RESUMEN
The proliferation and migration of vascular smooth muscle cells (VSMCs) after vascular injury lead to neointimal hyperplasia, thus aggravating vascular diseases. However, the molecular mechanisms underlying neointima formation are not fully elucidated. Extracellular vesicles (EVs) are mediators of various intercellular communications. The potential of EVs as regulators in cardiovascular diseases has raised significant interest. In the current study we investigated the role of circulating small extracellular vesicles (csEVs), the most abundant EVs (1010 EVs/mL serum) in VSMC functions. csEVs were prepared from bovine, porcine or rat serum. We showed that incubation with csEVs (0.5 × 1010-2 × 1010) dose-dependently enhanced the proliferation and migration of VSMCs via the membrane phosphatidylserine (PS). In rats with ligation of right carotid artery, we demonstrated that application of csEVs in the ligated vessels aggravated neointima formation via interaction of membrane PS with injury. Furthermore, incubation with csEVs markedly enhanced the phosphorylation of AXL and MerTK in VSMCs. Pretreatment with BSM777607 (pan-TAM inhibitor), bemcentinib (AXL inhibitor) or UNC2250 (MerTK inhibitor) blocked csEV-induced proliferation and migration of VSMCs. We revealed that csEV-activated AXL and MerTK shared the downstream signaling pathways of Akt, extracellular signal-regulated kinase (ERK) and focal adhesion kinase (FAK) that mediated the effects of csEVs. We also found that csEVs increased the expression of AXL through activation of transcription factor YAP, which might constitute an AXL-positive feedback loop to amplify the signals. Finally, we demonstrated that dual inhibition of AXL/MerTK by ONO-7475 (0.1 µM) effectively hindered csEV-mediated proliferation and migration of VSMCs in ex vivo mouse aorta injury model. Based on these results, we propose an essential role for csEVs in proliferation and migration of VSMCs and highlight the feasibility of dual AXL/MerTK inhibitors in the treatment of vascular diseases.
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Vesículas Extracelulares , Músculo Liso Vascular , Animales , Bovinos , Ratones , Ratas , Tirosina Quinasa c-Mer/metabolismo , Movimiento Celular , Proliferación Celular , Células Cultivadas , Vesículas Extracelulares/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Neointima/metabolismo , Porcinos , Enfermedades Vasculares/tratamiento farmacológico , Enfermedades Vasculares/metabolismoRESUMEN
While failure in resolution of inflammation is considered to increase the risk of tumorigenesis, there is paucity of experimental as well as clinical evidence supporting this association. Resolvin D1 (RvD1) is a representative pro-resolving lipid mediator that is endogenously generated from docosahexaenoic acid for the resolution of inflammation. Here, we report a decreased level of RvD1 in the blood from colorectal cancer patients and mice having inflammation-induced colon cancer, suggesting plasma RvD1 as a potential biomarker for monitoring colorectal cancer. Administration of RvD1 attenuated dextran sodium sulfate (DSS)-induced colitis and azoxymethane (AOM) plus DSS-induced colorectal carcinogenesis by suppressing the production of interleukin-6 (IL-6) and IL-6-mediated chromosomal instability. The protective effect of RvD1 against chromosomal instability is associated with downregulation of IL-6-induced Cyclin D1 expression, which appears to be mediated by blocking the Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) axis. RvD1 inhibited the STAT3 signaling pathway by interfering with the binding of IL-6 to its receptor (IL-6R), suggesting the novel function of RvD1 as a putative IL-6R antagonist. Together, our findings suggest that RvD1-mediated blockade of IL-6 signal transmission may contribute to inhibition of chromosomal instability and tumorigenesis.
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Carcinogénesis/patología , Colitis/complicaciones , Neoplasias del Colon/tratamiento farmacológico , Neoplasias Colorrectales/tratamiento farmacológico , Ácidos Docosahexaenoicos/farmacología , Interleucina-6/farmacología , Huso Acromático/efectos de los fármacos , Animales , Carcinogénesis/metabolismo , Estudios de Casos y Controles , Colitis/inducido químicamente , Colitis/patología , Neoplasias del Colon/etiología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Neoplasias Colorrectales/etiología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Humanos , Masculino , Ratones , Ratones Endogámicos ICR , Huso Acromático/patologíaRESUMEN
Acoustic Doppler current profilers (ADCPs) were developed to acquire water current velocities, as well as depth-dependent echo intensities. As the backscattering strength of an underwater object can be estimated from the measured echo intensity, the ADCP can be used to estimate plankton populations and distributions. In this study, the backscattering strength of bubble clusters in a water tank was estimated using the commercial ADCP as a proof-of-concept. Specifically, the temporal variations in the backscattering strength and the duration of bubble existence were quantitatively evaluated. Additionally, the PDSL (population density spectrum level) and VF (void fraction) of the artificial bubbles were characterized based on the obtained distribution characteristics using a PDPA (phase Doppler particle analyzer).
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Acústica , AguaRESUMEN
Acute ischemic stroke is the leading cause of morbidity and mortality worldwide. Recombinant tissue plasminogen activator (rtPA) is the only agent clinically approved by FDA for patients with acute ischemic stroke. However, delayed treatment of rtPA (e.g., more than 3 h after stroke onset) exacerbates ischemic brain damage by causing intracerebral hemorrhage and increasing neurotoxicity. In the present study, we investigated whether the neuroprotant otaplimastat reduced delayed rtPA treatment-evoked neurotoxicity in male Sprague Dawley rats subjected to embolic middle cerebral artery occlusion (eMCAO). Otaplimastat reduced cerebral infarct size and edema and improved neurobehavioral deficits. In particular, otaplimastat markedly reduced intracerebral hemorrhagic transformation and mortality triggered by delayed rtPA treatment, consequently extending the therapeutic time window of rtPA. We further found that ischemia-evoked extracellular matrix metalloproteases (MMPs) expression was closely correlated with cerebral hemorrhagic transformation and brain damage. In ischemic conditions, delayed rtPA treatment further increased brain injury via synergistic expression of MMPs in vascular endothelial cells. In oxygen-glucose-deprived endothelial cells, otaplimastat suppressed the activity rather than protein expression of MMPs by restoring the level of tissue inhibitor of metalloproteinase (TIMP) suppressed in ischemia, and consequently reduced vascular permeation. This paper shows that otaplimastat under clinical trials is a new drug which can inhibit stroke on its own and extend the therapeutic time window of rtPA, especially when administered in combination with rtPA.
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Isquemia Encefálica , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Acetamidas , Animales , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/metabolismo , Hemorragia Cerebral/tratamiento farmacológico , Hemorragia Cerebral/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Fibrinolíticos/uso terapéutico , Humanos , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/metabolismo , Masculino , Metaloproteinasas de la Matriz/metabolismo , Quinazolinas/uso terapéutico , Quinazolinonas , Ratas , Ratas Sprague-Dawley , Accidente Cerebrovascular/metabolismo , Terapia Trombolítica , Activador de Tejido PlasminógenoRESUMEN
OBJECTIVE: Otaplimastat is a neuroprotectant that inhibits matrix metalloprotease pathway, and reduces edema and intracerebral hemorrhage induced by recombinant tissue plasminogen activator (rtPA) in animal stroke models. We aimed to assess the safety and efficacy of otaplimastat in patients receiving rtPA. METHODS: This was a phase 2, 2-part, multicenter trial in stroke patients (19-80 years old) receiving rtPA. Intravenous otaplimastat was administered <30 minutes after rtPA. Stage 1 was a single-arm, open-label safety study in 11 patients. Otaplimastat 80 mg was administered twice daily for 3 days. Stage 2 was a randomized, double-blind, placebo-controlled study involving 69 patients, assigned (1:1:1) to otaplimastat 40 mg, otaplimastat 80 mg, or a placebo. The primary endpoint was the occurrence of parenchymal hematoma (PH) on day 1. Secondary endpoints included serious adverse events (SAEs), mortality, and modified Rankin scale (mRS) distribution at 90 days (clinicaltrials.gov identifier: NCT02787278). RESULTS: No safety issues were encountered in stage 1. The incidence of PH during stage 2 was comparable: 0 of 22 with the placebo, 0 of 22 with otaplimastat 40 mg, and 1 of 21 with the 80 mg dose. No differences in SAEs (13%, 17%, 14%) or death (8.3%, 4.2%, 4.8%) were observed among the 3 groups. Three adverse events (chills, muscle rigidity, hepatotoxicity) were judged to be related to otaplimastat. INTERPRETATION: Intravenous otaplimastat adjunctive therapy in patients receiving rtPA is feasible and generally safe. The functional efficacy of otaplimastat needs to be investigated with further large trials. ANN NEUROL 2020;87:233-245.
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Acetamidas/uso terapéutico , Isquemia Encefálica/tratamiento farmacológico , Quinazolinonas/uso terapéutico , Accidente Cerebrovascular/tratamiento farmacológico , Acetamidas/efectos adversos , Administración Intravenosa , Adulto , Anciano , Anciano de 80 o más Años , Isquemia Encefálica/complicaciones , Método Doble Ciego , Femenino , Fibrinolíticos/uso terapéutico , Humanos , Masculino , Persona de Mediana Edad , Fármacos Neuroprotectores/efectos adversos , Fármacos Neuroprotectores/uso terapéutico , Quinazolinonas/efectos adversos , Accidente Cerebrovascular/complicaciones , Activador de Tejido Plasminógeno/uso terapéutico , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: Gremlin-1 (GREM1), one of the bone morphogenetic protein antagonists, is involved in organogenesis, tissue differentiation and kidney development. However, the role of GREM1 in cancer progression and its underlying mechanisms remain poorly understood. METHODS: The role of GREM1 in breast cancer progression was assessed by measuring cell viability, colony formation, 3D tumour spheroid formation/invasion and xenograft tumour formation. Chromatin immunoprecipitation, a luciferase reporter assay and flow cytometry were performed to investigate the molecular events in which GREM1 is involved. RESULTS: GREM1 expression was elevated in breast cancer cells and tissues obtained from breast cancer patients. Its overexpression was associated with poor prognosis in breast cancer patients, especially those with oestrogen receptor (ER)-negative tumours. GREM1 knockdown inhibited the proliferation of breast cancer cells and xenograft mammary tumour growth, while its overexpression enhanced their viability, growth and invasiveness. Oestrogen-related receptor α (ERRα), an orphan nuclear hormone receptor, directly interacted with the GREM1 promoter and increased the expression of GREM1. GREM1 also enhanced the promoter activity of ESRRA encoding ERRα, comprising a positive feedback loop. Notably, GREM1 bound to and activated EGFR, a well-known upstream regulator of ERRα. CONCLUSIONS: Our study suggests that the GREM1-ERRα axis can serve as a potential therapeutic target in the management of cancer, especially ER-negative tumour.
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Neoplasias de la Mama/etiología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Receptores de Estrógenos/fisiología , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Línea Celular Tumoral , Progresión de la Enfermedad , Receptores ErbB/fisiología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Ratones Endogámicos BALB C , Transducción de Señal/fisiología , Receptor Relacionado con Estrógeno ERRalfaRESUMEN
The proviral integration site for Moloney murine leukemia virus (PIM) family of serine/threonine-specific kinases consist of three isoforms, that regulate proliferation, apoptosis, metabolism, invasion, and metastasis of cancer cells. Among these, abnormally elevated kinase activity of PIM-1 contributes to the progression of gastric cancer and predicts poor prognosis and a low survival rate in gastric cancer patients. In the present study, we found that resveratrol, one of the representative chemopreventive and anticarcinogenic phytochemicals, directly binds to PIM-1 and thereby inhibits its catalytic activity in human gastric cancer SNU-601 cells. This resulted in suppression of phosphorylation of the proapoptotic Bad, a known substrate of PIM-1. Resveratrol, by inactivating PIM-1, also inhibited anchorage-independent growth and proliferation of SNU-601 cells. To understand the molecular interaction between resveratrol and PIM-1, we conducted docking simulation and found that resveratrol directly binds to the PIM-1 at the ATP-binding pocket. In conclusion, the proapototic and anti-proliferative effects of resveratrol in gastric cancer cells are likely to be mediated through suppression of PIM-1 kinase activity, which may represent a novel mechanism underlying its chemopreventive and anticarcinogenic actions.
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Proliferación Celular/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Resveratrol/farmacología , Neoplasias Gástricas/tratamiento farmacológico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Neoplasias Gástricas/metabolismoRESUMEN
The corneal fibrotic responses to corneal damage often lead to severe corneal opacification thereby resulting in severe visual impairment or even blindness. The persistence of corneal opacity depends heavily on the activity of corneal myofibroblast. Myofibroblasts are opaque and synthesize a disorganized extracellular matrix (ECM) and thus promoting opacification. Cluster of differentiation 147 (CD147), a member of the immunoglobulin superfamily, is known to play important roles in the differentiation process from fibroblast to myofibroblast in damaged cornea and may therefore be an effective target for treatment of corneal opacity. Here, we examined the therapeutic efficacy of novel CD147 inhibiting verbenone derivative SP-8356 ((1S,5R)-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one) on corneal fibrosis. Topical SP-8356 significantly reduced corneal haze and fibrosis in the alkali-burned cornea. In detail, SP-8356 inhibited both alpha-smooth muscle actin (α-SMA) expressing myofibroblast and its ECM-related products, such as matrix-metalloproteinase-9 and collagen type III and IV. Similar to SP-8356, topical corticosteroid (prednisolone acetate, PA) also reduced the ECM-related products and opacification. However, prednisolone acetate failed to decrease the population of α-SMA-positive corneal myofibroblast. In conclusion, SP-8356 is capable enough to prevent corneal haze by preventing pathological fibrosis after severe corneal damage. Therefore, SP-8356 could be a potentially promising therapeutic drug for corneal fibrosis.
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Álcalis/efectos adversos , Basigina/antagonistas & inhibidores , Monoterpenos Bicíclicos/farmacología , Lesiones de la Cornea/etiología , Lesiones de la Cornea/patología , Quemaduras Oculares/etiología , Quemaduras Oculares/patología , Animales , Biopsia , Colágeno/metabolismo , Lesiones de la Cornea/tratamiento farmacológico , Citocinas/metabolismo , Modelos Animales de Enfermedad , Quemaduras Oculares/tratamiento farmacológico , Fibroblastos/metabolismo , Fibrosis , Inmunohistoquímica , Mediadores de Inflamación/metabolismo , Masculino , RatasRESUMEN
(1S,5R)-4-((E)-3,4-dihydroxy-5-methoxystryryl)-6,6-dimethylbicylco[3.1.1]hept-3-en-2-one (SP-8356) is a novel (1S)-(-)-verbenone derivative that is currently in preclinical development for the treatment of ischemic stroke and atherosclerosis. This report aimed at characterization of the metabolism and pharmacokinetic properties of SP-8356. Following intravenous dose in rats and dogs, plasma concentrations of SP-8356 declined rapidly with high clearance (CL) and short half-life; after oral administration in both species, its plasma levels were below the quantitation limit. Fourteen circulating metabolites, formed by mono-oxygenation, demethylation, glucuronidation, catechol O-methylation, sulfation and oxidation (bioactivation) followed by glutathione (GSH) conjugation, were tentatively identified in both species. Urinary excretion of SP-8356 appeared to be minimal in rats, compared to its metabolites. GSH conjugate of SP-8356 was also formed during incubation with rat liver S9 fraction consistent with oxidative bioactivation; this bioactivation was almost completely inhibited by the cofactors for glucuronidation, sulfation and methylation, indicating that it may be abolished by competing metabolic reactions in the body. The human pharmacokinetics of SP-8356 was predicted to be similar to that of the animals based on the current in vitro metabolic stability results. In summary, rapid phase II metabolism appears to be mainly responsible for its suboptimal pharmacokinetics, such as high CL and low oral absorption. Because of competing metabolic reactions, potential safety risks related to SP-8356 bioactivation may be low.
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Monoterpenos Bicíclicos/metabolismo , Monoterpenos Bicíclicos/farmacocinética , Hígado/efectos de los fármacos , Administración Intravenosa , Administración Oral , Animales , Monoterpenos Bicíclicos/administración & dosificación , Monoterpenos Bicíclicos/sangre , Cromatografía Líquida de Alta Presión , Perros , Glutatión/metabolismo , Semivida , Humanos , Hígado/metabolismo , Masculino , Tasa de Depuración Metabólica/fisiología , Farmacocinética , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Neointimal hyperplasia and its related arterial stiffness are the crucial pathophysiological features in atherosclerosis and in-stent restenosis. Cluster of differentiation 147 (CD147), a member of the immunoglobulin super family that induces the expression of matrix metalloproteinase-9 (MMP-9) by dimerization, may play important roles in neointimal hyperplasia and may therefore be an effective target for the treatment of this condition. Here, we investigated whether a novel CD147 inhibitor SP-8356 ((1S,5R)-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one) reduces neointimal hyperplasia and arterial stiffness in a rat model of partial carotid artery ligation. METHODS: Neointimal hyperplasia was induced in Sprague-Dawley rats by partial ligation of the right carotid artery combined with a high fat diet and vitamin D injection. Rats were subdivided into vehicle, SP-8356 (50 mg/kg), and rosuvastatin (10 mg/kg) groups. The drugs were administrated via intraperitoneal injections for 4 weeks. The elasticity of blood vessels was assessed by measuring pulse wave velocity using Doppler ultrasonography before sacrifice. Histomolecular analysis was carried out on harvested carotid arteries. RESULTS: SP-8356 significantly reduced MMP activity by inhibiting CD147 dimerization. SP-8356 reduced neointimal hyperplasia and prevented the deterioration of vascular elasticity. SP-8356 had a greater inhibitory effect on neointimal hyperplasia than did rosuvastatin. Furthermore, rosuvastatin did not improve vascular elasticity. SP-8356 increased the expression of smooth muscle myosin heavy chain (SM-MHC), but decreased the expression of collagen type III and MMP-9 in the neointimal region. In contrast to SP-8356, rosuvastatin did not alter the expression of SM-MHC or MMP-9. CONCLUSIONS: The ability of SP-8356 to reduce neointimal hyperplasia and improve arterial stiffness in affected carotid artery suggests that SP-8356 could be a promising therapeutic drug for vascular remodeling disorders involving neointimal hyperplasia and arterial stiffness.
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Basigina/antagonistas & inhibidores , Monoterpenos Bicíclicos/farmacología , Compuestos Bicíclicos con Puentes/farmacología , Arterias Carótidas/patología , Arterias Carótidas/fisiopatología , Neointima/patología , Rigidez Vascular/efectos de los fármacos , Animales , Basigina/metabolismo , Monoterpenos Bicíclicos/química , Compuestos Bicíclicos con Puentes/química , Línea Celular , Células Cultivadas , Colágeno Tipo III/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Descubrimiento de Drogas , Hiperplasia , Ligadura , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Músculo Liso Vascular/metabolismo , Fenotipo , Ratas Sprague-DawleyRESUMEN
A key event required for effective resolution of inflammation is efferocytosis, which is defined as phagocytic removal of apoptotic cells mostly by macrophages acquiring an alternatively activated phenotype (M2). c-Myc has been reported to play a role in alternative activation of human macrophages and is proposed as one of the M2 macrophage markers. We found that M2-like peritoneal macrophages from zymosan A-treated mice exhibited a marked accumulation of Myc-nick, a truncated protein generated by a Calpain-mediated proteolytic cleavage of full-length c-Myc. Further, ectopic expression of Myc-nick in murine bone marrow-derived macrophages promoted the M2 polarization and, consequently, enhanced their efferocytic capability. Notably, Myc-nick-induced efferocytosis was found to be tightly associated with α-tubulin acetylation by K acetyltransferase 2a (Kat2a/Gcn5) activity. These findings suggest Myc-nick as a novel proresolving mediator that has a fundamental function in maintaining homeostasis under inflammatory conditions.-Zhong, X., Lee, H.-N., Kim, S. H., Park, S.-A., Kim, W., Cha, Y.-N., Surh, Y.-J. Myc-nick promotes efferocytosis through M2 macrophage polarization during resolution of inflammation.
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Células de la Médula Ósea/inmunología , Macrófagos Peritoneales/inmunología , Proteínas Proto-Oncogénicas c-myc/inmunología , Acetilación , Animales , Antígenos de Diferenciación/inmunología , Células de la Médula Ósea/patología , Histona Acetiltransferasas/inmunología , Inflamación/inmunología , Inflamación/patología , Macrófagos Peritoneales/patología , Ratones , Tubulina (Proteína)/inmunología , Factores de Transcripción p300-CBP/inmunologíaRESUMEN
Resolution of inflammation that occurs after microbial infection or tissue damage is an important physiologic process in maintaining or restoring host homeostasis. Taurine chloramine (TauCl) is formed by a reaction between taurine and hypochlorite in leukocytes, and it is especially abundant in activated neutrophils that encounter an oxidative burst. As neutrophils undergo apoptosis, TauCl is released to the extracellular matrix at the inflamed sites, thereby affecting coexisting macrophages in the inflammatory microenvironment. In this study, we investigated the role of TauCl in phagocytosis by macrophages during resolution of fungal infection-induced inflammation. We found that exogenous TauCl substantially increased the phagocytic efficiency of macrophages through up-regulation of dectin-1, a receptor for fungal ß-1,3-glucans, which is present on the membrane of macrophages. Our previous studies demonstrated the induction of heme oxygenase-1 (HO-1) expression in murine peritoneal macrophages treated with TauCl. In the present study, knocking out HO-1 or pharmacologic inhibition of HO-1 with zinc protoporphyrin IX attenuated the TauCl-induced expression of dectin-1 and subsequent phagocytosis. Furthermore, carbon monoxide (CO), a by-product of the HO-1-catalyzed reaction, induced expression of dectin-1 and potentiated phagocytic capability of the macrophages, which appeared to be mediated through up-regulation of peroxisome proliferator-activated receptor γ. Taken together, induction of HO-1 expression and subsequent CO production by TauCl are essential for phagocytosis of fungi by macrophages. Our results suggest that TauCl has important roles in host defense against fungal infection and has therapeutic potential in the management of inflammatory diseases.-Kim, S. H., Zhong, X., Kim, W., Kim, K., Suh, Y.-G., Kim, C., Joe, Y., Chung, H. T., Cha, Y.-N., Surh, Y.-J. Taurine chloramine potentiates phagocytic activity of peritoneal macrophages through up-regulation of dectin-1 mediated by heme oxygenase-1-derived carbon monoxide.
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Inhibidores Enzimáticos/farmacología , Lectinas Tipo C/metabolismo , Macrófagos Peritoneales/efectos de los fármacos , Fagocitosis , Taurina/análogos & derivados , Regulación hacia Arriba , Animales , Candida albicans/patogenicidad , Monóxido de Carbono/metabolismo , Células Cultivadas , Hemo-Oxigenasa 1/antagonistas & inhibidores , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Lectinas Tipo C/genética , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/microbiología , Ratones , Ratones Endogámicos C57BL , PPAR gamma , Taurina/farmacologíaRESUMEN
Interactions between CD147 and cyclophilin A (CypA) promote plaque rupture that causes atherosclerosis-related cardiovascular events, such as myocardial infarction and stroke. Here, we investigated whether SP-8356 ((1S,5R)-4-(3,4-dihydroxy-5-methoxystyryl)-6,6-dimethylbicyclo[3.1.1]hept-3-en-2-one), a novel drug, can exert therapeutic effects against plaque progression and instability through disruption of CD147-CypA interactions in apolipoprotein E-deficient (ApoE KO) mice. Immunocytochemistry and immunoprecipitation analyses were performed to assess the effects of SP-8356 on CD147-CypA interactions. Advanced plaques were induced in ApoE KO mice via partial ligation of the right carotid artery coupled with an atherogenic diet, and SP-8356 (50 mg/kg) orally administrated daily one day after carotid artery ligation for three weeks. The anti-atherosclerotic effect of SP-8356 was assessed using histological and molecular approaches. SP-8356 interfered with CD147-CypA interactions and attenuated matrix metalloproteinase-9 activation. Moreover, SP-8356 induced a decreased in atherosclerotic plaque size in ApoE KO mice and stabilized plaque vulnerability by reducing the necrotic lipid core, suppressing macrophage infiltration, and enhancing fibrous cap thickness through increasing the content of vascular smooth muscle cells. SP-8356 exerts remarkable anti-atherosclerotic effects by suppressing plaque development and improving plaque stability through inhibiting CD147-CypA interactions. Our novel findings support the potential utility of SP-8356 as a therapeutic agent for atherosclerotic plaque.
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Antiinflamatorios/uso terapéutico , Basigina/metabolismo , Monoterpenos Bicíclicos/uso terapéutico , Ciclofilina A/metabolismo , Placa Aterosclerótica/tratamiento farmacológico , Animales , Antiinflamatorios/farmacología , Apolipoproteínas E/genética , Basigina/antagonistas & inhibidores , Monoterpenos Bicíclicos/farmacología , Arterias Carótidas/efectos de los fármacos , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Línea Celular , Células Cultivadas , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Placa Aterosclerótica/genética , Unión Proteica , Ratas , Ratas Sprague-DawleyRESUMEN
Phagocytosis of pathogens by macrophages is crucial for the successful resolution of inflammation induced by microbial infection. Taurine chloramine (TauCl), an endogenous anti-inflammatory and antioxidative substance, is produced by reaction between taurine and hypochlorous acid by myeloperoxidase activity in neutrophils under inflammatory conditions. In the present study, we investigated the effect of TauCl on resolution of acute inflammation caused by fungal infection using a zymosan A-induced murine peritonitis model. TauCl administration reduced the number of the total peritoneal leukocytes, while it increased the number of peritoneal monocytes. Furthermore, TauCl promoted clearance of pathogens remaining in the inflammatory environment by macrophages. When the macrophages isolated from thioglycollate-treated mice were treated with TauCl, their phagocytic capability was enhanced. In the murine macrophage-like RAW264.7 cells treated with TauCl, the proportion of macrophages clearing the zymosan A particles was also increased. TauCl administration resulted in elevated expression of heme oxygenase-1 (HO-1) in the peritoneal macrophages. Pharmacologic inhibition of HO-1 activity or knockdown of HO-1 in the murine macrophage RAW264.7 cells abolished the TauCl-induced phagocytosis, whereas the overexpression of HO-1 augmented the phagocytic ability of macrophages. Moreover, peritoneal macrophages isolated from HO-1 null mice failed to mediate TauCl-induced phagocytosis. Our results suggest that TauCl potentiates phagocytic activity of macrophages through upregulation of HO-1 expression.
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Hemo-Oxigenasa 1/metabolismo , Hemo-Oxigenasa 1/fisiología , Taurina/análogos & derivados , Animales , Antioxidantes , Inflamación , Macrófagos/fisiología , Macrófagos Peritoneales , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Peritonitis/inducido químicamente , Peritonitis/fisiopatología , Fagocitos , Fagocitosis/fisiología , Células RAW 264.7 , Taurina/metabolismo , Taurina/farmacología , Regulación hacia Arriba , Zimosan/farmacologíaRESUMEN
Variants of the SHANK3 gene, which encodes a core scaffold protein of the postsynaptic density of excitatory synapses, have been causally associated with numerous brain disorders. Shank3 proteins directly bind zinc ions through their C-terminal sterile α motif domain, which enhances the multimerization and synaptic localization of Shank3, to regulate excitatory synaptic strength. However, no studies have explored whether zinc affects the protein interactions of Shank3, which might contribute to the synaptic changes observed after zinc application. To examine this, we first purified Shank3 protein complexes from mouse brain synaptosomal lysates that were incubated with different concentrations of ZnCl2, and analyzed them with mass spectrometry. We used strict criteria to identify 71 proteins that specifically interacted with Shank3 when extra ZnCl2 was added to the lysate. To characterize the zinc-induced Shank3 interactome, we performed various bioinformatic analyses that revealed significant associations of the interactome with subcellular compartments, including mitochondria, and brain disorders, such as bipolar disorder and schizophrenia. Together, our results showing that zinc affected the Shank3 protein interactions of in vitro mouse synaptosomes provided an additional link between zinc and core synaptic proteins that have been implicated in multiple brain disorders.
Asunto(s)
Encefalopatías/metabolismo , Cloruros/administración & dosificación , Enfermedades Mitocondriales/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Mapas de Interacción de Proteínas/fisiología , Proteoma/metabolismo , Sinaptosomas/metabolismo , Compuestos de Zinc/administración & dosificación , Animales , Relación Dosis-Respuesta a Droga , Humanos , Metaboloma/efectos de los fármacos , Metaboloma/fisiología , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos , Proteínas del Tejido Nervioso/genética , Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas/efectos de los fármacos , Proteoma/efectos de los fármacos , Sinaptosomas/efectos de los fármacosRESUMEN
Astrocytes play a key role in brain homeostasis, protecting neurons against neurotoxic stimuli such as oxidative stress. Therefore, the neuroprotective therapeutics that enhance astrocytic functionality has been regarded as a promising strategy to reduce brain damage. We previously reported that ciclopirox, a well-known antifungal N-hydroxypyridone compound, protects astrocytes from oxidative stress by enhancing mitochondrial function. Using the N-hydroxypyridone scaffold, we have synthesized a series of cytoprotective derivatives. Mitochondrial activity assay showed that N-hydroxypyridone derivatives with biphenyl group have comparable to better protective effects than ciclopirox in astrocytes exposed to H2O2. N-hydroxypyridone derivatives, especially 11g, inhibited H2O2-induced deterioration of mitochondrial membrane potential and oxygen consumption rate, and significantly improved cell viability of astrocytes. The results indicate that the N-hydroxypyridone motif can provide a novel cytoprotective scaffold for astrocytes via enhancing mitochondrial functionality.
Asunto(s)
Astrocitos/efectos de los fármacos , Descubrimiento de Drogas , Peróxido de Hidrógeno/antagonistas & inhibidores , Mitocondrias/efectos de los fármacos , Piridonas/farmacología , Animales , Muerte Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Peróxido de Hidrógeno/farmacología , Mitocondrias/metabolismo , Estructura Molecular , Piridonas/síntesis química , Piridonas/química , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Helicobacter pylori (H. pylori) infection has been known to be implicated in human gastric carcinogenesis. Snail, the zinc-finger transcription factor known as a key inducer of changes in the cell shape and morphogenetic movement, is aberrantly overexpressed and correlates with lymph node metastasis in gastric cancer. In the present study, we investigated whether H. pylori could induce Snail activation to provoke these changes. Using a cell scatter assay, we noticed that human gastric cancer AGS cells infected with H. pylori underwent morphological changes as well as disruption of cell-cell interaction, which was then reversed by silencing of Snail by use of small interfering RNA (siRNA). In addition, infection with H. pylori resulted in an increased intracellular level of Snail in gastric cancer cells, which was abrogated in the presence of U0126 and LY294002, inhibitors of MEK/Erk and PI3K/Akt pathways, respectively. Cycloheximide pulse-chase experiments coupled with immunocytochemical analysis revealed that the induction of Snail by H. pylori was regulated at multiple levels, including increased transcription of Snail mRNA, inhibition of protein degradation, and enhancement of nuclear translocation of Snail. Pre-treatment of AGS cells with N-acetylcysteine, a well-known reactive oxygen species (ROS) scavenger, attenuated the H. pylori-induced activation of Erk, its binding to Snail promoter, inactivation of GSK-3ß, and accumulation of Snail. Collectively, these findings suggest that the upregulation of Snail expression induced by H. pylori and transformation to a spindle-like shape as a consequence in gastric cancer cells are attributable to ROS-mediated activation of Erk and the inhibition of GSK-3ß signaling. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Glucógeno Sintasa Quinasa 3 beta/metabolismo , Infecciones por Helicobacter/complicaciones , Helicobacter pylori/fisiología , Sistema de Señalización de MAP Quinasas , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción de la Familia Snail/genética , Neoplasias Gástricas/virología , Regulación hacia Arriba , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/metabolismo , Infecciones por Helicobacter/virología , Humanos , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismoRESUMEN
Activated caspases play a central role in the execution of apoptosis by cleaving endogenous substrates. Here, we developed a high throughput screening method to identify novel substrates for caspase-3 in a neuronal cell line. Critical steps in our strategy consist of two-dimensional electrophoresis-based protein separation and in vitro caspase-3 incubation of immobilized proteins to sort out direct substrates. Among 46 putative substrates identified in MN9D neuronal cells, we further evaluated whether caspase-3-mediated cleavage of anamorsin, a recently recognized cell death-defying factor in hematopoiesis, is a general feature of apoptosis. In vitro and cell-based cleavage assays indicated that anamorsin was specifically cleaved by caspase-3 but not by other caspases, generating 25- and 10-kDa fragments. Thus, in apoptosis of neuronal and non-neuronal cells induced by various stimuli including staurosporine, etoposide, or 6-hydroxydopamine, the cleavage of anamorsin was found to be blocked in the presence of caspase inhibitor. Among four tetrapeptide consensus DXXD motifs existing in anamorsin, we mapped a specific cleavage site for caspase-3 at DSVD(209)↓L. Intriguingly, the 25-kDa cleaved fragment of anamorsin was also detected in post-mortem brains of Alzheimer and Parkinson disease patients. Although the RNA interference-mediated knockdown of anamorsin rendered neuronal cells more vulnerable to staurosporine treatment, reintroduction of full-length anamorsin into an anamorsin knock-out stromal cell line made cells resistant to staurosporine-induced caspase activation, indicating the antiapoptotic function of anamorsin. Taken together, our approach seems to be effective to identify novel substrates for caspases and has the potential to provide meaningful insights into newly identified substrates involved in neurodegenerative processes.
Asunto(s)
Caspasa 3/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Degeneración Nerviosa/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Apoptosis/fisiología , Sitios de Unión , Estudios de Casos y Controles , Línea Celular , Células Cultivadas , Femenino , Técnicas de Silenciamiento del Gen , Ensayos Analíticos de Alto Rendimiento , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Ratones , Persona de Mediana Edad , Datos de Secuencia Molecular , Degeneración Nerviosa/etiología , Degeneración Nerviosa/patología , Neuronas/citología , Neuronas/metabolismo , Enfermedad de Parkinson/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Especificidad por SustratoRESUMEN
Calpains are a family of calcium-dependent cysteine proteases that are ubiquitously expressed in mammals and play critical roles in neuronal death by catalyzing substrate proteolysis. Here, we developed two-dimensional gel electrophoresis-based protease proteomics to identify putative calpain substrates. To accomplish this, cellular lysates from neuronal cells were first separated by pI, and the immobilized sample on a gel strip was incubated with a recombinant calpain and separated by molecular weight. Among 25 altered protein spots that were differentially expressed by at least 2-fold, we confirmed that arsenical pump-driving ATPase, optineurin, and peripherin were cleaved by calpain using in vitro and in vivo cleavage assays. Furthermore, we found that all of these substrates were cleaved in MN9D cells treated with either ionomycin or 1-methyl-4-phenylpyridinium, both of which cause a calcium-mediated calpain activation. Their cleavage was blocked by calcium chelator or calpain inhibitors. In addition, calpain-mediated cleavage of these substrates and its inhibition by calpeptin were confirmed in a middle cerebral artery occlusion model of cerebral ischemia, as well as a stereotaxic brain injection model of Parkinson disease. Transient overexpression of each protein was shown to attenuate 1-methyl-4-phenylpyridinium-induced cell death, indicating that these substrates may confer protection of varying magnitudes against dopaminergic injury. Taken together, the data indicate that our protease proteomic method has the potential to be applicable for identifying proteolytic substrates affected by diverse proteases. Moreover, the results described here will help us decipher the molecular mechanisms underlying the progression of neurodegenerative disorders where protease activation is critically involved.
Asunto(s)
Calpaína/metabolismo , Neuronas Dopaminérgicas/metabolismo , Proteoma/metabolismo , 1-Metil-4-fenilpiridinio/farmacología , Animales , ATPasas Transportadoras de Arsenitos/genética , ATPasas Transportadoras de Arsenitos/metabolismo , Calpaína/antagonistas & inhibidores , Muerte Celular , Línea Celular , Dipéptidos/farmacología , Dipéptidos/uso terapéutico , Neuronas Dopaminérgicas/efectos de los fármacos , Electroforesis en Gel Bidimensional/métodos , Glicina/análogos & derivados , Glicina/farmacología , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/metabolismo , Ionomicina/farmacología , Periferinas/genética , Periferinas/metabolismo , Proteómica/métodos , Ratas , Ratas Sprague-DawleyRESUMEN
The type 2 cannabinoid receptor (CB2R) was recently shown to mediate neuroprotection in ischemic injury. However, the role of CB2Rs in the central nervous system, especially neuronal and glial CB2Rs in the cortex, remains unclear. We, therefore, investigated anti-ischemic mechanisms of cortical CB2R activation in various ischemic models. In rat cortical neurons/glia mixed cultures, a CB2R agonist, trans-caryophyllene (TC), decreased neuronal injury and mitochondrial depolarization caused by oxygen-glucose deprivation/re-oxygenation (OGD/R); these effects were reversed by the selective CB2R antagonist, AM630, but not by a type 1 cannabinoid receptor antagonist, AM251. Although it lacked free radical scavenging and antioxidant enzyme induction activities, TC reduced OGD/R-evoked mitochondrial dysfunction and intracellular oxidative stress. Western blot analysis demonstrated that TC enhanced phosphorylation of AMP-activated protein kinase (AMPK) and cAMP responsive element-binding protein (CREB), and increased expression of the CREB target gene product, brain-derived neurotrophic factor. However, TC failed to alter the activity of either Akt or extracellular signal-regulated kinase, two major CB2R signaling pathways. Selective AMPK and CREB inhibitors abolished the neuroprotective effects of TC. In rats, post-ischemic treatment with TC decreased cerebral infarct size and edema, and increased phosphorylated CREB and brain-derived neurotrophic factor expression in neurons. All protective effects of TC were reversed by co-administration with AM630. Collectively, these data demonstrate that cortical CB2R activation by TC ameliorates ischemic injury, potentially through modulation of AMPK/CREB signaling, and suggest that cortical CB2Rs might serve as a putative therapeutic target for cerebral ischemia.