RESUMO
Atrial fibrillation (AF) is the most common arrhythmia, and atrial fibrosis is a pathological hallmark of structural remodeling in AF. Prostaglandin I2 (PGI2) can prevent the process of fibrosis in various tissues via cell surface Prostaglandin I2 receptor (IP). However, the role of PGI2 in AF and atrial fibrosis remains unclear. The present study aimed to clarify the role of PGI2 in angiotensin II (Ang II)-induced AF and the underlying molecular mechanism. PGI2 content was decreased in both plasma and atrial tissue from patients with AF and mice treated with Ang II. Treatment with the PGI2 analog, iloprost, reduced Ang II-induced AF and atrial fibrosis. Iloprost prevented Ang II-induced atrial fibroblast collagen synthesis and differentiation. RNA-sequencing analysis revealed that iloprost significantly attenuated transcriptome changes in Ang II-treated atrial fibroblasts, especially mitogen-activated protein kinase (MAPK)-regulated genes. We demonstrated that iloprost elevated cAMP levels and then activated protein kinase A, resulting in a suppression of extracellular signal-regulated kinase1/2 and P38 activation, and ultimately inhibiting MAPK-dependent interleukin-6 transcription. In contrast, cardiac fibroblast-specific IP-knockdown mice had increased Ang II-induced AF inducibility and aggravated atrial fibrosis. Together, our study suggests that PGI2/IP system protects against atrial fibrosis and that PGI2 is a therapeutic target for treating AF.The prospectively registered trial was approved by the Chinese Clinical Trial Registry. The trial registration number is ChiCTR2200056733. Data of registration was 2022/02/12.
Assuntos
Angiotensina II , Fibrilação Atrial , Remodelamento Atrial , Epoprostenol , Camundongos Endogâmicos C57BL , Transdução de Sinais , Animais , Fibrilação Atrial/metabolismo , Fibrilação Atrial/patologia , Fibrilação Atrial/induzido quimicamente , Fibrilação Atrial/prevenção & controle , Camundongos , Humanos , Masculino , Transdução de Sinais/efeitos dos fármacos , Remodelamento Atrial/efeitos dos fármacos , Epoprostenol/metabolismo , Fibrose , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Átrios do Coração/efeitos dos fármacos , Iloprosta/farmacologia , Receptores de Epoprostenol/metabolismo , Receptores de Epoprostenol/genética , FemininoRESUMO
It has been reported that, in the spontaneously hypertensive rat (SHR) model of hypertension, different components of the G-protein/adenylate cyclase (AC)/Calcium-activated potassium channel of high conductance (BK) channel signaling pathway are altered differently. In the upstream part of the pathway (G-protein/AC), a comparatively low efficacy has been established, whereas downstream BK currents seem to be increased. Thus, the overall performance of this signaling pathway in SHR is elusive. For a better understanding, we focused on one aspect, the direct targeting of the BK channel by the G-protein/AC pathway and tested the hypothesis that the comparatively low AC pathway efficacy in SHR results in a reduced agonist-induced stimulation of BK currents. This hypothesis was investigated using freshly isolated smooth muscle cells from WKY and SHR rat tail artery and the patch-clamp technique. It was observed that: (1) single BK channels have similar current-voltage relationships, voltage-dependence and calcium sensitivity; (2) BK currents in cells with a strong buffering of the BK channel activator calcium have similar current-voltage relationships; (3) the iloprost-induced concentration-dependent increase of the BK current is larger in WKY compared to SHR; (4) the effects of activators of the PKA pathway, the catalytic subunit of PKA and the potent and selective cAMP-analogue Sp-5,6-DCl-cBIMPS on BK currents are similar. Thus, our data suggest that the lower iloprost-induced stimulation of the BK current in freshly isolated rat tail artery smooth muscle cells from SHR compared with WKY is due to the lower efficacy of upstream elements of the G-Protein/AC/BK channel pathway.
Assuntos
Cálcio , Hipertensão , Iloprosta , Canais de Potássio Ativados por Cálcio de Condutância Alta , Músculo Liso Vascular , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Vasodilatadores , Animais , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/citologia , Ratos , Cálcio/metabolismo , Iloprosta/farmacologia , Hipertensão/metabolismo , Hipertensão/tratamento farmacológico , Vasodilatadores/farmacologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Masculino , Artérias/efeitos dos fármacos , Artérias/metabolismo , Cauda/irrigação sanguínea , Transdução de Sinais/efeitos dos fármacosRESUMO
Thrombospondin-1 (TSP-1) is released by platelets upon activation and can increase platelet activation, but its role in hemostasis in vivo is unclear. We show that TSP-1 is a critical mediator of hemostasis that promotes platelet activation by modulating inhibitory cyclic adenosine monophosphate (cAMP) signaling. Genetic deletion of TSP-1 did not affect platelet activation in vitro, but in vivo models of hemostasis and thrombosis showed that TSP-1-deficient mice had prolonged bleeding, defective thrombosis, and increased sensitivity to the prostacyclin mimetic iloprost. Adoptive transfer of wild-type (WT) but not TSP-1-/- platelets ameliorated the thrombotic phenotype, suggesting a key role for platelet-derived TSP-1. In functional assays, TSP-1-deficient platelets showed an increased sensitivity to cAMP signaling, inhibition of platelet aggregation, and arrest under flow by prostacyclin (PGI2). Plasma swap experiments showed that plasma TSP-1 did not correct PGI2 hypersensitivity in TSP-1-/- platelets. By contrast, incubation of TSP-1-/- platelets with releasates from WT platelets or purified TSP-1, but not releasates from TSP-1-/- platelets, reduced the inhibitory effects of PGI2. Activation of WT platelets resulted in diminished cAMP accumulation and downstream signaling, which was associated with increased activity of the cAMP hydrolyzing enzyme phosphodiesterase 3A (PDE3A). PDE3A activity and cAMP accumulation were unaffected in platelets from TSP-1-/- mice. Platelets deficient in CD36, a TSP-1 receptor, showed increased sensitivity to PGI2/cAMP signaling and diminished PDE3A activity, which was unaffected by platelet-derived or purified TSP-1. This scenario suggests that the release of TSP-1 regulates hemostasis in vivo through modulation of platelet cAMP signaling at sites of vascular injury.
Assuntos
Plaquetas/fisiologia , AMP Cíclico/fisiologia , Transtornos Hemorrágicos/genética , Hemostasia/fisiologia , Trombospondina 1/fisiologia , Animais , Tempo de Sangramento , Plaquetas/efeitos dos fármacos , Antígenos CD36/deficiência , Antígenos CD36/fisiologia , Células Cultivadas , Cloretos/toxicidade , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Grânulos Citoplasmáticos/metabolismo , Epoprostenol/fisiologia , Compostos Férricos/toxicidade , Humanos , Iloprosta/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Transfusão de Plaquetas , Sistemas do Segundo Mensageiro/fisiologia , Trombose/induzido quimicamente , Trombose/prevenção & controle , Trombospondina 1/deficiência , Trombospondina 1/farmacologiaRESUMO
OBJECTIVE: Iloprost (ILO) is recommended for the treatment of systemic sclerosis (SSc) microangiopathy, but there is no common consensus on its optimal dosage. The aim of this study is to evaluate the kinetics of response to ILO administered in a daily outpatient scheme in SSc subjects using laser speckle contrast analysis (LASCA). METHOD: Adult SSc patients in stable therapy with ILO administered for 6 h for 2 consecutive days every 4 weeks were enrolled. Peripheral finger perfusion was assessed by LASCA. Each patient underwent five LASCA evaluations: before and after each day of ILO (D1pre, D1post, D2pre, and D2post) and after 4 weeks (D30). RESULTS: Twenty-seven SSc patients (77.8% female, mean age 61.5 years) were enrolled. LASCA showed an increase in perfusion at the end of each ILO course, but on the second day (both D1pre vs D2pre and D2pre vs D2post) the increase was no longer significant in half of the fingers. Moreover, compared to D1post, at the beginning of the second ILO day most of the fingers had already shown a significant reduction in perfusion. After 1 month, there were no statistically significant differences between the perfusion values of D1pre and D30. CONCLUSION: This LASCA study highlights the transience of the vasoactive effect of ILO, with a perfusion benefit that is completely lost after 1 month. The brevity of the perfusion effect of ILO and the use of LASCA are elements to consider in the design of future SSc trials to determine the optimal ILO dosage.
Assuntos
Iloprosta , Escleroderma Sistêmico , Adulto , Humanos , Feminino , Pessoa de Meia-Idade , Masculino , Iloprosta/farmacologia , Iloprosta/uso terapêutico , Dedos , Capilares , Escleroderma Sistêmico/tratamento farmacológico , LasersRESUMO
Inhalational prostacyclins act as strong vasodilators, potentially improving oxygenation by reducing shunt fraction and ventilation-perfusion mismatch. As prostacyclin receptors are known to be present on human erythrocytes, possible direct effects on hemoglobin oxygen transport were further explored by examining the sole in vitro influence of prostacyclins on hemoglobin oxygen (Hb-O2) affinity. Venous blood samples from 20 healthy volunteers were exposed in vitro to supramaximal doses of epoprostenol, iloprost, and compared with control. By high-throughput measurements, hemoglobin oxygen dissociation curves (ODCs) were derived. Hb-O2 affinity, expressed by P50 and Hill coefficient, was determined and analyzed for three subgroups: males (n = 10), females not taking oral contraceptives (n = 4), and females taking oral contraceptives (n = 6). Epoprostenol significantly decreased P50 in all (males, females without contraceptives, and females taking oral contraceptives) [27.5 (26.4-28.6) mmHg (control) vs. 24.2 (22.7-25.3) mmHg; P < 0.001. median (interquartile range, IQR)] thereby increasing Hb-O2 affinity. Inversely, iloprost only showed significant effects in females taking oral contraceptives where P50 was markedly increased and therefore Hb-O2 affinity decreased [28.4 (27.9-28.9) mmHg (control) vs. 34.4 (32.2-36.0) mmHg; P < 0.001]. Prostacyclin-receptor stimulation and subsequent cAMP-mediated ATP release from erythrocytes are discussed as a possible underlying mechanism for the effect of epoprostenol on Hb-O2 affinity. The reason for the sex hormone-modified iloprost effect remains unclear. Being aware of potentially differing effects on Hb-O2 affinity might help select the right prostacyclin (epoprostenol vs. iloprost) depending on the patient and the underlying disease (e.g., acute respiratory distress syndrome vs. peripheral arterial disease).
Assuntos
Epoprostenol , Iloprosta , Anticoncepcionais Orais , Epoprostenol/farmacologia , Feminino , Hemoglobinas , Humanos , Iloprosta/farmacologia , Masculino , Oxigênio , Prostaglandinas IRESUMO
Hematopoietic stem cells (HSCs) with superior reconstitution potential are reported to be enriched in the endosteal compared to central bone marrow (BM) region. To investigate whether specific factors at the endosteum may contribute to HSC potency, we screened for candidate HSC niche factors enriched in the endosteal compared to central BM regions. Together with key known HSC supporting factors Kitl and Cxcl12, we report that prostacyclin/prostaglandin I2 (PGI2 ) synthase (Ptgis) was one of the most highly enriched mRNAs (>10-fold) in endosteal compared to central BM. As PGI2 signals through receptors distinct from prostaglandin E2 (PGE2 ), we investigated functional roles for PGI2 at the endosteal niche using therapeutic PGI2 analogs, iloprost, and cicaprost. We found PGI2 analogs strongly reduced HSC differentiation in vitro. Ex vivo iloprost pulse treatment also significantly boosted long-term competitive repopulation (LT-CR) potential of HSCs upon transplantation. This was associated with increased tyrosine-phosphorylation of transducer and activator of transcription-3 (STAT3) signaling in HSCs but not altered cell cycling. In vivo, iloprost administration protected BM HSC potential from radiation or granulocyte colony-stimulating factor-induced exhaustion, and restored HSC homing potential with increased Kitl and Cxcl12 transcription in the BM. In conclusion, we propose that PGI2 is a novel HSC regulator enriched in the endosteum that promotes HSC regenerative potential following stress.
Assuntos
Medula Óssea , Epoprostenol , Epoprostenol/farmacologia , Células-Tronco Hematopoéticas , Iloprosta/farmacologia , Nicho de Células-Tronco/fisiologiaRESUMO
BACKGROUND: Pulmonary arterial hypertension (PAH) is a severe, life-threatening disorder despite the availability of specific drug therapy. A lack of endogenous prostacyclin secondary to downregulation of prostacyclin synthase in PAH may contribute to vascular pathologies. Therefore, prostacyclin and its analogs including inhaled iloprost may decrease pulmonary arterial pressure and ventricular pressure. METHODS: Here, we studied that acute effects of iloprost used in pulmonary vasoreactivity testing on the intracardiac conduction system in patients with PAH. A total of 35 (15 idiopathic PAH, 20 congenital heart disease) patients with PAH were included in this prospective study. Patients were divided into two groups: 22 patients with negative pulmonary vasoreactivity in group 1 and 13 with positive pulmonary vasoreactivity in group 2. Electrophysiological parameters including basic cycle length, atrium-His (AH) interval, His-ventricle (HV) interval, PR interval, QT interval, QRS duration, Wenckebach period, and sinus node recovery time (SNRT) were evaluated before and after pulmonary vasoreactivity testing in both groups. RESULTS: The AH interval (81 [74-93]; 80 [65.5-88], pâ¯= 0.019) and SNRT (907.7⯱ 263.4; 854.0⯱ 288.04, pâ¯= 0.027) was significantly decreased after pulmonary vasoreactivity testing. Mean right atrium pressure was found to be correlated with baseline AH (râ¯= 0.371, pâ¯= 0.031) and SNRT (râ¯= 0.353, pâ¯= 0.037). CONCLUSION: Inhaled iloprost can improve cardiovascular performance in the presence of PAH, primarily through a reduction in right ventricular afterload and interventricular pressure. Decreased pressure on the interventricular septum and ventricles leads to conduction system normalization including of the AH interval and SNRT due to resolution of inflammation and edema.
Assuntos
Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Administração por Inalação , Humanos , Hipertensão Pulmonar/complicações , Iloprosta/farmacologia , Iloprosta/uso terapêutico , Estudos Prospectivos , VasodilatadoresRESUMO
The prostacyclin analogue iloprost is used to treat vascular alterations and digital ulcers, the early derangements manifesting in systemic sclerosis (SSc), an autoimmune disease leading to skin and organ fibrosis. Bioindicator(s) of SSc onset and progress are still lacking and the therapeutic approach remains a challenge. The T helper 1 (Th1) chemokine interferon (IFN)γ-induced protein 10 (IP-10/CXCL10) associates with disease progression and worse prognosis. Endothelial cells and fibroblasts, under Th1-dominance, release CXCL10, further enhancing SSc's detrimental status. We analyzed the effect of iloprost on CXCL10 in endothelial cells, dermal fibroblasts, and in the serum of SSc patients. Human endothelial cells and dermal fibroblasts activated with IFNγ/Tumor Necrosis Factor (TNF)α, with/without iloprost, were investigated for CXCL10 secretion/expression and for intracellular signaling cascade underlying chemokine release (Signal Transducer and Activator of Transcription 1, STAT1; Nuclear Factor kappa-light-chain-enhancer of activated B cells, NF-kB; c-Jun NH2-terminal kinase, JNK: Phosphatidyl-Inositol 3-kinase (PI3K)/protein kinase B, AKT; Extracellular signal-Regulated Kinase 1/2, ERK1/2). CXCL10 was quantified in sera from 25 patients taking iloprost, satisfying the American College of Rheumatology (ACR)/European Alliance of Associations for Rheumatology (EULAR) 2013 classification criteria for SSc, and in sera from 20 SSc sex/age-matched subjects without therapy, previously collected. In human endothelial cells and fibroblasts, iloprost targeted CXCL10, almost preventing IFNγ/TNFα-dependent cascade activation in endothelial cells. In SSc subjects taking iloprost, serum CXCL10 was lower. These in vitro and in vivo data suggest a potential role of iloprost to limit CXCL10 at local vascular/dermal and systemic levels in SSc and warrant further translational research aimed to ameliorate SSc understanding/management.
Assuntos
Iloprosta , Escleroderma Sistêmico , Quimiocina CXCL10/metabolismo , Quimiocinas/metabolismo , Células Endoteliais/metabolismo , Epoprostenol/metabolismo , Humanos , Iloprosta/metabolismo , Iloprosta/farmacologia , Iloprosta/uso terapêutico , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Endothelial cell injury is an early event in systemic sclerosis (SSc) pathogenesis and several studies indicate oxidative stress as the trigger of SSc-associated vasculopathy. Here, we show that circulating factors present in sera of SSc patients increased reactive oxygen species (ROS) production and collagen synthesis in human pulmonary microvascular endothelial cells (HPMECs). In addition, the possibility that iloprost, a drug commonly used in SSc therapy, might modulate the above-mentioned biological phenomena has been also investigated. In this regard, as compared to sera of SSc patients, sera of iloprost-treated SSc patients failed to increased ROS levels and collagen synthesis in HPMEC, suggesting a potential antioxidant mechanism of this drug.
Assuntos
Colágeno/biossíntese , Células Endoteliais/efeitos dos fármacos , Iloprosta/farmacologia , Microvasos/citologia , Estresse Oxidativo/efeitos dos fármacos , Escleroderma Sistêmico/sangue , Soro/metabolismo , Adulto , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Feminino , Humanos , Masculino , Espécies Reativas de Oxigênio/metabolismoRESUMO
G protein-coupled receptors (GPCRs) are the largest class of transmembrane receptors and serve as signal mediators to transduce information from extracellular signals such as neurotransmitters, hormones, or drugs to cellular responses. They are exposed to the strong electrical field of the plasma membrane. In the last decade voltage modulation of ligand-induced GPCR activity has been reported for several GPCRs. Using Foerster resonance energy transfer-based biosensors in patch clamp experiments, we discovered a robust voltage dependence of the thromboxane receptor (TP receptor) on the receptor level as well as on downstream signaling. TP receptor activity doubled upon depolarization from -90 to +60 mV in the presence of U46619, a stable analog of prostaglandin H2 Half-maximal effective potential (V0.5) determined for TP receptor was -46 mV, which is within the physiologic range. We identified that depolarization affected the agonist affinity for the TP receptor. Depolarization enhanced responses of several structural analogs of U46619 with modifications to a similar extent all around the molecule, indicating that voltage modulates the general conformation of TP receptor. By means of site direct mutagenesis, we identified TP receptor R2957.40, which showed alteration of voltage sensitivity of TP receptor upon mutation. Voltage sensitivity was not limited to TP receptor because prostaglandin F receptor activated with U46619 and prostaglandin E2 receptor subtype 3 activated with iloprost showed a similar reaction to depolarization as TP receptor. However, prostacyclin receptor activated with iloprost showed no detectable voltage dependence. SIGNIFICANCE STATEMENT: Prostanoids mediate many of their physiological effects via transmembrane receptors expressed in the plasma membrane of excitable cells. We found that agonist-mediated activation of prostaglandin F receptors and prostaglandin E2 receptors as well as thromboxane receptors are activated upon depolarization, whereas prostacyclin receptors are not. The voltage-induced modulation of thromboxane receptor activity was observed on the level of receptor conformation and downstream signaling. The range of voltage dependence was restricted by R2957.40 in the agonist-binding pocket.
Assuntos
Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Receptores de Prostaglandina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Arginina/genética , Sítios de Ligação/genética , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Iloprosta/farmacologia , Ligantes , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Receptores de Epoprostenol/metabolismo , Receptores de Prostaglandina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Adenosine diphosphate (ADP) enhances platelet activation by virtually any other stimulant to complete aggregation. It binds specifically to the G-protein-coupled membrane receptors P2Y1 and P2Y12, stimulating intracellular signaling cascades, leading to integrin αIIbß3 activation, a process antagonized by endothelial prostacyclin. P2Y12 inhibitors are among the most successful antiplatelet drugs, however, show remarkable variability in efficacy. We reasoned whether a more detailed molecular understanding of ADP-induced protein phosphorylation could identify (1) critical hubs in platelet signaling toward aggregation and (2) novel molecular targets for antiplatelet treatment strategies. We applied quantitative temporal phosphoproteomics to study ADP-mediated signaling at unprecedented molecular resolution. Furthermore, to mimic the antagonistic efficacy of endothelial-derived prostacyclin, we determined how Iloprost reverses ADP-mediated signaling events. We provide temporal profiles of 4797 phosphopeptides, 608 of which showed significant regulation. Regulated proteins are implicated in well-known activating functions such as degranulation and cytoskeletal reorganization, but also in less well-understood pathways, involving ubiquitin ligases and GTPase exchange factors/GTPase-activating proteins (GEF/GAP). Our data demonstrate that ADP-triggered phosphorylation occurs predominantly within the first 10 seconds, with many short rather than sustained changes. For a set of phosphorylation sites (eg, PDE3ASer312, CALDAG-GEFISer587, ENSASer109), we demonstrate an inverse regulation by ADP and Iloprost, suggesting that these are central modulators of platelet homeostasis. This study demonstrates an extensive spectrum of human platelet protein phosphorylation in response to ADP and Iloprost, which inversely overlap and represent major activating and inhibitory pathways.
Assuntos
Difosfato de Adenosina/metabolismo , Plaquetas/metabolismo , Ativação Plaquetária/fisiologia , Transdução de Sinais/fisiologia , Plaquetas/efeitos dos fármacos , Western Blotting , Humanos , Iloprosta/farmacologia , Fosforilação , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Proteômica/métodosRESUMO
BACKGROUND: The glycoprotein (GP) Ib-IX-V complex is a unique platelet plasma membrane receptor, which is essential for platelet adhesion and thrombus formation. GPIbα, part of the GPIb-IX-V complex, has several physiological ligands such as von Willebrand factor (vWF), thrombospondin and distinct coagulation factors, which trigger platelet activation. Despite having an important role, intracellular GPIb-IX-V signaling and its regulation by other pathways are not well defined. Our aim was to establish the intracellular signaling response of selective GPIbα activation in human platelets, in particular the role of the tyrosine kinase Syk and its regulation by cAMP/PKA and cGMP/PKG pathways, respectively. We addressed this using echicetin beads (EB), which selectively bind to GPIbα and induce platelet aggregation. METHODS: Purified echicetin from snake Echis carinatus venom was validated by mass spectrometry. Washed human platelets were incubated with EB, in the presence or absence of echicetin monomers (EM), Src family kinase (SFK) inhibitors, Syk inhibitors and the cAMP- and cGMP-elevating agents iloprost and riociguat, respectively. Platelet aggregation was analyzed by light transmission aggregometry, protein phosphorylation by immunoblotting. Intracellular messengers inositolmonophosphate (InsP1) and Ca2+i were measured by ELISA and Fluo-3 AM/FACS, respectively. RESULTS: EB-induced platelet aggregation was dependent on integrin αIIbß3 and secondary mediators ADP and TxA2, and was antagonized by EM. EB stimulated Syk tyrosine phosphorylation at Y352, which was SFK-dependent and Syk-independent, whereas Y525/526 phosphorylation was SFK-dependent and partially Syk-dependent. Furthermore, phosphorylation of both Syk Y352 and Y525/526 was completely integrin αIIbß3-independent but, in the case of Y525/526, was partially ADP/TxA2-dependent. Syk activation, observed as Y352/ Y525/Y526 phosphorylation, led to the phosphorylation of direct substrates (LAT Y191, PLCγ2 Y759) and additional targets (Akt S473). PKA/PKG pathways inhibited EB-induced platelet aggregation and Akt phosphorylation but, surprisingly, enhanced Syk and LAT/PLCγ2 tyrosine phosphorylation. A similar PKA/PKG effect was confirmed with convulxin-/GPVI-stimulated platelets. EB-induced InsP1 accumulation/InsP3 production and Ca2+-release were Syk-dependent, but only partially inhibited by PKA/PKG pathways. CONCLUSION: EB and EM are specific agonists and antagonists, respectively, of GPIbα-mediated Syk activation leading to platelet aggregation. The cAMP/PKA and cGMP/PKG pathways do not inhibit but enhance GPIbα-/GPVI-initiated, SFK-dependent Syk activation, but strongly inhibit further downstream responses including aggregation. These data establish an important intracellular regulatory network induced by GPIbα.
Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Quinase Syk/metabolismo , Difosfato de Adenosina/metabolismo , Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Iloprosta/farmacologia , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Fosforilação/efeitos dos fármacos , Pirazóis/farmacologia , Pirimidinas/farmacologiaRESUMO
Myocardial ischemia-reperfusion injury continues to be observed during open heart surgery. Various experimental models have been developed to overcome this injury and to increase postoperative prognosis. This study was conducted to assess the effect that iloprost, a prostacyclin analogue, can have on myocardial ischemia-reperfusion injury. We evaluated tissue damage by measuring the levels of malonyldialdehyde (MDA), glutathione, and nitric oxide (NO) in tissue and perfusates. In this study, 20 guinea pig hearts were prepared by using the modified Langendorff perfusion apparatus to form control (n = 10) and experimental study groups (n = 10). Following a preischemic period of perfusion and an ischemic period of 20 minutes, control hearts were perfused with KrebsHenseleit solution. In the experimental group, iloprost (0.45 µg/kg per hour) was included in the perfusates for the last 10 minutes of the preischemic phase. Following cardiac stabilization, heart rate (pulse/min), contractility (mm), and aortic pressure (mmHg) values were recorded at the end of preischemia, postischemia, and reperfusion. Perfusate and tissue analyses for glutathione, MDA, and NO levels were made in each group at the end of experiments. Iloprost was found to have protective effects against myocardial ischemia by means of increased myocardial contractility, decreased tissue/perfusate glutathione levels and inhibited rise of tissue/perfusate MDA observed in the iloprost-treated experimental group. Future investigations on myocardial ischemia-reperfusion injury must evaluate iloprost-related mechanisms.
Assuntos
Pressão Arterial/efeitos dos fármacos , Epoprostenol/análise , Frequência Cardíaca/efeitos dos fármacos , Iloprosta/farmacologia , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Animais , Modelos Animais de Doenças , Feminino , Cobaias , Masculino , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/metabolismo , Miocárdio/patologia , Óxido Nítrico/metabolismo , Resultado do Tratamento , Vasodilatadores/farmacologiaRESUMO
Cyclooxygenase-2 (COX-2/PTGS2) mediates hyperoxia-induced impairment of lung development in newborn animals and is increased in the lungs of human infants with bronchopulmonary dysplasia (BPD). COX-2 catalyzes the production of cytoprotective prostaglandins, such as prostacyclin (PGI2), as well as proinflammatory mediators, such as thromboxane A2. Our objective was to determine whether iloprost, a synthetic analog of PGI2, would attenuate hyperoxia effects in the newborn mouse lung. To test this hypothesis, newborn C57BL/6 mice along with their dams were exposed to normoxia (21% O2) or hyperoxia (85% O2) from 4 to 14 days of age in combination with daily intraperitoneal injections of either iloprost 200 µg·kg-1·day-1, nimesulide (selective COX-2 antagonist) 100 mg·kg-1·day-1, or vehicle. Alveolar development was estimated by radial alveolar counts and mean linear intercepts. Lung function was determined on a flexiVent, and multiple cytokines and myeloperoxidase (MPO) were quantitated in lung homogenates. Lung vascular and microvascular morphometry was performed, and right ventricle/left ventricle ratios were determined. We determined that iloprost (but not nimesulide) administration attenuated hyperoxia-induced inhibition of alveolar development and microvascular density in newborn mice. Iloprost and nimesulide both attenuated hyperoxia-induced, increased lung resistance but did not improve lung compliance that was reduced by hyperoxia. Iloprost and nimesulide reduced hyperoxia-induced increases in MPO and some cytokines (IL-1ß and TNF-α) but not others (IL-6 and KC/Gro). There were no changes in pulmonary arterial wall thickness or right ventricle/left ventricle ratios. We conclude that iloprost improves lung development and reduces lung inflammation in a newborn mouse model of BPD.
Assuntos
Displasia Broncopulmonar/tratamento farmacológico , Hiperóxia/fisiopatologia , Iloprosta/farmacologia , Pneumonia/prevenção & controle , Alvéolos Pulmonares/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Displasia Broncopulmonar/etiologia , Displasia Broncopulmonar/metabolismo , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia/etiologia , Pneumonia/metabolismo , Gravidez , Alvéolos Pulmonares/irrigação sanguínea , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologia , Vasodilatadores/farmacologiaRESUMO
OBJECTIVES: Iloprost plays an important role in the treatment of Raynaud's phenomenon (RP), but has transient vasodilatory effects owing to its very short half-time. We aimed to evaluate short- and medium-term haemodynamic effects of iloprost by measuring dorsal finger microvessel blood flow using laser Doppler flowmetry (LDF), in patients with RP associated with systemic sclerosis (SSc). METHOD: In 24 consecutive SSc patients with RP (disease duration 10.5 ± 1.3 years), LDF with heating probes was used to measure blood flow in four fingers by occlusive and heating tests, at baseline, after 3 consecutive days of iloprost infusion, and at 24 h and 7 days after last iloprost infusion. Nailfold videocapillaroscopy (NVC) patterns of microvascular damage were investigated. Sixteen healthy controls were studied to compare baseline flows. RESULTS: Compared to controls, SSc patients showed significantly impaired axon reflex vasoregulation and nitric oxide responses at baseline (p = 0.001 and p = 0.03, respectively). After iloprost, a prompt but transient significant improvement in endothelial-dependent vasodilation (occlusive test) was seen only in SSc patients with an 'active' NVC pattern (p ≤ 0.05). The iloprost effects vanished within 7 days after the last infusion. No significant differences were found, in the whole study, between patients with and without digital ulcers. CONCLUSIONS: Microcirculatory blood flow increases following 3 days of iloprost infusion but fades shortly after treatment. Although iloprost is effective in reducing the severity of RP in SSc, the most suitable regimen and timing to obtain longer lasting vasodilatory benefits remain to be established.
Assuntos
Dedos/irrigação sanguínea , Iloprosta/farmacologia , Microcirculação/efeitos dos fármacos , Fluxo Sanguíneo Regional/efeitos dos fármacos , Escleroderma Sistêmico/tratamento farmacológico , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Dedos/diagnóstico por imagem , Humanos , Iloprosta/uso terapêutico , Infusões Intravenosas , Fluxometria por Laser-Doppler , Estudos Longitudinais , Masculino , Angioscopia Microscópica , Pessoa de Meia-Idade , Escleroderma Sistêmico/fisiopatologia , Vasodilatadores/uso terapêuticoRESUMO
PURPOSE: The hypothesis that locally-released iloprost, a synthetic prostacyclin analog, affects macrophage phenotype at a microdialysis implant in the subcutaneous space of rats was tested. Macrophage activation towards alternatively-activated phenotypes using pharmaceutical release is of interest to improve integration of implants and direct the foreign body reaction toward a successful outcome. METHODS: Macrophage cell culture was used to test iloprost macrophage activation in vitro. Microdialysis sampling probes were implanted into the subcutaneous space of Sprague-Dawley rats to locally deliver iloprost in awake- and freely-moving rats. Monocyte chemoattractant protein -1 (CCL2) was quantified from collected dialysates using ELISA. Immunohistochemical staining was used to determine the presence of CD163+ macrophages in explanted tissues. RESULTS: Iloprost reduced CCL2 concentrations in NR8383 macrophages stimulated with lipopolysaccharide. CCL2 concentrations in collected dialysates were similarly reduced in the presence of iloprost. Iloprost caused an increase in CD163+ cells in explanted tissue surrounding implanted microdialysis probes at two days post probe implantation. CONCLUSIONS: Localized delivery of iloprost caused macrophage activation at the tissue interface of a microdialysis subcutaneous implant in rat. This model system may be useful for testing other potential macrophage modulators in vivo.
Assuntos
Quimiocina CCL2/metabolismo , Iloprosta/química , Iloprosta/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Técnicas de Cultura de Células , Linhagem Celular , Liberação Controlada de Fármacos , Humanos , Iloprosta/administração & dosagem , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Masculino , Microdiálise , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/metabolismo , Distribuição TecidualRESUMO
Recent studies have shown that a bioactive lipid prostacyclin (PGI2) plays a role in various cancers, including lung cancer. However, the specific function of PGI2 in ovarian cancer progression has not been determined. This study investigated the effects of PGI2 on cell growth, migration, and invasion in ovarian cancer cells using iloprost, a stable PGI2 analog. Iloprost significantly inhibited migration and invasion, but not cell growth, in a dose-dependent manner in human ovarian cancer cells (A2780 and SKOV3). Interestingly, the cell surface Gs protein-coupled PGI2 receptor IP was enhanced in human ovarian cancer cells. The inhibitory effect of iloprost on migration and invasion was entirely reversed by an IP antagonist (CAY10449) and IP siRNA, whereas the knockdown of peroxisome proliferator-activated receptor δ (PPARδ), a nuclear receptor of PGI2, did not rescue the effect of iloprost. Additionally, iloprost markedly decreased the expression of matrix metallopeptidase-2 and -9 (MMP-2 and MMP-9), which may be induced in the process of ovarian cancer metastasis. IP siRNA inhibited iloprost-reduced MMP-2 expression but not MMP-9 expression. Moreover, inhibition of protein kinase A (PKA) and overexpression of Akt and p38 rescued the inhibition of invasion and the reduction of MMP-2 expression by iloprost. Furthermore, iloprost-induced activation of PKA was associated with PKA-mediated Akt and p38 inactivation in ovarian cancer cells. Taken together, these results demonstrate that iloprost inhibits ovarian cancer cell invasion by downregulating MMP-2 expression via the IP-mediated PKA pathway. This study is the first to reveal a novel role for iloprost and to clarify its underlying mechanism in human ovarian cancer cells.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Epoprostenol/análogos & derivados , Iloprosta/farmacologia , Metaloproteinase 2 da Matriz/metabolismo , Neoplasias Ovarianas/patologia , Receptores de Epoprostenol/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Feminino , Humanos , Iloprosta/análogos & derivados , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND AND OBJECTIVE: Involvement of pulmonary vascular remodelling is a characteristic sign in COPD. Vascular mediators such as vascular endothelial growth factor (VEGF) and prostacyclin may regulate fibroblast activity. The objective was to study the synthesis of VEGF and interactions with prostacyclin and transforming growth factor (TGF)-ß1 in lung fibroblasts from patients with COPD and healthy control subjects. To further explore the autocrine role of synthesized VEGF on fibroblast activity, studies were performed in human lung fibroblasts (HFL-1). METHODS: Primary distal lung fibroblast cultures were established from healthy individuals and from COPD patients (GOLD stage IV). Lung fibroblasts were stimulated with the prostacyclin analogue iloprost and the profibrotic stimuli TGF-ß1 . VEGF synthesis was measured in the cell culture medium. Changes in proliferation rate, migration and synthesis of the extracellular matrix (ECM) proteins proteoglycans were analysed after stimulations with VEGF-A isoform 165 (VEGF165 ; 1-10 000 pg/mL) in HFL-1. RESULTS: Iloprost and TGF-ß1 significantly increased VEGF synthesis in both fibroblasts from COPD patients and control subjects. TGF-ß1 -induced VEGF synthesis was significantly reduced by the cyclooxygenase inhibitor indomethacin in fibroblasts from COPD patients. VEGF significantly increased proliferation rate and migration capacity in HFL-1. VEGF also significantly increased synthesis of the ECM proteins biglycan and perlecan. The VEGF receptors (VEGFR), VEGFR1, VEGFR2 and VEGFR3, were all expressed in primary lung fibroblasts and HFL-1. CONCLUSION: VEGF is synthesized in high amounts by distal lung fibroblasts and may have a crucial role in ongoing vascular remodelling processes in the distal lung compartments.
Assuntos
Fibroblastos/efeitos dos fármacos , Iloprosta/farmacologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Remodelação Vascular , Idoso , Biglicano/biossíntese , Movimento Celular , Proliferação de Células , Células Cultivadas , Inibidores de Ciclo-Oxigenase/farmacologia , Fibroblastos/metabolismo , Proteoglicanas de Heparan Sulfato/biossíntese , Humanos , Indometacina/farmacologia , Pulmão/citologia , Pulmão/metabolismo , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/patologia , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Periodontal ligament is considered a rich source of mesenchymal stem cells for pulp regeneration. This study investigated the effect of iloprost, a prostacyclin analog, on cell proliferation and the expression of angiogenesis-related genes in human periodontal ligament cells (hPDLs) in vitro. The hPDLs were treated with 10-9-10-6 M iloprost for 1, 6 and 24 h. Cell proliferation was determined by an MTT assay. Vascular endothelial growth factor (VEGF), alpha-1 type I collagen (COL1), and basic fibroblast growth factor (bFGF) mRNA expression were determined by semi-quantitative and qPCR. ELISA was employed for assessment of VEGF expression. Immunofluorescence staining for COL1 protein expression was performed. A prostacyclin receptor (IP) antagonist was used to verify the signaling pathway. The Kruskal-Wallis and Tukey significant difference tests were used to analyze the results. From the results, iloprost treatment did not affect cell morphology or proliferation. Iloprost induced the upregulation of VEGF and COL1 mRNA levels as shown by PCR. The effect of iloprost on bFGF mRNA expression was not observed. The immunofluorescence assay revealed that COL1 protein expression was increased in the iloprost groups. Pretreating the hPDLs with the IP antagonist significantly suppressed the enhancing effect of iloprost on VEGF and COL1 mRNA expression and suppressed COL1 protein expression. In conclusion, iloprost promoted mRNA and protein expression of VEGF and COL1, but not of bFGF in hPDL cells. The increased effect of iloprost was abolished by IP receptor antagonist pretreatment. Iloprost might be a promising agent in dentin-pulp regeneration.
Assuntos
Proliferação de Células/efeitos dos fármacos , Iloprosta/farmacologia , Ligamento Periodontal/citologia , Benzofenonas/farmacologia , Células Cultivadas , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Ensaio de Imunoadsorção Enzimática , Fator 2 de Crescimento de Fibroblastos/metabolismo , Imunofluorescência , Expressão Gênica , Humanos , Imidazóis/farmacologia , Técnicas In Vitro , Dente Serotino , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Bone marrow oedema (BMO) is a multifactorial condition. Various conservative treatment options include analgesic therapy, immobilisation of the affected joint and/or systemic intravenous iloprost or bisphosphonate therapy. Many studies confirm the positive effect of iloprost therapy in larger joints, e.g. the hip and knee joint, after short-term follow up. The objective of this study was to investigate that treatment with iloprost leads to positive long-term functional and radiological outcomes for BMO of the hip joint. Nineteen patients with BMO of the hip joint, ARCO stage 1-2, were included in this study. The Harris Hip Score, the SF-36, the WOMAC score and a visual analogue pain scale (VAS) were evaluated before and 29 ± 11 months after Ilomedin therapy. All patients underwent MRI for radiological follow-up monitoring three months after treatment. Significant improvements were found in the WOMAC Index and the VAS. In 79% of patients, follow-up MRI after three months showed complete regression of the oedema. Based on the positive results of our study, we support treatment with iloprost for BMO of the hip joint at ARCO stage 1-2.