RESUMEN
Perimenstrual worsening of asthma occurs in up to 40% of women with asthma, leading to increased acute exacerbations requiring clinical care. The role of sex hormones during these times remains unclear. In the current study, we used a translational approach to determine whether progesterone exacerbates allergic inflammation in the traditional chicken egg ovalbumin (OVA) model in BALB/c mice. Simultaneously, we used peripheral blood mononuclear cells (PBMC) from healthy human donors to assess the effects of progesterone on circulating group 2 innate lymphoid cells (ILC2). Briefly, lungs of ovariectomized (OVX) or sham-operated female (F-Sham) controls were implanted with a progesterone (P4, 25 mg) (OVX-P4) or placebo pellet (OVX-Placebo), followed by sensitization and challenge with ovalbumin (OVA). Progesterone increased total inflammatory histologic scores, increased hyper-responsiveness to methacholine (MCh), increased select chemokines in the bronchoalveolar lavage (BAL) and serum, and increased ILC2 and neutrophil numbers, along the airways compared with F-Sham-OVA and OVX-Placebo-OVA animals. Lung ILC2 were sorted from F-Sham-OVA, OVX-Placebo-OVA and OVX-P4-OVA treated animals and stimulated with IL-33. OVX-P4-OVA lung ILC2 were more responsive to interleukin 33 (IL-33) compared with F-Sham-OVA treated, producing more IL-13 and chemokines following IL-33 stimulation. We confirmed the expression of the progesterone receptor (PR) on human ILC2, and showed that P4 + IL-33 stimulation also increased IL-13 and chemokine production from human ILC2. We establish that murine ILC2 are capable of responding to P4 and thereby contribute to allergic inflammation in the lung. We confirmed that human ILC2 are also hyper-responsive to P4 and IL-33 and likely contribute to airway exacerbations following allergen exposures in asthmatic women with increased symptoms around the time of menstruation.NEW & NOTEWORTHY There is a strong association between female biological sex and severe asthma. We investigated the allergic immune response, lung pathology, and airway mechanics in the well-described chicken egg ovalbumin (OVA) model with steady levels of progesterone delivered throughout the treatment period. We found that progesterone enhances the activation of mouse group 2 innate lymphoid cells (ILC2). Human ILC2 are also hyper-responsive to progesterone and interleukin 33 (IL-33), and likely contribute to airway exacerbations following allergen exposures in women with asthma.
Asunto(s)
Asma , Pulmón , Linfocitos , Ratones Endogámicos BALB C , Ovalbúmina , Progesterona , Progesterona/farmacología , Animales , Femenino , Linfocitos/inmunología , Linfocitos/metabolismo , Humanos , Asma/inmunología , Asma/patología , Asma/metabolismo , Ratones , Ovalbúmina/inmunología , Pulmón/patología , Pulmón/inmunología , Pulmón/metabolismo , Inmunidad Innata/efectos de los fármacos , Interleucina-33/metabolismo , Hipersensibilidad/inmunología , Hipersensibilidad/patología , Hipersensibilidad/metabolismo , Inflamación/patología , Inflamación/inmunología , Inflamación/metabolismo , Modelos Animales de EnfermedadRESUMEN
Immunotherapies including checkpoint blockade immunotherapy (CBI) and chimeric antigen receptor T cells (CAR-T) have revolutionized cancer treatment for patients with certain cancers. However, these treatments are not effective for all cancers, and even for those cancers that do respond, not all patients benefit. Most cancer patients have elevated levels of myeloid-derived suppressor cells (MDSCs) that are potent inhibitors of antitumor immunity, and clinical and animal studies have demonstrated that neutralization of MDSCs may restore immune reactivity and enhance CBI and CAR-T immunotherapies. MDSCs are homeostatically regulated in that elimination of mature circulating and intratumoral MDSCs results in increased production of MDSCs from bone marrow progenitor cells. Therefore, targeting MDSC development may provide therapeutic benefit. The pro-inflammatory molecules S100A8/A9 and high mobility group box protein 1 (HMGB1) and their receptor RAGE are strongly associated with the initiation and progression of most cancers. This article summarizes the literature demonstrating that these molecules are integrally involved in the early development, accumulation, and suppressive activity of MDSCs, and postulates that S100A8/A9 and HMGB1 serve as early biomarkers of disease and in conjunction with RAGE are potential targets for reducing MDSC levels and enhancing CBI and CAR-T immunotherapies.
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RATIONALE: Asthma is a chronic airway condition that occurs more often in women than men during reproductive years. Population studies have collectively shown that long-term use of oral contraceptives decreased the onset of asthma in women of reproductive age. In the current study, we hypothesized that steady-state levels of estrogen would reduce airway inflammation and airway hyperresponsiveness to methacholine challenge. METHODS: Ovariectomized BALB/c mice (Ovx) were implanted with subcutaneous hormone pellets (estrogen, OVX-E2) that deliver consistent levels of estrogen [68 ± 2 pg/mL], or placebo pellets (OVX-Placebo), followed by ovalbumin sensitization and challenge. In conjunction with methacholine challenge, immune phenotyping was performed to correlate inflammatory proteins and immune populations with better or worse pulmonary outcomes measured by invasive pulmonary mechanics techniques. RESULTS: Histologic analysis showed an increase in total cell infiltration and mucus staining around the airways leading to an increased inflammatory score in ovarectomized (OVX) animals with steady-state estrogen pellets (OVX-E2-OVA) as compared to other groups including female-sham operated (F-INTACT-OVA) and OVX implanted with a placebo pellet (OVX-Pl-OVA). Airway resistance (Rrs) and lung elastance (Ers) were increased in OVX-E2-OVA in comparison to F-INTACT-OVA following aerosolized intratracheal methacholine challenges. Immune phenotyping revealed that steady-state estrogen reduced CD3+ T cells, CD19+ B cells, ILC2 and eosinophils in the BAL across all experiments. While these commonly described allergic cells were reduced in the BAL, or airways, we found no changes in neutrophils, CD3+ T cells or CD19+ B cells in the remaining lung tissue. Similarly, inflammatory cytokines (IL-5 and IL-13) were also decreased in OVX-E2-OVA-treated animals in comparison to Female-INTACT-OVA mice in the BAL, but in the lung tissue IL-5, IL-13 and IL-33 were comparable in OVX-E2-OVA and F-INTACT OVA mice. ILC2 were sorted from the lungs and stimulated with exogenous IL-33. These ILC2 had reduced cytokine and chemokine expression when they were isolated from OVX-E2-OVA animals, indicating that steady-state estrogen suppresses IL-33-mediated activation of ILC2. CONCLUSIONS: Therapeutically targeting estrogen receptors may have a limiting effect on eosinophils, ILC2 and potentially other immune populations that may improve asthma symptoms in those females that experience perimenstrual worsening of asthma, with the caveat, that long-term use of estrogens or hormone receptor modulators may be detrimental to the lung microenvironment over time.
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Asma , Interleucina-33 , Femenino , Animales , Ratones , Interleucina-33/uso terapéutico , Estradiol/farmacología , Estradiol/uso terapéutico , Inmunidad Innata , Interleucina-13/uso terapéutico , Cloruro de Metacolina/farmacología , Cloruro de Metacolina/uso terapéutico , Alérgenos/uso terapéutico , Resistencia de las Vías Respiratorias , Interleucina-5/uso terapéutico , Líquido del Lavado Bronquioalveolar , Linfocitos/metabolismo , Linfocitos/patología , Pulmón/metabolismo , Asma/tratamiento farmacológico , Asma/metabolismo , Citocinas , Estrógenos/uso terapéuticoRESUMEN
The mechanisms underlying muscle dysfunction with Chronic Obstructive Pulmonary Disease (COPD) are poorly understood. Indirect evidence has recently suggested a role of Advanced Glycation End Products (AGEs) and their receptor (RAGE) in the pathophysiology of COPD. Accordingly, this study aimed to examine the redox balance and mitochondrial alterations in the skeletal muscle of a mouse model deficient in the receptor for AGE (RAGE-KO) and wild-type C57BL/6 exposed to cigarette smoke for 8-months using immunoblotting, spectrophotometry, and high-resolution respirometry. Cigarette smoke exposure increased by two-fold 4-HNE levels (P < 0.001), a marker of oxidative stress, and markedly downregulated contractile proteins, mitochondrial respiratory complexes, and uncoupling proteins levels (P < 0.001). Functional alterations with cigarette smoke exposure included a greater reliance on complex-I supported respiration (P < 0.01) and lower relative respiratory capacity for fatty acid (P < 0.05). RAGE knockout resulted in 47% lower 4-HNE protein levels than the corresponding WT control mice exposed to cigarette smoke (P < 0.05), which was partly attributed to increased Complex III protein levels. Independent of cigarette smoke exposure, RAGE KO decreased mitochondrial specific maximal respiration (P < 0.05), resulting in a compensatory increase in mitochondrial content measured by citrate synthase activity (P < 0.001) such that muscle respiratory capacity remained unaltered. Together, these findings suggest that knockout of RAGE protected the skeletal muscle against oxidative damage induced by 8 months of cigarette smoke exposure. In addition, this study supports a role for RAGE in regulating mitochondrial content and function and can thus serve as a potential therapeutic target.
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Fumar Cigarrillos , Enfermedad Pulmonar Obstructiva Crónica , Ratones , Animales , Receptor para Productos Finales de Glicación Avanzada , Fumar Cigarrillos/efectos adversos , Ratones Noqueados , Ratones Endogámicos C57BL , Estrés Oxidativo , Mitocondrias/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Productos Finales de Glicación Avanzada/genética , Productos Finales de Glicación Avanzada/metabolismoRESUMEN
Asthmatic women tend to develop severe airway disease in their reproductive years, and 30%-40% of asthmatic women have peri-menstrual worsening of asthma symptoms. This indicates that fluctuations in ovarian hormones are involved in advancement of asthmatic disease and exacerbation of symptoms. Group 2 innate lymphoid cells, or ILC2, are readily detected in allergic conditions, such as rhinosinusitis, in individuals that develop nasal polyps do to allergen exposures, and in allergic asthma. ILC2 are airway localized immune cells activated by IL-33, an innate cytokine that perpetuates allergic inflammation by driving the production of IL-5 and IL-13. We have previously shown that ILC2 are highly activated in naïve and ovalbumin (OVA) challenged, female BALB/c mice in comparison to male mice following stimulation with IL-33. Here, we investigated the effect of steady-state ovarian hormones on ILC2 and the NF-κB signaling pathway following OVA sensitization and challenge. We found that estrogen-treated ovariectomized mice (OVX-E2) that had been challenged with OVA had reduced IL-5 and IL-13 production by lung ILC2 as compared to lung ILC2 isolated from intact male and female sham-operated controls that had been treated with OVA. ILC2 were isolated from untreated animals and co-cultured ex vivo with and without estrogen plus IL-33. Those estrogen-treated ILC2 similarly produced less IL-5 and IL-13 in comparison to untreated, and had reduced NF-κB activation. Single-cell RNA sequencing showed that 120 genes were differentially expressed in male and female ILC2, and Nfkb1 was found among top-ranked regulatory interactions. Together, these results provide new insight into the suppressive effect of estrogen on ILC2 which may be protective in female asthmatics. Understanding further how estrogen modulates ILC2 may provide therapeutic targets for the treatment of allergic diseases.
RESUMEN
Apoptosis is a physiological and anti-inflammatory form of cell death that is indispensable for normal physiology and homeostasis. Several studies have reported aberrant activation of apoptosis in various tissues at the onset of hypertension. However, the functional significance of apoptosis during essential hypertension remains largely undefined. The current study was designed to test the hypothesis that apoptosis contributes to sex differences in blood pressure and the T cell profile in spontaneously hypertensive rats (SHR). Apoptosis was measured in kidney, aorta and spleen of 13-week-old adult hypertensive male and female SHR. Female SHR had greater renal and aortic apoptosis compared to age-matched males; apoptosis in the spleen was comparable between the sexes. Based on well-established sex differences in hypertension, we tested the hypothesis that greater apoptosis in female SHR contributes to the lower BP and pro-inflammatory profile compared to males. Male and female SHR were randomized to receive vehicle or ZVAD-FMK, a cell permeable pan-caspase inhibitor, in established hypertension from 13 to 15 weeks of age or at the onset of hypertension from 6 to 12 weeks or age. Treatment with ZVAD-FMK lowered renal apoptosis in both studies, yet neither BP nor renal T cells were altered in either male or female SHR. These results suggest that apoptosis does not contribute to the control or maintenance of BP in male or female SHR or sex differences in renal T cells.
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Because patients with chronic obstructive pulmonary disease (COPD) are often physically inactive, it is still unclear whether the lower respiratory capacity in the locomotor muscles of these patients is due to cigarette smoking per se or is secondary to physical deconditioning. Accordingly, the purpose of this study was to examine mitochondrial alterations in the quadriceps muscle of 10 mice exposed to 8 mo of cigarette smoke, a sedentary mouse model of emphysema, and 9 control mice, using immunoblotting, spectrophotometry, and high-resolution respirometry in permeabilized muscle fibers. Mice exposed to smoke displayed a twofold increase in the oxidative stress marker, 4-HNE, (P < 0.05) compared with control mice. This was accompanied by significant decrease in protein expression of UCP3 (65%), ANT (58%), and mitochondrial complexes II-V (â¼60%-75%). In contrast, maximal ADP-stimulated respiration with complex I and II substrates (CON: 23.6 ± 6.6 and SMO: 19.2 ± 8.2 ρM·mg-1·s-1) or octanoylcarnitine (CON: 21.8 ± 9.0 and SMO: 16.5 ± 6.6 ρM·mg-1·s-1) measured in permeabilized muscle fibers, as well as citrate synthase activity, were not significantly different between groups. Collectively, our findings revealed that sedentary mice exposed to cigarette smoke for 8 mo, which is typically associated with pulmonary inflammation and emphysema, exhibited a preserved mitochondrial respiratory capacity for various substrates, including fatty acid, in the skeletal muscle. However, the mitochondrial adaptations induced by cigarette smoke favored the development of chronic oxidative stress, which can indirectly contribute to augment the susceptibility to muscle fatigue and exercise intolerance.NEW & NOTEWORTHY It is unclear whether the exercise intolerance and skeletal muscle mitochondrial dysfunction observed in patients with COPD is due to cigarette smoke exposure, per se, or if they are secondary consequences to inactivity. Herein, while long-term exposure to cigarette smoke induces oxidative stress and an altered skeletal muscle phenotype, cigarette smoke does not directly contribute to mitochondrial dysfunction. With this evidence, we demonstrate the critical role of physical inactivity in cigarette smoke-related skeletal muscle dysfunction.
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Adaptación Fisiológica/efectos de los fármacos , Mitocondrias Musculares/metabolismo , Músculo Esquelético/ultraestructura , Nicotiana , Humo/efectos adversos , Animales , Citrato (si)-Sintasa/metabolismo , Modelos Animales de Enfermedad , Enfisema/patología , Femenino , Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Musculares/genética , Estrés Oxidativo , Consumo de Oxígeno , Músculo Cuádriceps/ultraestructura , Conducta SedentariaRESUMEN
Metastatic cancer has a poor prognosis. Novel pharmacologic targets need to be identified. The receptor for advanced glycation endproducts (RAGE) is a pattern recognition receptor constitutively expressed in the lungs. Absence of overt disease in RAGE null mice suggests that RAGE is unnecessary or redundant in health. We report that RAGE null tumor-bearing mice have reduced lung metastasis and improved survival. Bone marrow chimera studies suggest that hematopoietic cell RAGE is an important contributor to these effects. Deletion of RAGE reduces both the quantity and suppressive activity of tumor-induced MDSC. Protein and mRNA studies suggest that RAGE contributes to the generation and function of MDSC including expression of the alarmins S100A8/A9 and activity of inducible nitric oxide synthase, arginase-1, and NF-κB. These findings demonstrate the important role of RAGE in determining the quantity and function of tumor-associated MDSC and suggest RAGE as a pharmacologic target for patients with metastatic disease.
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Pulmón/patología , Melanoma/metabolismo , Células Supresoras de Origen Mieloide/inmunología , Neoplasias Experimentales/metabolismo , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Animales , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Diferenciación Celular , Humanos , Tolerancia Inmunológica , Melanoma/inmunología , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Metástasis de la Neoplasia , Neoplasias Experimentales/inmunología , Receptor para Productos Finales de Glicación Avanzada/genética , Microambiente TumoralRESUMEN
CASE PRESENTATION: A 73-year-old man presented to the ED of an outside hospital with asymptomatic chest wall swelling 10 h after discharge from our hospital. Four days earlier, he had presented to our hospital with increased dyspnea, cough, and sputum production. His history was notable for severe COPD with bullous emphysema. Chest imaging demonstrated bilateral opacities and a collection of gas and liquid in the major fissure of the left lung. A catheter was placed into the collection of gas and liquid under imaging guidance. After 4 days, the catheter was removed without event and the patient was discharged from the hospital with an extended course of antibiotics. Imaging performed in the ED revealed gas in the tissues of the chest wall and no evidence of a pneumothorax. He was transported back to our hospital by helicopter.
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Fístula Bronquial/complicaciones , Catéteres/efectos adversos , Fístula Cutánea/complicaciones , Descompresión Quirúrgica/instrumentación , Remoción de Dispositivos/efectos adversos , Enfisema Subcutáneo/etiología , Anciano , Fístula Bronquial/diagnóstico , Fístula Bronquial/cirugía , Fístula Cutánea/diagnóstico , Fístula Cutánea/cirugía , Humanos , Masculino , Enfisema Subcutáneo/diagnóstico , Enfisema Subcutáneo/cirugía , Tomografía Computarizada por Rayos XRESUMEN
The receptor for advanced glycation end products (RAGE), a cell membrane receptor, recognizes ligands produced by cigarette smoke (CS) and has been implicated in the pathogenesis of COPD. We demonstrate that deletion or pharmacologic inhibition of RAGE prevents development of CS-induced emphysema. To identify molecular pathways by which RAGE mediates smoking related lung injury we performed unbiased gene expression profiling of alveolar macrophages (AM) obtained from RAGE null and C57BL/6 WT mice exposed to CS for one week or four months. Pathway analysis of RNA expression identified a number of genes integral to the pathogenesis of COPD impacted by the absence of RAGE. Altered expression of antioxidant response genes and lung protein 4-HNE immunostaining suggest attenuated oxidative stress in the RAGE null mice despite comparable CS exposure and lung leukocyte burden as the WT mice. Reduced endoplasmic reticulum stress in response to CS exposure also was observed in the AM from RAGE null mice. These findings provide novel insight into the sources of oxidative stress, macrophage activation, and the pathogenesis of lung disease due to CS exposure.
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Fumar Cigarrillos/efectos adversos , Enfisema/fisiopatología , Pulmón/patología , Activación de Macrófagos , Macrófagos Alveolares/inmunología , Estrés Oxidativo , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Animales , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor para Productos Finales de Glicación Avanzada/deficiencia , Humo/efectos adversosRESUMEN
Pulmonary innate immune responses involve a highly regulated multicellular network to defend the enormous surface area of the lung. Disruption of these responses renders the host susceptible to pneumonia. Alveolar epithelial cells (AEC) are a critical source of innate immune molecules such as GM-CSF, which determine the functional maturation of alveolar macrophages. In many pulmonary diseases, heterogeneous ventilation leads to regional hypoxia in the lung. The effect of hypoxia on AEC innate immune function is unknown. We now report that exposure of primary murine AEC to hypoxia (1% oxygen) for 24 h results in significant suppression of key innate immune molecules, including GM-CSF, CCL2, and IL-6. This exposure did not cause toxicity but did induce stabilization of hypoxia-inducible factor 1α protein (HIF-1α) and shift to glycolytic metabolism. Focusing on GM-CSF, we found that hypoxia greatly decreased the rate of GM-CSF transcription. Hypoxia both decreased NF-κB signaling in AEC and induced chromosomal changes, resulting in decreased accessibility in the GM-CSF proximal promoter of target sequences for NF-κB binding. In mice exposed to hypoxia in vivo (12% oxygen for 2 d), lung GM-CSF protein expression was reduced. In vivo phagocytosis of fluorescent beads by alveolar macrophages was also suppressed, but this effect was reversed by treatment with GM-CSF. These studies suggest that in critically ill patients, local hypoxia may contribute to the susceptibility of poorly ventilated lung units to infection through complementary effects on several pathways, reducing AEC expression of GM-CSF and other key innate immune molecules.
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Células Epiteliales Alveolares/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Hipoxia/inmunología , Pulmón/patología , Macrófagos Alveolares/inmunología , Animales , Células Cultivadas , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Inmunidad Innata , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B/genética , FN-kappa B/metabolismo , Fagocitosis , Regiones Promotoras Genéticas/genética , Transducción de SeñalRESUMEN
Patients with acute lung injury almost always require supplemental oxygen during treatment; however, elevated oxygen itself is toxic. Receptors for advanced glycation end-products (RAGE) are multi-ligand cell surface receptors predominantly localized to alveolar type I cells that influence development and cigarette smoke-induced inflammation, but studies that address the role of RAGE in acute lung injury are insufficient. In the present investigation, we test the hypothesis that RAGE signaling functions in hyperoxia-induced inflammation. RAGE-null mice exposed to hyperoxia survived 3 days longer than age-matched wild-type mice. After 4 days in hyperoxia, RAGE-null mice had less total cell infiltration into the airway, decreased total protein leak, diminished alveolar damage in hematoxylin and eosin-stained lung sections, and a lower lung wet-to-dry weight ratio. An inflammatory cytokine antibody array revealed decreased secretion of several proinflammatory molecules in lavage fluid obtained from RAGE knockout mice when compared with wild-type control animals. Real-time RT-PCR and immunoblotting revealed that hyperoxia induced RAGE expression in primary alveolar epithelial cells, and immunohistochemistry identified increased RAGE expression in the lungs of mice after exposure to hyperoxia. These data reveal that RAGE targeting leads to a diminished hyperoxia-induced pulmonary inflammatory response. Further research into the role of RAGE signaling in the lung should identify novel targets likely to be important in the therapeutic alleviation of lung injury and associated persistent inflammation.
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Hiperoxia/complicaciones , Lesión Pulmonar/etiología , Lesión Pulmonar/prevención & control , Receptores Inmunológicos/metabolismo , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/patología , Animales , Líquido del Lavado Bronquioalveolar/citología , Células Cultivadas , Femenino , Hiperoxia/metabolismo , Hiperoxia/patología , Mediadores de Inflamación/metabolismo , Lesión Pulmonar/metabolismo , Lesión Pulmonar/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor para Productos Finales de Glicación Avanzada , Análisis de SupervivenciaRESUMEN
Persistent hypoxia can cause pulmonary arterial hypertension that may be associated with significant remodeling of the pulmonary arteries, including smooth muscle cell proliferation and hypertrophy. We previously demonstrated that the NADPH oxidase homolog NOX4 mediates human pulmonary artery smooth muscle cell (HPASMC) proliferation by transforming growth factor-beta1 (TGF-beta1). We now show that hypoxia increases HPASMC proliferation in vitro, accompanied by increased reactive oxygen species generation and NOX4 gene expression, and is inhibited by antioxidants, the flavoenzyme inhibitor diphenyleneiodonium (DPI), and NOX4 gene silencing. HPASMC proliferation and NOX4 expression are also observed when media from hypoxic HPASMC are added to HPASMC grown in normoxic conditions, suggesting autocrine stimulation. TGF-beta1 and insulin-like growth factor binding protein-3 (IGFBP-3) are both increased in the media of hypoxic HPASMC, and increased IGFBP-3 gene expression is noted in hypoxic HPASMC. Treatment with anti-TGF-beta1 antibody attenuates NOX4 and IGFBP-3 gene expression, accumulation of IGFBP-3 protein in media, and proliferation. Inhibition of IGFBP-3 expression with small interfering RNA (siRNA) decreases NOX4 gene expression and hypoxic proliferation. Conversely, NOX4 silencing does not decrease hypoxic IGFBP-3 gene expression or secreted protein. Smad inhibition does not but the phosphatidylinositol 3-kinase (PI3K) signaling pathway inhibitor LY-294002 does inhibit NOX4 and IGFBP-3 gene expression, IGFBP-3 secretion, and cellular proliferation resulting from hypoxia. Immunoblots from hypoxic HPASMC reveal increased TGF-beta1-mediated phosphorylation of the serine/threonine kinase (Akt), consistent with hypoxia-induced activation of PI3K/Akt signaling pathways to promote proliferation. We conclude that hypoxic HPASMC produce TGF-beta1 that acts in an autocrine fashion to induce IGFBP-3 through PI3K/Akt. IGFBP-3 increases NOX4 gene expression, resulting in HPASMC proliferation. These observations add to our understanding hypoxic pulmonary vascular remodeling.
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Hipoxia de la Célula/fisiología , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/biosíntesis , Miocitos del Músculo Liso/metabolismo , NADPH Oxidasas/metabolismo , Arteria Pulmonar/metabolismo , Factor de Crecimiento Transformador beta1/biosíntesis , Comunicación Autocrina , Hipoxia de la Célula/genética , Proliferación Celular , Células Cultivadas , Expresión Génica , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Modelos Biológicos , Miocitos del Músculo Liso/citología , NADPH Oxidasa 4 , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Arteria Pulmonar/citología , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
Multiple critical roles within mitosis have been assigned to Polo-like kinase 1 (Plk1), making it an attractive candidate for mitotic targeting of cancer cells. Plk1 contains two domains amenable for targeted interference: a kinase domain responsible for the enzymatic function and a polo box domain necessary for substrate recognition and subcellular localization. Here, we compare two approaches for targeted interference with Plk1 function, either by a Plk1 small-molecule enzyme inhibitor or by inducible overexpression of the polo box in human cancer cell lines. Inducible expression of the Plk1 polo box resulted in growth inhibition of RKOp27 human colon adenocarcinoma cells without obvious signs of mitotic abnormalities. A Plk1 kinase inhibitor in the same cell line arrested cells in mitosis with subsequent onset of apoptosis. Similarly, PC-3 human prostate cancer cells were growth inhibited on expression of the polo box. Prolonged expression of the polo box in these cells resulted in the occurrence of binucleated or multinucleated cells. In contrast, U2OS human osteosarcoma cells responded to overexpression of the polo box with a massive mitotic accumulation coinciding with the onset of apoptosis. Comparison of spindle formation revealed very similar mitotic abnormalities in polo box-overexpressing U2OS cells compared with U2OS cells treated with the Plk1 kinase inhibitor. We conclude that interference with polo box function and inhibition of Plk1 kinase activity can exert very similar phenotypic effects in certain cell lines but highly contrasting effects in others. This may point to subtle differences in the molecular machinery of mitosis regulation in cancer cells.
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Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Adenocarcinoma/enzimología , Adenocarcinoma/patología , Línea Celular Tumoral , Neoplasias del Colon/enzimología , Neoplasias del Colon/patología , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Humanos , Masculino , Osteosarcoma/enzimología , Osteosarcoma/patología , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Quinasa Tipo Polo 1Asunto(s)
Hipertensión Pulmonar/metabolismo , Hipoxia/fisiopatología , NADPH Oxidasas/fisiología , Óxido Nítrico/metabolismo , Animales , División Celular , Enfermedad Crónica , Transporte de Electrón , Retículo Endoplásmico/metabolismo , Furina/fisiología , Humanos , Hipertensión Pulmonar/etiología , Hipertrofia , Hipoxia/complicaciones , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Pulmón/irrigación sanguínea , Miocitos del Músculo Liso/patología , NADPH Oxidasa 4 , NADPH Oxidasas/metabolismo , Oxidorreductasas/metabolismo , Oxígeno/metabolismo , Arteria Pulmonar/metabolismo , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Factor de Crecimiento Transformador beta1/fisiologíaRESUMEN
Advanced second generation inhibitors of histone deacetylases (HDAC) are currently used in clinical development. This study aimed at comparing the pharmacological properties of selected second generation HDAC inhibitors with the hydroxamate and benzamide head group, namely SAHA, LAQ824/LBH589, CI994, MS275 and MGCD0103. In biochemical assays using recombinant HDAC1, 3, 6 and 8 isoenzymes, SAHA and LAQ824/LBH589 behave as quite unselective HDAC inhibitors. In contrast, the benzamides CI994, MS275 and MGCD0103 are more selective, potent inhibitors of at least HDAC1 and HDAC3. All HDAC inhibitors induce histone H3 hyperacetylation, correlating with inhibition of proliferation, induction of cell differentiation and apoptosis. A broad cytotoxicity is seen across cell lines from different tumor entities with LAQ824/LBH589 being the most potent agents. The apoptosis inducing activity is evident in arrested and proliferating RKO colon cancer cells with inducible, heterologous p21(waf1) expression, indicative for a cell-cycle independent mode-of-action. Differentiation of MDA-MB468 breast cancer cells is induced by benzamide and hydroxamate analogs. The reversibility of drug action was evaluated by pulse treatment of A549 lung cancer cells. Whereas paclitaxel induced irreversible cell cycle alterations already after 6 hr treatment, HDAC inhibitor action was retarded and irreversible after >16 hr treatment. Interestingly, pulse treatment was equally effective as continous treatment. Finally, the efficacy of LAQ824, SAHA and MS275 in A549 nude mice xenografts was comparable to that of paclitaxel at well tolerated doses. We conclude that despite a different HDAC isoenzyme inhibition profile, hydroxamate and benzamide analogs as studied display similar cellular profiles.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Isoenzimas/antagonistas & inhibidores , Acetilación , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Carcinoma de Pulmón de Células no Pequeñas/patología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Genes Reporteros , Histona Desacetilasas/metabolismo , Humanos , Isoenzimas/metabolismo , Luciferasas/genética , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patologíaRESUMEN
Transforming growth factor-beta1 (TGF-beta1) plays a pivotal role in increasing airway smooth muscle mass in severe asthma by inducing proliferation and hypertrophy of human airway smooth muscle. The mechanism(s) for these effects of TGF-beta1 have not been fully elucidated. In this study, we demonstrate that TGF-beta1 is a potent inducer of expression of the nonphagocyte NAD(P)H oxidase catalytic homolog Nox4, diphenylene iodonium-inhibitable reactive oxygen species production, proliferation, and hypertrophy in cultured human airway smooth muscle cells. By confocal microscopy, TGF-beta1-induced Nox4 was localized with the endoplasmic reticulum and the nucleus, implying a role for Nox4 in regulation of both the cell cycle and protein synthesis. Consistent with this hypothesis, TGF-beta1 increased retinoblastoma protein phosphorylation at both Ser807/811 and Ser780. Silencing Nox4 prevented TGF-beta1-mediated retinoblastoma protein phosphorylation, proliferation, and cell hypertrophy. TGF-beta1 also increased phosphorylation of eukaryotic translation initiation factor 4E binding protein-1 at Thr37/46, and this was likewise blocked by silencing Nox4. This is the first report to suggest a functional role for Nox4 in cell cycle transition and to demonstrate that Nox4 influences the pathobiochemistry of asthma by generating reactive oxygen species that promote TGF-beta1-induced proliferation and hypertrophy of human airway smooth muscle.
Asunto(s)
Asma/metabolismo , Bronquios/citología , Miocitos del Músculo Liso/enzimología , NADPH Oxidasas/metabolismo , Proteína de Retinoblastoma/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Activinas/metabolismo , Activinas/farmacología , Asma/patología , Proteína Quinasa CDC2/metabolismo , División Celular/efectos de los fármacos , División Celular/fisiología , Núcleo Celular/enzimología , Células Cultivadas , Factor 4E Eucariótico de Iniciación/metabolismo , Humanos , Hipertrofia , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , NADPH Oxidasa 4 , NADPH Oxidasas/genética , Oxidación-Reducción , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Biosíntesis de Proteínas/fisiología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteína smad3/metabolismo , Transfección , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
Multiple roles within mitosis have been assigned to Polo-like kinase 1 (Plk1), making it an attractive candidate for mitotic targeting of cancer cells. We have employed chimeric antisense oligonucleotides to investigate the molecular alterations after targeted interference with Plk1 in RKO human colon adenocarcinoma and PC3 prostate cancer cells. Suppression of Plk1 mRNA resulted in a dramatic increase of the mitotic index followed by the onset of apoptosis. Mitotically arrested cells displayed randomly separated condensed chromosomes and the occurrence of multiple spindle poles with well-formed asters. Induction of apoptosis was strictly dependent on cell cycle progression: Genetically engineered RKO cells with inducible expression of the cyclin-dependent kinase inhibitor p27(Kip1) were completely refractory to Plk1 depletion-induced apoptosis when they were arrested in the G1 phase of the cell cycle. Various mitotic markers, including MPM-2, cdc25c, cyclin B1, or phosphorylated histone H3, were investigated to explore the molecular consequences of Plk1 depletion. Whereas most marker proteins showed similar alterations compared with treatment with paclitaxel, cdc25c was fully phosphorylated solely in paclitaxel-treated cells but only partially phosphorylated in Plk1-depleted cells, although both treatments caused a profound mitotic arrest. This differential phosphorylation of cdc25c was used to test whether a pharmacologic inhibitor of Plk1 would exert the same cellular effects as interference with Plk1 on a mRNA level. It was found that the differential electrophoretic mobility of cdc25c can serve as a reliable molecular marker to track inhibition of Plk1 by small-molecule inhibitors within a cell.
Asunto(s)
Proteínas de Ciclo Celular/genética , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Oligonucleótidos Antisentido/uso terapéutico , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , Secuencia de Bases , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Cartilla de ADN , Humanos , Mitosis/efectos de los fármacos , Mitosis/genética , Índice Mitótico , ARN Mensajero/efectos de los fármacos , Supresión Genética/efectos de los fármacos , Transfección , Fosfatasas cdc25/metabolismo , Quinasa Tipo Polo 1RESUMEN
Several phosphorothioate antisense oligodeoxynucleotides (ODN) are developed to target factors potentially involved in tumor growth and apoptosis suppression. Among them, the 18-mer G3139 (Oblimersen), which targets Bcl-2, is currently being tested in phase II and phase III clinical trials for various tumors in combination with chemotherapy. On the other hand, ODNs containing CpG dinucleotides (CpG-ODN) within specific-sequence contexts (CpG motifs) have been shown to activate rodent or primate immune cells via toll-like receptor 9 (TLR9) and have demonstrated remarkable T cell-dependent antitumor efficacy in a series of murine tumor models. However, immune cell activation by CpG-ODN is largely diminished upon C-5 methylation at CpG cytosine. As G3139 contains CpG motifs, we questioned whether the antitumor effects seen in human tumor xenografts might be abrogated by cytosine C-5 methylation of G3139, which retained the ability of G3139 to suppress Bcl-2 expression in tissue culture, or by similar derivatization of other phosphorothioate ODNs developed for the immune activation of rodent or human cells. The in vivo antitumor efficacy of the immunostimulatory H1826 and H2006 ODNs was compared with that of G3139. Bcl-2 suppression achieved by G3139 purportedly sensitizes tumor cells toward cytotoxic agents, and some of the experiments employed combinations of ODN with such drugs as cisplatin or etoposide. H1826, H2006, and G3139 all produced similar, striking, growth inhibitory effects on either H69 SCLC, A2780 ovarian carcinoma, or A549 lung adenocarcinoma human tumor xenografts at doses of 0.3 mg/kg and 1 mg/kg (H1826, H2006) or 12 mg/kg (G3139) per day. In contrast, the H2006-mC (1 mg/kg) or G3139-mC (12 mg/kg) derivatives demonstrated no significant antitumor effects. The combination of G3139 (12 mg/kg) with cisplatin produced some additive antitumor efficacy, which was not seen in combinations of G3139-mC (12 mg/kg) or H1826 (1 mg/kg) with cisplatin. G3139, at a dose of 12 mg/kg, alone induced extensive enlargement of the spleen. Immunostimulation was evaluated in vitro by flow cytometric measurements of the CD80 and CD86 activation markers found on CD19+ murine splenocytes. The CpG-ODN producing strong antitumor effects in vivo also induced these activation markers in vitro, in contrast to the in vivo inactive G3139-mC. Our data indicate a significant contribution of the immunostimulatory properties of CpG-ODN (including G3139) to the antitumor effects observed in nude mouse xenograft models. This is in contrast to previous data presented by other authors indicating that the activity of G3139 in human tumor xenografts was Bcl-2 specific. Furthermore, as nude mice are devoid of T cells, a T cell-mediated immune response apparently is not required for the potent antitumor responses observed here; innate immune responses are sufficient.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Oligodesoxirribonucleótidos/uso terapéutico , Tionucleótidos/uso terapéutico , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/química , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Linfocitos B/efectos de los fármacos , Carcinoma/tratamiento farmacológico , Carcinoma/inmunología , Línea Celular Tumoral , Citosina/química , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Activación de Linfocitos , Metilación , Ratones , Ratones Desnudos , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/química , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Tionucleótidos/administración & dosificación , Tionucleótidos/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Transforming growth factor-beta1 (TGF-beta1) is abundantly expressed in pulmonary hypertension, but its effect on the pulmonary circulation remains unsettled. We studied the consequences of TGF-beta1 stimulation on freshly isolated human pulmonary artery smooth muscle cells (HPASMC). TGF-beta1 initially promoted differentiation, with upregulated expression of smooth muscle contractile proteins. TGF-beta1 also induced expression of Nox4, the only NAD(P)H oxidase membrane homolog found in HPASMC, through a signaling pathway involving Smad 2/3 but not mitogen-activated protein (MAP) kinases. TGF-beta1 likewise increased production of reactive oxygen species (ROS), an effect significantly reduced by the NAD(P)H oxidase flavoprotein inhibitor diphenylene iodonium (DPI) and by Nox4 siRNAs. In the absence of TGF-beta1, Nox4 was present in freshly cultured cells but progressively lost with each passage in culture, paralleling a decrease in ROS production by HPASMC over time. At a later time point (72 h), TGF-beta1 promoted HPASMC proliferation in a manner partially inhibited by Nox4 small interfering RNA and dominant negative Smad 2/3, indicating that TGF-beta1 stimulates HPASMC growth in part by a redox-dependent mechanism mediated through induction of Nox4. HPASMC activation of the MAP kinases ERK1/2 was reduced by the NAD(P)H oxidase inhibitors DPI and 4-(2-aminoethyl)benzenesulfonyl fluoride, suggesting that TGF-beta1 may facilitate proliferation by upregulating Nox4 and ROS production, with transient oxidative inactivation of phosphatases and augmentation of growth signaling cascades. These findings suggest that Nox4 is the relevant Nox homolog in HPASMC. This is the first observation that TGF-beta1 regulates Nox4, with important implications for mechanisms of pulmonary vascular remodeling.