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BACKGROUND: Dickkopf-related protein 1 (DKK1) is a Wingless-related integrate site (Wnt) signaling modulator that is upregulated in prostate cancers (PCa) with low androgen receptor expression. DKN-01, an IgG4 that neutralizes DKK1, delays PCa growth in pre-clinical DKK1-expressing models. These data provided the rationale for a clinical trial testing DKN-01 in patients with metastatic castration-resistant PCa (mCRPC). METHODS: This was an investigator-initiated parallel-arm phase 1/2 clinical trial testing DKN-01 alone (monotherapy) or in combination with docetaxel 75 mg/m2 (combination) for men with mCRPC who progressed on ≥1 AR signaling inhibitors. DKK1 status was determined by RNA in-situ expression. The primary endpoint of the phase 1 dose escalation cohorts was the determination of the recommended phase 2 dose (RP2D). The primary endpoint of the phase 2 expansion cohorts was objective response rate by iRECIST criteria in patients treated with the combination. RESULTS: 18 pts were enrolled into the study-10 patients in the monotherapy cohorts and 8 patients in the combination cohorts. No DLTs were observed and DKN-01 600 mg was determined as the RP2D. A best overall response of stable disease occurred in two out of seven (29%) evaluable patients in the monotherapy cohort. In the combination cohort, five out of seven (71%) evaluable patients had a partial response (PR). A median rPFS of 5.7 months was observed in the combination cohort. In the combination cohort, the median tumoral DKK1 expression H-score was 0.75 and the rPFS observed was similar between patients with DKK1 H-score ≥1 versus H-score = 0. CONCLUSION: DKN-01 600 mg was well tolerated. DKK1 blockade has modest anti-tumor activity as a monotherapy for mCRPC. Anti-tumor activity was observed in the combination cohorts, but the response duration was limited. DKK1 expression in the majority of mCRPC is low and did not clearly correlate with anti-tumor activity of DKN-01 plus docetaxel.
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BACKGROUND AND AIMS: Dickkopf-1 (DKK1) is associated with poor prognosis in intrahepatic cholangiocarcinoma (iCCA), but the mechanisms behind this are unclear. Here, we show that DKK1 plays an immune regulatory role in vivo and inhibition reduces tumour growth. METHODS: Various in vivo GEMM mouse models and patient samples were utilized to assess the effects of tumour specific DKK1 overexpression in iCCA. DKK1-driven changes to the tumour immune microenvironment were characterized by immunostaining and gene expression analysis. DKK1 overexpressing and damage-induced models of iCCA were used to demonstrate the therapeutic efficacy of DKK1 inhibition in these contexts using the anti-DKK1 therapeutic, DKN-01. RESULTS: DKK1 overexpression in mouse models of iCCA drives an increase in chemokine and cytokine signalling, the recruitment of regulatory macrophages, and promotes the formation of a tolerogenic niche with higher numbers of regulatory T cells. We show a similar association of DKK1 with FOXP3 and regulatory T cells in patient tissue and gene expression data, demonstrating these effects are relevant to human iCCA. Finally, we demonstrate that inhibition of DKK1 with the monoclonal antibody mDKN-01 is effective at reducing tumour burden in two distinct mouse models of the disease. CONCLUSION: DKK1 promotes tumour immune evasion in iCCA through the recruitment of immune suppressive macrophages. Targeting DKK1 with a neutralizing antibody is effective at reducing tumour growth in vivo. As such, DKK1 targeted and immune modulatory therapies may be an effective strategy in iCCA patients with high DKK1 tumour expression or tolerogenic immune phenotypes.
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Neoplasias de los Conductos Biliares , Colangiocarcinoma , Péptidos y Proteínas de Señalización Intercelular , Animales , Humanos , Ratones , Neoplasias de los Conductos Biliares/tratamiento farmacológico , Neoplasias de los Conductos Biliares/genética , Neoplasias de los Conductos Biliares/metabolismo , Conductos Biliares Intrahepáticos/patología , Colangiocarcinoma/tratamiento farmacológico , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/genética , Fenotipo , Microambiente TumoralRESUMEN
PURPOSE: TRX518 is a mAb engaging the glucocorticoid-induced TNF receptor-related protein (GITR). This open-label, phase I study (TRX518-003) evaluated the safety and efficacy of repeated dose TRX518 monotherapy and in combination with gemcitabine, pembrolizumab, or nivolumab in advanced solid tumors. PATIENTS AND METHODS: TRX518 monotherapy was dose escalated (Part A) and expanded (Part B) up to 4 mg/kg loading, 1 mg/kg every 3 weeks. Parts C-E included dose-escalation (2 and 4 mg/kg loading followed by 1 mg/kg) and dose-expansion (4 mg/kg loading) phases with gemcitabine (Part C), pembrolizumab (Part D), or nivolumab (Part E). Primary endpoints included incidence of dose-limiting toxicities (DLT), serious adverse events (SAE), and pharmacokinetics. Secondary endpoints were efficacy and pharmacodynamics. RESULTS: A total of 109 patients received TRX518: 43 (Parts A+B), 30 (Part C), 26 (Part D), and 10 (Part E), respectively. A total of 67% of patients in Parts D+E had received prior anti-PD(L)1 or anti-CTLA-4. No DLTs, treatment-related SAEs, and/or grade 4 or 5 AEs were observed with TRX518 monotherapy. In Parts C-E, no DLTs were observed, although TRX518-related SAEs were reported in 3.3% (Part C) and 10.0% (Part E), respectively. Objective response rate was 3.2%, 3.8%, 4%, and 12.5% in Parts A+B, C, D, and E, respectively. TRX518 affected peripheral and intratumoral regulatory T cells (Treg) with different kinetics depending on the combination regimen. Responses with TRX518 monotherapy+anti-PD1 combination were associated with intratumoral Treg reductions and CD8 increases and activation after treatment. CONCLUSIONS: TRX518 showed an acceptable safety profile with pharmacodynamic activity. Repeated dose TRX518 monotherapy and in combination resulted in limited clinical responses associated with immune activation. See related commentary by Hernandez-Guerrero and Moreno, p. 3905.
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Antineoplásicos , Neoplasias , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales Humanizados/administración & dosificación , Antineoplásicos/uso terapéutico , Desoxicitidina/análogos & derivados , Humanos , Neoplasias/patología , Nivolumab/administración & dosificación , GemcitabinaRESUMEN
GITR is a TNF receptor, and its activation promotes immune responses and drives antitumor activity. The receptor is activated by the GITR ligand (GITRL), which is believed to cluster receptors into a high-order array. Immunotherapeutic agonist antibodies also activate the receptor, but their mechanisms are not well characterized. We solved the structure of full-length mouse GITR bound to Fabs from the antibody DTA-1. The receptor is a dimer, and each subunit binds one Fab in an orientation suggesting that the antibody clusters receptors. Binding experiments with purified proteins show that DTA-1 IgG and GITRL both drive extensive clustering of GITR. Functional data reveal that DTA-1 and the anti-human GITR antibody TRX518 activate GITR in their IgG forms but not as Fabs. Thus, the divalent character of the IgG agonists confers an ability to mimic GITRL and cluster and activate GITR. These findings will inform the clinical development of this class of antibodies for immuno-oncology.
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Dickkopf-1 (DKK1) is a secreted modulator of Wnt signaling that is frequently overexpressed in tumors and associated with poor clinical outcomes. DKN-01 is a humanized monoclonal therapeutic antibody that binds DKK1 with high affinity and has demonstrated clinical activity in gastric/gastroesophageal junction (G/GEJ) patients with elevated tumoral expression of DKK1. Here we report on the validation of a DKK1 RNAscope chromogenic in situ hybridization assay to assess DKK1 expression in G/GEJ tumor tissue. To reduce pathologist time, potential pathologist variability from manual scoring and support pathologist decision making, a digital image analysis algorithm that identifies tumor cells and quantifies the DKK1 signal was developed. Following CLIA guidelines the DKK1 RNAscope chromogenic in situ hybridization assay and digital image analysis algorithm were successfully validated for sensitivity, specificity, accuracy, and precision. The DKK1 RNAscope assay in conjunction with the digital image analysis solution is acceptable for prospective screening of G/GEJ adenocarcinoma patients. The work described here will further advance the companion diagnostic development of our DKK1 RNAscope assay and could generally be used as a guide for the validation of RNAscope assays with digital image quantification.
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Adenocarcinoma/diagnóstico , Biomarcadores de Tumor/análisis , Neoplasias Esofágicas/diagnóstico , Procesamiento de Imagen Asistido por Computador/métodos , Péptidos y Proteínas de Señalización Intercelular/análisis , Neoplasias Gástricas/diagnóstico , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patología , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Ensayos Clínicos Fase II como Asunto , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Unión Esofagogástrica/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Hibridación in Situ/métodos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Persona de Mediana Edad , Estudios Multicéntricos como Asunto , Estudios Prospectivos , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Estudios Retrospectivos , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/genéticaRESUMEN
Dickkopf-1 (DKK1), a secreted modulator of Wnt signaling, is overexpressed in many cancers, is often associated with worse clinical outcomes, and has been shown to have immunosuppressive effects. DKN-01 is an IgG4 clinical stage antibody that potently and specifically neutralizes human and murine DKK1 and has recently completed a promising study in combination with pembrolizumab in patients with gastric/gastroesophageal junction cancer. The purpose of this study is to characterize a murine version of DKN-01 (mDKN-01) and to better understand its mechanism of action. We examined the efficacy of mDKN-01 in both melanoma and metastatic breast cancer models. Immune depletion experiments revealed a requirement for natural killer (NK) but not B and T cells for tumor growth inhibition. mDKN-01 treatment promotes the induction of the NK-activating cytokines IL15 and IL33 as well as an enhanced recruitment of CD45+ cells. Other treatment-related changes include a reduction of Gr-1+CD11b+ myeloid-derived suppressor cells (MDSC) in the tumor and spleen and the upregulation of PD-L1 on MDSCs. In addition, mDKN-01 has a marked effect at reducing pulmonary metastases in the mouse 4T1 breast cancer model. Finally, the mDKN-01/anti-PD-1 combination was more effective at inhibiting melanoma growth than mDKN-01 alone. Taken together, our data demonstrate that mDKN-01 has efficacy by blocking the immunosuppressive effects of DKK1 in the tumor microenvironment (TME) and provides insight into the clinical activity observed with DKN-01-based treatment. IMPLICATIONS: mDKN-01 reverses a DKK1-mediated innate immune suppression in the TME and has additive efficacy with a PD-1 inhibitor.
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Anticuerpos Monoclonales/uso terapéutico , Inmunidad Innata/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular/uso terapéutico , Neoplasias/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/farmacología , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Ratones , Neoplasias/patología , Microambiente TumoralRESUMEN
PURPOSE: Metastatic castration-resistant prostate cancer (mCRPC) with low androgen receptor (AR) and without neuroendocrine signaling, termed double-negative prostate cancer (DNPC), is increasingly prevalent in patients treated with AR signaling inhibitors and is in need of new biomarkers and therapeutic targets. METHODS: Candidate genes enriched in DNPC were determined using differential gene expression analysis of discovery and validation cohorts of mCRPC biopsies. Laboratory studies were carried out in human mCRPC organoid cultures, prostate cancer (PCa) cell lines, and mouse xenograft models. Epigenetic studies were carried out in a rapid autopsy cohort. RESULTS: Dickkopf-1 (DKK1) expression is increased in DNPC relative to prostate-specific antigen (PSA)-expressing mCRPC in the Stand Up to Cancer/Prostate Cancer Foundation discovery cohort (11.2 v 0.28 reads per kilobase per million mapped reads; q < 0.05; n = 117) and in the University of Washington/Fred Hutchinson Cancer Research Center cohort (9.2 v 0.99 fragments per kilobase of transcript per million mapped reads; P < .0001). DKK1 expression can be regulated by activated Wnt signaling in vitro and correlates with activating canonical Wnt signaling mutations and low PSA mRNA in mCRPC biopsies (P < .05). DKK1 hypomethylation was associated with increased DKK1 mRNA expression (Pearson r = -0.66; P < .0001) in a rapid autopsy cohort (n = 7). DKK1-high mCRPC biopsies are infiltrated with significantly higher numbers of quiescent natural killer (NK) cells (P < .005) and lower numbers of activated NK cells (P < .0005). Growth inhibition of the human PCa model PC3 by the anti-DKK1 monoclonal antibody DKN-01 depends on the presence of NK cells in a severe combined immunodeficient xenograft mouse model. CONCLUSION: These results support DKK1 as a contributor to the immunosuppressive tumor microenvironment of DNPC. These data have provided the rationale for a clinical trial targeting DKK1 in mCRPC (ClinicalTrials.gov identifier: NCT03837353).
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Modulating T cell homeostatic mechanisms with checkpoint blockade can efficiently promote endogenous anti-tumor T cell responses1-11. However, many patients still do not benefit from checkpoint blockade12, highlighting the need for targeting of alternative immune pathways13. Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) is an attractive target for immunotherapy, owing to its capacity to promote effector T cell (Teff) functions14,15 and hamper regulatory T cell (Treg) suppression16-20. On the basis of the potent preclinical anti-tumor activity of agonist anti-GITR antibodies, reported by us and others16,21,22, we initiated the first in-human phase 1 trial of GITR agonism with the anti-GITR antibody TRX518 ( NCT01239134 ). Here, we report the safety profile and immune effects of TRX518 monotherapy in patients with advanced cancer and provide mechanistic preclinical evidence to rationally combine GITR agonism with checkpoint blockade in future clinical trials. We demonstrate that TRX518 reduces circulating and intratumoral Treg cells to similar extents, providing an easily assessable biomarker of anti-GITR activity. Despite Treg reductions and increased Teff:Treg ratios, substantial clinical responses were not seen. Similarly, in mice with advanced tumors, GITR agonism was not sufficient to activate cytolytic T cells due to persistent exhaustion. We demonstrate that T cell reinvigoration with PD-1 blockade can overcome resistance of advanced tumors to anti-GITR monotherapy. These findings led us to start investigating TRX518 with PD-1 pathway blockade in patients with advanced refractory tumors ( NCT02628574 ).
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Anticuerpos Monoclonales Humanizados/uso terapéutico , Proteína Relacionada con TNFR Inducida por Glucocorticoide/agonistas , Inmunoterapia/métodos , Animales , Biomarcadores de Tumor/inmunología , Diseño de Fármacos , Proteína Relacionada con TNFR Inducida por Glucocorticoide/antagonistas & inhibidores , Proteína Relacionada con TNFR Inducida por Glucocorticoide/inmunología , Humanos , Melanoma Experimental/inmunología , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos C57BL , Neoplasias/inmunología , Neoplasias/terapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Linfocitos T Reguladores/inmunologíaRESUMEN
BACKGROUND: Acute coronary syndrome is the leading cause of mortality worldwide. However, treatment of acute coronary occlusion inevitably results in ischemia-reperfusion injury. Circulating natural IgM has been shown to play a significant role in mouse models of ischemia-reperfusion injury. A highly conserved self-antigen, nonmuscle myosin heavy chain II, has been identified as a target of pathogenic IgM. We hypothesized that a monoclonal antibody (m21G6) directed against nonmuscle myosin heavy chain II may inhibit IgM binding and reduce injury in a preclinical model of myocardial infarction. Thus, our objective was to evaluate the efficacy of intravenous m21G6 treatment in limiting infarct expansion, troponin release, and left ventricular dysfunction in a swine myocardial infarction model. METHODS AND RESULTS: Massachusetts General Hospital miniature swine underwent occlusion of the midleft anterior descending coronary artery for 60 minutes, followed by 1 hour, 5-day, or 21-day reperfusion. Specificity and localization of m21G6 to injured myocardium were confirmed using fluorescently labeled m21G6. Treatment with m21G6 before reperfusion resulted in a 49% reduction in infarct size (P<0.005) and a 61% reduction in troponin-T levels (P<0.05) in comparison with saline controls at 5-day reperfusion. Furthermore, m21G6-treated animals recovered 85.4% of their baseline left ventricular function as measured by 2-dimensional transthoracic echocardiography in contrast to 67.1% in controls at 21-day reperfusion (P<0.05). CONCLUSIONS: Treatment with m21G6 significantly reduced infarct size and troponin-T release, and led to marked preservation of cardiac function in our study. Overall, these findings suggest that pathogenic IgM blockade represents a valid therapeutic strategy in mitigating myocardial ischemia-reperfusion injury.
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Anticuerpos Antiidiotipos/farmacología , Anticuerpos Monoclonales/farmacología , Inmunoglobulina M/inmunología , Infarto del Miocardio/tratamiento farmacológico , Miocardio/patología , Disfunción Ventricular Izquierda/prevención & control , Función Ventricular Izquierda/fisiología , Animales , Vasos Coronarios , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Ecocardiografía , Estudios de Seguimiento , Infarto del Miocardio/complicaciones , Infarto del Miocardio/fisiopatología , Miocardio/metabolismo , Cadenas Pesadas de Miosina/inmunología , Porcinos , Porcinos Enanos , Troponina T/metabolismo , Disfunción Ventricular Izquierda/etiología , Disfunción Ventricular Izquierda/fisiopatología , Función Ventricular Izquierda/efectos de los fármacosRESUMEN
High-mobility group box 1 protein (HMGB1), a DNA-binding nuclear and cytosolic protein, is a pro-inflammatory cytokine released by monocytes and macrophages. HMGB1 as well as its B box domain induce maturation of human dendritic cells (DCs). This report demonstrates that the B box domain induces phenotypic maturation of murine bone marrow-derived dendritic cells (BM-DCs) as evidenced by increased CD86, CD40 and MHC-II expression. The B box domain enhanced secretion of pro-inflammatory cytokines and chemokines: IL-1beta, IL-2, IL-5, IL-8, IL-12 and tumor necrosis factor (TNF)-alpha, but not IL-6 and IL-10. Furthermore, four peptides whose sequences correspond to different regions of HMGB1 induced production of IL-1beta, IL-2 and IL-12 (p70), but not IL-10 and IL-6 in mouse BM-DCs. Interestingly, these peptides differed in their capacity to induce TNF-alpha, IL-5, IL-18 and IL-8. B box domain as well as peptide-activated DCs acted as potent stimulators of allogeneic T cells in a mixed leukocyte reaction. DCs exposed to HMGB1 peptides induced proliferation of ovalbumin-specific syngeneic T cells. These DC-activating peptides could serve as an adjuvant in immunotherapeutic or vaccine context and the selective activity of these different peptides suggests a means to customize the functional properties of DCs.
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Citocinas/metabolismo , Células Dendríticas/efectos de los fármacos , Proteína HMGB1/genética , Proteína HMGB1/farmacología , Péptidos/inmunología , Secuencia de Aminoácidos , Animales , Línea Celular , Células Dendríticas/inmunología , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia MolecularRESUMEN
Formation of a neointima is associated with grafted artery or vein, angioplasty, and stent and inferior vena cava filter (IVCF) implantation. Contributing to the neointima is a population of vascular smooth muscle cells (SMC) that migrates from media and subsequently proliferates within intima. The purpose of this present study was to culture SMC from normal vessel wall and from neointima and to compare migration and growth of these cells. Neointima was stimulated in the vena cava of pigs by placement of an IVCF for 30 days. Tissue was taken from the thickened wall between the struts and from a normal segment of the IVCF. After removal of the endothelium and adventitia, explants were placed in culture dishes and were observed for the migration of cells. Immunoassay for smooth muscle alpha-actin was used to identify cell origin. Proliferation was determined by cell counting. The cell cycle regulator cyclin D1 was detected by Western blot analysis. SMC phenotype was confirmed by positive immunostaining for smooth muscle alpha-actin. Cells migrated from the neointimal explants (NI-SMC) more rapidly than cells from explants of normal media (NM-SMC). Proliferation of NI-SMC was also more rapid than NM-SMC with or without exogenous mitogens. NI-SMC expressed more cyclin D1 than NM-SMC. Injury to the vena cava triggered neointima formation characterized by the expansion of a population of SMC with increased migration and replication compared with SMC from normal regions of the vessel.
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Movimiento Celular/fisiología , Proliferación Celular , Miocitos del Músculo Liso/fisiología , Túnica Íntima/patología , Filtros de Vena Cava , Vena Cava Inferior/patología , Animales , Implantación de Prótesis Vascular , Ciclina D1/metabolismo , Porcinos , Túnica Íntima/metabolismo , Vena Cava Inferior/cirugíaRESUMEN
Severe sepsis, a lethal syndrome after infection or injury, is the third leading cause of mortality in the United States. The pathogenesis of severe sepsis is characterized by organ damage and accumulation of apoptotic lymphocytes in the spleen, thymus, and other organs. To examine the potential causal relationships of apoptosis to organ damage, we administered Z-VAD-FMK, a broad-spectrum caspase inhibitor, to mice with sepsis. We found that Z-VAD-FMK-treated septic mice had decreased levels of high mobility group box 1 (HMGB1), a critical cytokine mediator of organ damage in severe sepsis, and suppressed apoptosis in the spleen and thymus. In vitro, apoptotic cells activate macrophages to release HMGB1. Monoclonal antibodies against HMGB1 conferred protection against organ damage but did not prevent the accumulation of apoptotic cells in the spleen. Thus, our data indicate that HMGB1 production is downstream of apoptosis on the final common pathway to organ damage in severe sepsis.
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Apoptosis/inmunología , Proteína HMGB1/fisiología , Sepsis/mortalidad , Sepsis/patología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Anticuerpos Monoclonales/uso terapéutico , Apoptosis/efectos de los fármacos , Inhibidores de Caspasas , Femenino , Proteína HMGB1/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Sepsis/inmunología , Sepsis/terapiaRESUMEN
Migration and proliferation of vascular smooth muscle cells (VSMCs) are two events involved in atherosclerosis, restenosis after balloon angioplasty, and stenosis of grafted vessels. Platelet-derived growth factor (PDGF) found in stenotic vessels is known to induce migration of VSMCs. VSMCs express both alpha- and beta-adrenergic receptors on their surface, and blood vessels are innervated by the adrenergic nervous system and exposed to circulating epinephrine. We examined the role of these receptors on PDGF-induced migration of VSMCs. VSMCs were cultured from saphenous vein segments. Migration was stimulated by PDGF. Effect of pretreatment of VSMCs with the beta-agonist isoproterenol, the alpha-agonist phenylephrine, or forskolin on PDGF-induced migration was examined with a modified Boyden chamber. Cell migration was quantitated by spectrophotometry. Intracellular cyclic AMP was determined by radioimmunoassay. PDGF significantly induced VSMC migration. Isoproterenol (0.1 and 1.0 microM) inhibited PDGF-induced migration by 30 per cent and 50 per cent, respectively. Forskolin (10 microM) completely blocked PDGF-induced migration. The migration inhibition by isoproterenol or forskolin was associated with a significant elevation of intracellular cyclic AMP. In contrast, phenylephrine had no effect on PDGF-induced migration or on cyclic AMP. Activation of beta-adrenergic receptors and the consequent rise in intracellular cyclic AMP inhibits migration of VSMCs induced by PDGF. These results are consistent with the notion that adrenergic agonists with substantial beta-receptor affinity, such as isoproterenol, can inhibit smooth muscle cell migration.
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Agonistas Adrenérgicos beta/farmacología , Movimiento Celular/fisiología , Músculo Liso Vascular/citología , Receptores Adrenérgicos beta/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colforsina/farmacología , AMP Cíclico/metabolismo , Humanos , Técnicas In Vitro , Líquido Intracelular/metabolismo , Isoproterenol/farmacología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Fenilefrina/farmacología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Radioinmunoensayo , Receptores Adrenérgicos beta/metabolismo , Vena Safena/citología , EspectrofotometríaRESUMEN
BACKGROUND: Therapeutic and prophylactic vena cava filters (VCFs) are used to prevent pulmonary embolism. Concerns exist over placing a permanent filter in a young trauma patient. Recently, retrievable VCFs have become available. One such filter is the OptEase, which has a recommended time of removal of up to 23 days after insertion. Data supporting this recommendation are sparse. Many trauma patients will need filters for more than 2 weeks, and there are no data evaluating the safety of removal after extended time periods. The purpose of this study was to determine the safety, feasibility, and reaction of the vena cava when removing the OptEase retrievable VCF at different time intervals. METHODS: Twenty Yorkshire cross pigs (80-113 kg) underwent general anesthesia with tiletamine and zolazepam. Filters were placed in the infrarenal vena cava (VC) through the femoral vein under fluoroscopic guidance. Animals were then divided into four groups. In group 1, filters were removed at 14 days; in group 2, at 30 days; in group 3, at 60 days; and in group 4, at 90 days. Removal was attempted using a snare-and-sheath technique through the femoral vein. Animals with successful filter removal were allowed to recover; then, the animals underwent autopsy (gross and microscopic VC examination) 2 months later. Animals with unsuccessful filter removal underwent autopsy immediately after attempted removal. Venacavograms were taken at filter insertion, at removal, and before autopsy to evaluate any VC abnormalities. RESULTS: Successful removal of the filter in all five pigs (100%) was reliably performed only in the 14-day group. In this group, the initial VC transverse diameter was 19.4 +/- 0.8 mm and was significantly reduced to 9.8 +/- 1.1 mm (p < 0.05) immediately after removal. Sixty days later, before autopsy, VC diameter had increased to 15.3 +/- 1.9 mm, which was significantly larger than at removal (p < 0.05) but not different from the initial value. In the 30-day group, removal was successful in only one of five animals. Although removal was successful in the one pig, autopsy at 2 months postremoval revealed total occlusion of the VC. Filters could not be removed from 60- and 90-day groups. At autopsy, the VCF struts were embedded or protruded through the VC wall. Microscopic examination of the VC revealed significant scarring underneath and between the struts. CONCLUSION: Removal of the retrievable OptEase VCF may be successfully performed up to 14 days after insertion. Strut protrusion through the VC wall prohibited successful and safe removal at extended time intervals.
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Embolia Pulmonar/prevención & control , Filtros de Vena Cava , Vena Cava Inferior/lesiones , Trombosis de la Vena/complicaciones , Animales , Falla de Equipo , Embolia Pulmonar/etiología , Porcinos , Factores de Tiempo , Vena Cava Inferior/patologíaRESUMEN
The monoclonal theory of atherosclerosis postulates that a subpopulation of vascular smooth muscle cells (VSMCs) is selectively expanded in response to pathologic stimuli and accumulates in vascular intima. The purpose of this research was to clone VSMC, determine growth rates of the clones and their ability to release the mitogenic cytokine tumor necrosis factor-alpha (TNF-alpha). With approval of the institutional animal care and use committee, VSMCs were isolated and cultured from the thoracic aortas of three rats. To confirm that the cells in primary culture were of smooth muscle origin, they were immunostained with anti-alpha-smooth muscle-actin antibodies. Single cell-derived individual colonies with uniform appearance were surrounded by cloning rings, released with trypsin, and expanded. Growth rates of the clones were assessed by the mitochondrial dependent reduction of methyltetrazolium (MTT) to formazan after 24-hour stimulation with 10 per cent serum. Additionally, cloned cells were stimulated with 0.1, 1, 10, and 20 microg/mL lipopolysaccharide (LPS) for 24 hours, and TNF-alpha was determined in the culture medium. Data were analyzed by ANOVA. Two clones were isolated that could be divided into categories based on distinctly different morphology: 1) spindle-shaped (SP) or 2) epithelioid-shaped (EP) VSMCs. The SP clone had a growth rate that was 25 per cent higher than the EP clone (P < 0.05). Also, the SP clone had significantly higher release of TNF-alpha in response to LPS. For instance, TNF-alpha released in response to 0.1 microg/mL of LPS in the SP clone was 157 +/- 45 pg/mL versus 21 +/- 8.5 pg/mL in the EP clone (P < 0.05). Primary cultures of rat VSMCs are heterogeneous and consist of at least two morphologically distinct cell types. These clones are different in growth rate and cytokine production. It is possible that selective expansion of one of these clones contributes to formation of stenotic vascular lesions.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Clonación de Organismos , Lipopolisacáridos/farmacología , Músculo Liso Vascular/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Análisis de Varianza , Animales , Aorta Torácica , Western Blotting , Células Cultivadas , Masculino , Modelos Animales , Músculo Liso Vascular/citología , Fenotipo , Probabilidad , Ratas , Ratas Sprague-Dawley , Sensibilidad y Especificidad , Factor de Necrosis Tumoral alfa/análisisRESUMEN
Tumor necrosis factor (TNF)-alpha is implicated in development of restenotic and atherosclerotic vascular lesions, which are pathological processes involving both proliferation and apoptosis of vascular smooth muscle cells (VSMCs). Human VSMCs were recently found to contain heterogeneous subpopulations. We therefore examined whether TNF has different effects on distinct subpopulations of VSMCs. With the use of cloning techniques, two stable subpopulations of VSMCs were isolated from human saphenous vein: spindle- and epithelioid-shaped smooth muscle cells (Sp- and Ep-SMCs, respectively). We found that TNF stimulated growth in Sp-SMCs but had a toxic effect on Ep-SMCs. TNF did not induce apoptosis in Sp-SMCs as determined by nuclear staining and cellular DNA electrophoresis. In contrast, the reduction of viability in Ep-SMCs was associated with induction of apoptosis as characterized by cellular DNA fragmentation and nuclear condensation. Higher levels of the TNF-R1 receptor subtype were detected in membrane preparations from Ep-SMCs than in membranes from Sp-SMCs. Activation of caspase-3 was also selectively induced in Ep-SMCs but not in Sp-SMCs. Cycloheximide, an inhibitor of protein synthesis, enhanced the toxicity of TNF in Ep-SMCs. This effect of cycloheximide was not seen in Sp-SMCs. The data presented here demonstrate for the first time that TNF either promotes growth or induces apoptosis in human VSMCs depending on phenotype.
Asunto(s)
Apoptosis/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Vena Safena/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Caspasa 3 , Caspasas/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cicloheximida/farmacología , Activación Enzimática/efectos de los fármacos , Humanos , Masculino , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/fisiología , Fenotipo , Inhibidores de la Síntesis de la Proteína/farmacología , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Proteínas Recombinantes/farmacología , Vena Safena/citología , Vena Safena/fisiologíaRESUMEN
Remodeling of cerebral arteries in hypertension produces thickened vessel walls associated with atherosclerotic plaque formation. In both thickening and plaque formation, proliferation of vascular smooth muscle cells is a hallmark. Genetically hypertensive rats treated with an angiotensin II (Ang II) AT1 receptor antagonist inhibited thickening of cerebral arteries suggesting a mitogenic action of Ang II on cerebral arterial VSMC (CVSMC). However, in studies using smooth muscle cells cultured from peripheral arteries, Ang II causes cell hypertrophy, but not proliferation. We determined the effect of Ang II on proliferation of cultured human CVSMC. CVSMC were cultured from the basilar artery obtained at autopsy. Ang II (10(-7) M) stimulated proliferation determined by counting cells and mitochondrial activity assay. Synthesis and release of basic fibroblast growth factor (bFGF) was essential for Ang II-stimulated proliferation. These findings are consistent with the notion that Ang II stimulates CVSMC proliferation thereby contributing to vessel remodeling.
Asunto(s)
Angiotensina II/metabolismo , Arterias Cerebrales/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Angiotensina II/farmacología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Arterias Cerebrales/metabolismo , Humanos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismoRESUMEN
Reperfusion after cardiopulmonary bypass causes induction of reactive oxygen species (ROS), elevated plasma levels of bacterial lipopolysaccharide (LPS), and production of tumor necrosis factor-alpha (TNF) by the heart. Nuclear factor-kappaB (NF-kappaB) regulates the expression of TNF. Because NF-kappaB is activated by both LPS and ROS, we hypothesized that an inhibitor of NF-kappaB, pyrrolidine dithiocarbamate (PDTC), would block release of TNF from the heart stimulated by these two agents. With Institutional animal care and use committee (IACUC) approval, rat hearts were perfused Langendorf style. LPS was infused and ROS were generated with a hypoxanthine/xanthine oxidase system. PDTC was added to the perfusion buffer. Other hearts were treated with forskolin in order to elevate cyclic AMP. Timed collections of coronary effluent were made for the determination of coronary flow and measurement of TNF. LPS stimulated TNF release to a maximum of 2247 +/- 133 pg/min at 150 minutes. PDTC inhibited LPS-stimulated TNF release. For instance, at 150 minutes, LPS-stimulated TNF release was 449 +/- 49 pg/min with 100 microM PDTC and was 70 +/- 65 pg/mL with 250 microM PDTC (P < 0.05 vs LPS alone). ROS stimulated TNF release was 1494 +/- 130 pg/min at 150 minutes and was not affected by PDTC. Forskolin almost completely blocked TNF release stimulated by LPS or ROS. These data are consistent with the notion that inhibitors of NF-kappaB block cytokine production stimulated by some agents but not others.
Asunto(s)
Lipopolisacáridos/metabolismo , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Antioxidantes/farmacología , Quimioterapia del Cáncer por Perfusión Regional/métodos , Técnicas In Vitro , Masculino , Modelos Animales , Miocardio/metabolismo , FN-kappa B/antagonistas & inhibidores , Pirrolidinas/farmacología , Ratas , Ratas Sprague-Dawley , Tiocarbamatos/farmacología , Factor de Necrosis Tumoral alfa/efectos de los fármacosRESUMEN
Neointimal proliferation with plaque formation is the principal cause of coronary artery disease. In the neointima, inflammatory cytokines like tumor necrosis factor-alpha (TNF-alpha) are expressed by vascular smooth muscle cells (VSMCs). These cytokines stimulate proliferation and migration of VSMCs, events that are crucial to neointima formation. Stents, liberating rapamycin, have been shown to reduce neointima formation in human coronary arteries. The purpose of this study was to determine if rapamycin could inhibit the production of TNF-alpha by VSMCs. With institutional review board approval, VSMCs were cultured from saphenous vein segments obtained from five patients. Cells were identified as VSMC by immunostaining for smooth muscle alpha-actin. Cells were exposed to bacterial lipopolysaccharide (LPS), LPS plus rapamycin, or LPS plus isoproterenol for 24 hours. Cells with no treatment served as controls. The culture medium was then removed and analyzed for TNF-alpha. Additionally, the effect of treatment on viability was determined by assay of mitochondrial activity. TNF-alpha released into the culture medium is expressed as pg TNF-alpha/mg cell protein. Statistical analysis was by ANOVA. In control cells, TNF-alpha was undetectable in the culture medium. The addition of LPS (10 microg/mL) increased TNF-alpha release to 4312 +/- 705 pg/mg at 24 hours. The addition of 1 ng/mL rapamycin with LPS reduced TNF-alpha production 50 per cent (P < 0.01 vs LPS alone). A similar reduction of TNF-alpha release was seen with 1 microM isoproterenol. LPS, rapamycin, or isoproterenol did not affect cell viability. These data show that rapamycin effectively inhibits the release of TNF-alpha from VSMCs stimulated with inflammatory mediators like LPS. Rapamycin is as effective as agents that raise intracellular cyclic AMP (e.g., isoproterenol). Therefore, a potential mechanism for the effectiveness of rapamycin-releasing stents is reduction of inflammatory cytokine expression by VSMCs.