly for solid tumors. Besides measuring platelet count, it has become popular to assess platelet function in the context of malignant diseases during the last decade. Malignant gliomas differ tremendously from malignancies outside the central nervous system because they virtually never form distant metastases. This review summarizes the current understanding of the platelet-immune cell communication and its potential role in glioma resistance and progression. Particularly, we focus on platelet-derived proinflammatory modulators, such as sphingosine-1-phosphate (S1P). The multifaceted interaction with immune cells puts the platelet into an interesting perspective regarding the recent advances in immunotherapeutic approaches in malignant glioma.

6.
Cardiovasc Res ; 115(13): 1886-1906, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30924864

RESUMO

AIMS: Inflammation is a key driver of atherosclerosis and myocardial infarction (MI), and beyond proteins and microRNAs (miRs), long noncoding RNAs (lncRNAs) have been implicated in inflammation control. To obtain further information on the possible role of lncRNAs in the context of atherosclerosis, we obtained comprehensive transcriptome maps of circulating immune cells (peripheral blood mononuclear cells, PBMCs) of early onset MI patients. One lncRNA significantly suppressed in post-MI patients was further investigated in a murine knockout model. METHODS AND RESULTS: Individual RNA-sequencing (RNA-seq) was conducted on PBMCs from 28 post-MI patients with a history of MI at age ≤50 years and stable disease ≥3 months before study participation, and from 31 healthy individuals without manifest cardiovascular disease or family history of MI as controls. RNA-seq revealed deregulated protein-coding transcripts and lncRNAs in post-MI PBMCs, among which nuclear enriched abundant transcript (NEAT1) was the most highly expressed lncRNA, and the only one significantly suppressed in patients. Multivariate statistical analysis of validation cohorts of 106 post-MI patients and 85 controls indicated that the PBMC NEAT1 levels were influenced (P = 0.001) by post-MI status independent of statin intake, left ventricular ejection fraction, low-density lipoprotein or high-density lipoprotein cholesterol, or age. We investigated NEAT1-/- mice as a model of NEAT1 deficiency to evaluate if NEAT1 depletion may directly and causally alter immune regulation. RNA-seq of NEAT1-/- splenocytes identified disturbed expression and regulation of chemokines/receptors, innate immunity genes, tumour necrosis factor (TNF) and caspases, and increased production of reactive oxygen species (ROS) under baseline conditions. NEAT1-/- spleen displayed anomalous Treg and TH cell differentiation. NEAT1-/- bone marrow-derived macrophages (BMDMs) displayed altered transcriptomes with disturbed chemokine/chemokine receptor expression, increased baseline phagocytosis (P < 0.0001), and attenuated proliferation (P = 0.0013). NEAT1-/- BMDMs responded to LPS with increased (P < 0.0001) ROS production and disturbed phagocytic activity (P = 0.0318). Monocyte-macrophage differentiation was deregulated in NEAT1-/- bone marrow and blood. NEAT1-/- mice displayed aortic wall CD68+ cell infiltration, and there was evidence of myocardial inflammation which could lead to severe and potentially life-threatening structural damage in some of these animals. CONCLUSION: The study indicates distinctive alterations of lncRNA expression in post-MI patient PBMCs. Regarding the monocyte-enriched NEAT1 suppressed in post-MI patients, the data from NEAT1-/- mice identify NEAT1 as a novel lncRNA-type immunoregulator affecting monocyte-macrophage functions and T cell differentiation. NEAT1 is part of a molecular circuit also involving several chemokines and interleukins persistently deregulated post-MI. Individual profiling of this circuit may contribute to identify high-risk patients likely to benefit from immunomodulatory therapies. It also appears reasonable to look for new therapeutic targets within this circuit.

7.
Clin Cancer Res ; 25(9): 2874-2886, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30670496

RESUMO

PURPOSE: Apoptotic dysregulation, redox adaptive mechanisms, and resilience to hypoxia are major causes of glioblastoma (GBM) resistance to therapy. Commonly known as crucial factors in energy metabolism, OCTN2 (SLC22A5) and its substrate L-carnitine (LC) are increasingly recognized as actors in cytoprotection. This study provides a comprehensive expression and survival analysis of the OCTN2/LC system in GBM and clarifies the system's impact on GBM progression. EXPERIMENTAL DESIGN: OCTN2 expression and LC content were measured in 121 resected human GBM specimens and 10 healthy brain samples and analyzed for prognostic significance. Depending on LC administration, the effects of hypoxic, metabolic, and cytotoxic stress on survival and migration of LN18 GBM cells were further studied in vitro. Finally, an orthotopic mouse model was employed to investigate inhibition of the OCTN2/LC system on in vivo GBM growth. RESULTS: Compared with healthy brain, OCTN2 expression was increased in primary and even more so in recurrent GBM on mRNA and protein level. High OCTN2 expression was associated with a poor overall patient survival; the unadjusted HR for death was 2.7 (95% CI, 1.47-4.91; P < 0.001). LC administration to GBM cells increased their tolerance toward cytotoxicity, whereas siRNA-mediated OCTN2 silencing led to a loss of tumor cell viability. In line herewith, OCTN2/LC inhibition by meldonium resulted in reduced tumor growth in an orthotopic GBM mouse model. CONCLUSIONS: Our data indicate a potential role of the OCTN2/LC system in GBM progression and resistance to therapy, and suggests OCTN2 as a prognostic marker in patients with primary GBM.

8.
Cardiovasc Res ; 115(2): 302-314, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30101304

RESUMO

Aims: The immune system is considered a key driver of atherosclerosis, and beyond proteins and microRNAs (miRs), long non-coding RNAs (lncRNAs) are implicated in immune control. We previously described that lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is involved in cardiac innate immunity in a myocarditis model. Here, we investigated the impact of MALAT1 deficiency upon atherosclerosis development. Methods and results: Heterozygous MALAT1-deficient ApoE-/- mice displayed massive immune system dysregulation and atherosclerosis within 2 months even when kept on normal diet. Aortic plaque area (P < 0.05) and aortic root plaque size (P < 0.001) were increased in MALAT1-deficient vs. MALAT1-wildtype ApoE-/- mice. Serum levels of interferon-γ (IFN-γ), tumour necrosis factor (TNF), and interleukin 6 (IL6) were elevated (P < 0.001) in MALAT1-deficient animals. MALAT1-deficient bone marrow-derived macrophages showed enhanced expression of TNF (P = 0.001) and inducible NO synthase (NOS2) (P = 0.002), suppressed MMP9 (P < 0.001), and impaired phagocytic activity (P < 0.001) upon lipopolysaccharide stimulation. RNA-sequencing revealed grossly altered transcriptomes of MALAT1-deficient splenocytes already at baseline, with massive induction of IFN- γ, TNF, NOS2, and granzyme B; CC and CXC chemokines and CCR8; and innate immunity genes interferon-induced protein with tetratricopeptide repeats (IFIT)1/3, interferon-induced transmembrane protein (IFITM)1/3, ISG15. Multiple miRs were up to 45-fold upregulated. Further, selective ablation of the cytosolic part of the MALAT1 system only, the enzymatically MALAT1-derived mascRNA, resulted in massive induction of TNF (P = 0.004) and IL6 (P = 0.028) in macrophages. Northern analysis of post-myocardial infarction patient vs. control peripheral blood mononuclear cells showed reduced (P = 0.005) mascRNA in the patients. CHART-enriched RNA-sequencing reads at the genomic loci of MALAT1 and neighbouring nuclear enriched abundant transcript (NEAT1) documented direct interaction between these lncRNA transcripts. Conclusion: The data suggest a molecular circuit involving the MALAT1-mascRNA system, interactions between MALAT1 and NEAT1, and key immune effector molecules, cumulatively impacting upon the development of atherosclerosis. It appears reasonable to look for therapeutic targets in this circuit and to screen for anomalies in the NEAT1-MALAT1 region in humans, too, as possible novel disease risk factors.


Assuntos
Aorta/metabolismo , Doenças da Aorta/metabolismo , Aterosclerose/metabolismo , Citocinas/sangue , Mediadores da Inflamação/sangue , RNA Longo não Codificante/metabolismo , Animais , Aorta/imunologia , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/imunologia , Doenças da Aorta/patologia , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/patologia , Células Cultivadas , Citocinas/imunologia , Modelos Animais de Doenças , Progressão da Doença , Mediadores da Inflamação/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Placa Aterosclerótica , RNA Longo não Codificante/genética , RNA Longo não Codificante/imunologia , Baço/imunologia , Baço/metabolismo , Fatores de Tempo
9.
Biochem Pharmacol ; 161: 14-25, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30557554

RESUMO

The inositol phosphates, InsP5 and InsP6, have recently been identified as binding partners of fibrinogen, which is critically involved in hemostasis by crosslinking activated platelets at sites of vascular injury. Here, we investigated the putative physiological role of this interaction and found that platelets increase their InsP6 concentration upon stimulation with the PLC-activating agonists thrombin, collagen I and ADP and present a fraction of it at the outer plasma membrane. Cone and plate analysis in whole blood revealed that InsP6 specifically increases platelet aggregate size. This effect is fibrinogen-dependent, since it is inhibited by an antibody that blocks fibrinogen binding to platelets. Furthermore, InsP6 has only an effect on aggregate size of washed platelets when fibrinogen is present, while it has no influence in presence of von Willebrand factor or collagen. By employing blind docking studies we predicted the binding site for InsP6 at the bundle between the γ and ß helical subunit of fibrinogen. Since InsP6 is unable to directly activate platelets and it did not exhibit an effect on thrombin formation or fibrin structure, our data indicate that InsP6 might be a hemostatic agent that is produced by platelets upon stimulation with PLC-activating agonists to promote platelet aggregation by supporting crosslinking of fibrinogen and activated platelets.


Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Ácido Fítico/metabolismo , Ácido Fítico/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Plaquetas/química , Fibrinogênio/metabolismo , Humanos , Ácido Fítico/química , Agregação Plaquetária/fisiologia , Estrutura Secundária de Proteína
10.
Oncotarget ; 9(40): 25860-25876, 2018 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-29899827

RESUMO

Patients with glioblastoma multiforme (GBM) suffer from an increased incidence of vascular thrombotic events. However, key influencing factors of the primary hemostasis have not been characterized in GBM patients to date. Thus, the present study determines the activation level of circulating platelets in GBM patients, in-vitro reactivity to agonist-induced platelet stimulation and the formation of circulating platelet-leucocyte conjugates as well as the plasma levels of the proinflammatory lipid mediator sphingosine-1-phosphate (S1P). The endogenous thrombin potential (ETP) was determined as global marker for hemostasis. The 21 GBM patients and 21 gender and age matched healthy individuals enrolled in this study did not differ in mean total platelet count. Basal surface expression of platelet CD63 determined by flow cytometry was significantly increased in GBM patients compared to controls as was observed for the concentration of soluble P-selectin in the plasma of GBM patients. While the ETP was not affected, the immunomodulatory lipid S1P was significantly decreased in peripheral blood in GBM. Interestingly, monocyte expression of PSGL-1 (CD162) was decreased in GBM patient blood, possibly explaining the rather decreased formation of platelet-monocyte conjugates. Our study reveals an increased CD63 expression and P-selectin expression/ secretion of circulating platelets in GBM patients. In parallel a down-modulated PSGL-1 expression in circulating monocytes and a trend towards a decreased formation of heterotypic platelet-monocyte conjugates in GBM patients was seen. Whether this and the observed decreased plasma level of the immunomodulatory S1P reflects a systemic anti-inflammatory status needs to be addressed in future studies.

11.
Int J Mol Sci ; 19(6)2018 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-29795022

RESUMO

Both signaling by transforming growth factor-ß (TGF-ß) and agonists of the G Protein-coupled receptors proteinase-activated receptor-1 (PAR1) and -2 (PAR2) have been linked to tissue fibrosis and cancer. Intriguingly, TGF-ß and PAR signaling either converge on the regulation of certain matrix genes overexpressed in these pathologies or display mutual regulation of their signaling components, which is mediated in part through sphingosine kinases and sphingosine-1-phosphate and indicative of an intimate signaling crosstalk between the two pathways. In the first part of this review, we summarize the various regulatory interactions that have been discovered so far according to the organ/tissue in which they were described. In the second part, we highlight the types of signaling crosstalk between TGF-ß on the one hand and PAR2/PAR1 on the other hand. Both ligand⁻receptor systems interact at various levels and by several mechanisms including mutual regulation of ligand⁻ligand, ligand⁻receptor, and receptor⁻receptor at the transcriptional, post-transcriptional, and receptor transactivation levels. These mutual interactions between PAR2/PAR1 and TGF-ß signaling components eventually result in feed-forward loops/vicious cycles of matrix deposition and malignant traits that exacerbate fibrosis and oncogenesis, respectively. Given the crucial role of PAR2 and PAR1 in controlling TGF-ß receptor activation, signaling, TGF-ß synthesis and bioactivation, combining PAR inhibitors with TGF-ß blocking agents may turn out to be more efficient than targeting TGF-ß alone in alleviating unwanted TGF-ß-dependent responses but retaining the beneficial ones.


Assuntos
Cirrose Hepática/metabolismo , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Humanos , Rim/metabolismo , Rim/patologia , Pulmão/metabolismo , Pulmão/patologia , Receptor do Fator de Crescimento Transformador beta Tipo I
12.
Nat Med ; 24(5): 667-678, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29662200

RESUMO

Sphingosine-1-phosphate (S1P) signaling influences bone metabolism, but its therapeutic potential in bone disorders has remained unexplored. We show that raising S1P levels in adult mice through conditionally deleting or pharmacologically inhibiting S1P lyase, the sole enzyme responsible for irreversibly degrading S1P, markedly increased bone formation, mass and strength and substantially decreased white adipose tissue. S1P signaling through S1P2 potently stimulated osteoblastogenesis at the expense of adipogenesis by inversely regulating osterix and PPAR-γ, and it simultaneously inhibited osteoclastogenesis by inducing osteoprotegerin through newly discovered p38-GSK3ß-ß-catenin and WNT5A-LRP5 pathways. Accordingly, S1P2-deficient mice were osteopenic and obese. In ovariectomy-induced osteopenia, S1P lyase inhibition was as effective as intermittent parathyroid hormone (iPTH) treatment in increasing bone mass and was superior to iPTH in enhancing bone strength. Furthermore, lyase inhibition in mice successfully corrected severe genetic osteoporosis caused by osteoprotegerin deficiency. Human data from 4,091 participants of the SHIP-Trend population-based study revealed a positive association between serum levels of S1P and bone formation markers, but not resorption markers. Furthermore, serum S1P levels were positively associated with serum calcium , negatively with PTH , and curvilinearly with body mass index. Bone stiffness, as determined through quantitative ultrasound, was inversely related to levels of both S1P and the bone formation marker PINP, suggesting that S1P stimulates osteoanabolic activity to counteract decreasing bone quality. S1P-based drugs should be considered as a promising therapeutic avenue for the treatment of osteoporotic diseases.


Assuntos
Aldeído Liases/antagonistas & inibidores , Anabolizantes/uso terapêutico , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/enzimologia , Terapia de Alvo Molecular , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Aldeído Liases/metabolismo , Anabolizantes/farmacologia , Animais , Reabsorção Óssea/sangue , Reabsorção Óssea/diagnóstico por imagem , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Fêmur/diagnóstico por imagem , Fêmur/patologia , Deleção de Genes , Lisofosfolipídeos/sangue , Camundongos Knockout , Obesidade/sangue , Obesidade/patologia , Tamanho do Órgão , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteoporose/metabolismo , Osteoporose/patologia , Osteoprotegerina/sangue , Osteoprotegerina/metabolismo , PPAR gama/metabolismo , Transdução de Sinais , Fator de Transcrição Sp7/metabolismo , Esfingosina/análogos & derivados , Esfingosina/sangue , Microtomografia por Raio-X
13.
Stem Cells Int ; 2018: 9628289, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29535786

RESUMO

Patients with glioblastoma multiforme (GBM) are at high risk to develop a relapse despite multimodal therapy. Assumedly, glioma stem cells (GSCs) are responsible for treatment resistance of GBM. Identification of specific GSC markers may help to develop targeted therapies. Here, we performed expression analyses of stem cell (ABCG2, CD44, CD95, CD133, ELF4, Nanog, and Nestin) as well as differentiation and microglia markers (GFAP, Iba1, and Sparc) in GBM compared to nonmalignant brain. Furthermore, the role of these proteins for patient survival and their expression in LN18 stem-like neurospheres was analyzed. At mRNA level, ABCG2 and CD95 were reduced, GFAP was unchanged; all other investigated markers were increased in GBM. At protein level, CD44, ELF4, Nanog, Nestin, and Sparc were elevated in GBM, but only CD133 and Nestin were strongly associated with survival time. In addition, ABCG2 and GFAP expression was decreased in LN18 neurospheres whereas CD44, CD95, CD133, ELF4, Nanog, Nestin, and Sparc were upregulated. Altogether only CD133 and Nestin were associated with survival rates. This raises concerns regarding the suitability of the other target structures as prognostic markers, but makes both CD133 and Nestin candidates for GBM therapy. Nevertheless, a search for more specific marker proteins is urgently needed.

14.
Thromb Haemost ; 118(1): 132-142, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29304533

RESUMO

Sphingosine-1-phosphate (S1P) is a potent lipid mediator released from activated platelets by an adenosine triphosphate (ATP)-dependent export mechanism. A candidate transport protein is the multidrug resistance protein 4 (MRP4/ABCC4), an ATP-dependent transporter highly expressed in platelets. Furthermore, several statins are known to affect platelet functions and exhibit antithrombotic properties. This study determines the involvement of MRP4 in the transport of S1P and a possible interference by statins. Transport studies in membrane vesicles of Sf9 cells containing recombinant human MRP4 revealed that MRP4 mediates ATP-dependent transport of fluorescein- and tritium-labelled S1P. Also, ATP-dependent S1P transport in platelet membrane vesicles containing endogenous MRP4 was inhibited by the MRP inhibitor MK571 and the MRP4-selective compound Ceefourin-1. Confocal microscopy using fluorescein-labelled S1P as well as boron-dipyrromethene (BODIPY)-labelled sphingosine indicated association of S1P and MRP4 in human platelets. In MRP4-deficient mice, agonist-induced S1P secretion was reduced compared with matched wild-type C57Bl/6 mice and platelet S1P concentrations were lower. Fluvastatin and rosuvastatin interfered with MRP4 function inhibiting ATP-dependent cGMP (cyclic guanosine monophosphate) uptake into MRP4-containing vesicles, inhibited MRP4-mediated S1P transport in vitro and significantly attenuated endogenous S1P release from agonist-activated platelet ex vivo. These data suggest that release of S1P from platelets depends on MRP4 and statins can interfere with this transport process. Potentially, this may be relevant for the pleiotropic anti-inflammatory effects of statins and their effect on modulating atherothrombosis.


Assuntos
Plaquetas/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Lisofosfolipídeos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Esfingosina/análogos & derivados , Animais , Transporte Biológico , Compostos de Boro , Cromatografia Líquida , Fluvastatina/farmacologia , Voluntários Saudáveis , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Ativação Plaquetária/efeitos dos fármacos , Testes de Função Plaquetária , Proteínas Recombinantes/metabolismo , Rosuvastatina Cálcica/farmacologia , Células Sf9 , Esfingosina/metabolismo , Espectrometria de Massas em Tandem
15.
Pharmacology ; 101(1-2): 72-75, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29131082

RESUMO

BACKGROUND: Aspirin plays a crucial role in the prevention of cardiovascular diseases. We previously described that aspirin has effects beyond inhibition of platelet aggregation, as it inhibited thrombin-mediated release of sphingosine-1-phosphate (S1P) from human platelets. S1P is a bioactive lipid with important functions on inflammation and apoptosis. In endothelial cells (EC), S1P is a key regulator of cell migration. In this study, we aimed to analyze the effects of aspirin on platelet-induced EC migration. METHODS: Human umbilical EC migration was measured by Boyden chamber assay. EC migration was induced by platelet supernatants of thrombin receptor-activating peptide-1 (AP1) stimulated platelets. To investigate the S1P receptor subtype that promotes EC migration, specific inhibitors of S1P receptor subtypes were applied. RESULTS: S1P induced EC migration in a concentration-dependent manner. EC migration induced by AP1-stimulated platelet supernatants was reduced by aspirin. S1P1 receptor inhibition almost completely abolished EC migration induced by activated platelets. The inhibition of S1P2 or S1P3 receptor had no effect. CONCLUSION: Aspirin inhibits EC migration induced by activated platelets that is in part due to S1P and mediated by the endothelial S1P1 receptor. The clinical significance of this novel mechanism of aspirin action has to be investigated in future studies.


Assuntos
Aspirina/farmacologia , Plaquetas/fisiologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Lisofosfolipídeos/fisiologia , Receptores de Lisoesfingolipídeo/fisiologia , Esfingosina/análogos & derivados , Anilidas/farmacologia , Movimento Celular , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Organofosfonatos/farmacologia , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Esfingosina/fisiologia
16.
Int J Mol Sci ; 18(12)2017 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-29261154

RESUMO

BACKGROUND: Recently, the expression of proteinase-activated receptor 2 (PAR2) has been shown to be essential for activin receptor-like kinase 5 (ALK5)/SMAD-mediated signaling and cell migration by transforming growth factor (TGF)-ß1. However, it is not known whether activation of non-SMAD TGF-ß signaling (e.g., RAS-RAF-MEK-extracellular signal-regulated kinase (ERK) signaling) is required for cell migration and whether it is also dependent on PAR2. METHODS: RNA interference was used to deplete cells of PAR2, followed by xCELLigence technology to measure cell migration, phospho-immunoblotting to assess ERK1/2 activation, and co-immunoprecipitation to detect a PAR2-ALK5 physical interaction. RESULTS: Inhibition of ERK signaling with the MEK inhibitor U0126 blunted the ability of TGF-ß1 to induce migration in pancreatic cancer Panc1 cells. ERK activation in response to PAR2 agonistic peptide (PAR2-AP) was strong and rapid, while it was moderate and delayed in response to TGF-ß1. Basal and TGF-ß1-dependent ERK, but not SMAD activation, was blocked by U0126 in Panc1 and other cell types indicating that ERK activation is downstream or independent of SMAD signaling. Moreover, cellular depletion of PAR2 in HaCaT cells strongly inhibited TGF-ß1-induced ERK activation, while the biased PAR2 agonist GB88 at 10 and 100 µM potentiated TGF-ß1-dependent ERK activation and cell migration. Finally, we provide evidence for a physical interaction between PAR2 and ALK5. Our data show that both PAR2-AP- and TGF-ß1-induced cell migration depend on ERK activation, that PAR2 expression is crucial for TGF-ß1-induced ERK activation, and that the functional cooperation of PAR2 and TGF-ß1 involves a physical interaction between PAR2 and ALK5.


Assuntos
Movimento Celular , Sistema de Sinalização das MAP Quinases , Receptores Acoplados a Proteínas-G/metabolismo , Linhagem Celular Tumoral , Humanos , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
17.
Int J Mol Sci ; 18(11)2017 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-29165389

RESUMO

The G protein-coupled receptor proteinase-activated receptor 2 (PAR2) has been implicated in various aspects of cellular physiology including inflammation, obesity and cancer. In cancer, it usually acts as a driver of cancer progression in various tumor types by promoting invasion and metastasis in response to activation by serine proteinases. Recently, we discovered another mode through which PAR2 may enhance tumorigenesis: crosstalk with transforming growth factor-ß (TGF-ß) signaling to promote TGF-ß1-induced cell migration/invasion and invasion-associated gene expression in ductal pancreatic adenocarcinoma (PDAC) cells. In this chapter, we review what is known about the cellular TGF-ß responses and signaling pathways affected by PAR2 expression, the signaling activities of PAR2 required for promoting TGF-ß signaling, and the potential molecular mechanism(s) that underlie(s) the TGF-ß signaling-promoting effect. Since PAR2 is activated through various serine proteinases and biased agonists, it may couple TGF-ß signaling to a diverse range of other physiological processes that may or may not predispose cells to cancer development such as local inflammation, systemic coagulation and pathogen infection.


Assuntos
Neoplasias/metabolismo , Neoplasias/patologia , Receptor PAR-2/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Transformação Celular Neoplásica/metabolismo , Progressão da Doença , Humanos , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor PAR-2/química , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo
18.
Int J Mol Sci ; 18(11)2017 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-29149079

RESUMO

The multifunctional sphingosine-1-phosphate (S1P) is a lipid signaling molecule and central regulator in the development of several cancer types. In recent years, intriguing information has become available regarding the role of S1P in the progression of Glioblastoma multiforme (GBM), the most aggressive and common brain tumor in adults. S1P modulates numerous cellular processes in GBM, such as oncogenesis, proliferation and survival, invasion, migration, metastasis and stem cell behavior. These processes are regulated via a family of five G-protein-coupled S1P receptors (S1PR1-5) and may involve mainly unknown intracellular targets. Distinct expression patterns and multiple intracellular signaling pathways of each S1PR subtype enable S1P to exert its pleiotropic cellular actions. Several studies have demonstrated alterations in S1P levels, the involvement of S1PRs and S1P metabolizing enzymes in GBM pathophysiology. While the tumorigenic actions of S1P involve the activation of several kinases and transcription factors, the specific G-protein (Gi, Gq, and G12/13)-coupled signaling pathways and downstream mediated effects in GBM remain to be elucidated in detail. This review summarizes the recent findings concerning the role of S1P and its receptors in GBM. We further highlight the current insights into the signaling pathways considered fundamental for regulating the cellular processes in GMB and ultimately patient prognosis.


Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Lisofosfolipídeos/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/análogos & derivados , Adulto , Movimento Celular , Progressão da Doença , Proteínas de Ligação ao GTP/metabolismo , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Prognóstico , Esfingosina/metabolismo
19.
Thromb Haemost ; 117(11): 2013-2025, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-29044290

RESUMO

Thrombin triggers activation of platelets through protease-activated receptor 1 (PAR-1) and PAR-4. Both receptors are widely expressed and exert multiple platelet-independent functions. PAR signalling contributes to healing responses after injury, by promoting cytokine activity and cellular growth and mobility. Uncontrolled PAR activation, however, can prevent timely resolution of inflammation, enhance thrombogenic endothelial function and drive adverse remodelling. The specific role of PAR-4 in thromboinflammatory vascular disease has been largely underestimated, given the relatively limited expression of PAR-4 in non-platelet cells under healthy conditions. However, unlike PAR-1, PAR-4 expression adapts dynamically to numerous stimuli associated with thromboinflammation, including thrombin, angiotensin II, sphingosine-1-phosphate (S1P), high glucose and redox stress, suggesting expression is switched on 'at need'. Prostacyclin negatively regulates PAR-4 expression at the post-transcriptional level, which may serve to fine-tune thrombin responses and limit these to the injury site. PAR-4 elicits inflammatory, mitogenic and proliferative actions not only in response to thrombin but also to numerous other inflammatory proteases, and can cross-talk with other receptor systems such as S1P and adenosine receptors. Accordingly, PAR-4 has emerged as a candidate player in vessel disease and cardiac post-infarction remodelling. Currently, PAR-4 is a particularly promising target for safer anti-thrombotic therapies. Recent studies with the PAR-4 antagonist BMS-986120 lend support to the concept that selective antagonism of PAR-4 may offer both an effective and safe anti-thrombotic therapy in the acute thrombotic setting as well as an anti-inflammatory strategy to prevent long-term progressive atherosclerotic disease in high-risk cardiovascular patients.


Assuntos
Plaquetas/metabolismo , Células Endoteliais/metabolismo , Mediadores da Inflamação/metabolismo , Receptores de Trombina/metabolismo , Transdução de Sinais , Trombose/metabolismo , Vasculite/metabolismo , Animais , Anti-Inflamatórios/uso terapêutico , Plaquetas/efeitos dos fármacos , Plaquetas/imunologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Fibrinolíticos/uso terapêutico , Humanos , Mediadores da Inflamação/antagonistas & inibidores , Ligantes , Ativação Plaquetária , Receptores de Trombina/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Trombose/sangue , Trombose/tratamento farmacológico , Trombose/imunologia , Remodelação Vascular , Vasculite/sangue , Vasculite/tratamento farmacológico , Vasculite/imunologia
20.
Mol Pharmacol ; 92(5): 519-532, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28842394

RESUMO

Transforming growth factor-ß (TGF-ß), serine proteinases such as trypsin, and proteinase-activated receptor 2 (PAR2) promote tumor development by stimulating invasion and metastasis. Previously, we found that in cancer cells derived from pancreatic ductal adenocarcinoma (PDAC) PAR2 protein is necessary for TGF-ß1-dependent cell motility. Here, we show in the same cells that, conversely, the type I TGF-ß receptor activin receptor-like kinase 5 is dispensable for trypsin and PAR2 activating peptide (PAR2-AP)-induced migration. To reveal whether Gq-calcium signaling is a prerequisite for PAR2 to enhance TGF-ß signaling, we investigated the effects of PAR2-APs, PAR2 mutation and PAR2 inhibitors on TGF-ß1-induced migration, reporter gene activity, and Smad activation. Stimulation of cells with PAR2-AP alone failed to enhance basal or TGF-ß1-induced C-terminal phosphorylation of Smad3, Smad-dependent activity of a luciferase reporter gene, and cell migration. Consistently, in complementary loss of function studies, abrogation of the PAR2-Gq-calcium signaling arm failed to suppress TGF-ß1-induced cell migration, reporter gene activity, and Smad3 activation. Together, our findings suggest that the calcium-regulating motif is not required for PAR2 to synergize with TGF-ß1 to promote cell motility. Additional experiments in PDAC cells revealed that PAR2 and TGF-ß1 synergy may involve TGF-ß1 induction of enzymes that cause autocrine cleavage/activation of PAR2, possibly through a biased signaling function. Our results suggest that although reducing PAR2 protein expression may potentially block TGF-ß's prooncogenic function, inhibiting PAR2-Gq-calcium signaling alone would not be sufficient to achieve this effect.


Assuntos
Sinalização do Cálcio/fisiologia , Movimento Celular/fisiologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Células HEK293 , Humanos , Oligopeptídeos/farmacologia , Receptor do Fator de Crescimento Transformador beta Tipo I
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