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1.
J Am Soc Nephrol ; 27(1): 159-70, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26015452

RESUMEN

Macrophages are a heterogeneous cell type implicated in injury, repair, and fibrosis after AKI, but the macrophage population associated with each phase is unclear. In this study, we used a renal bilateral ischemia-reperfusion injury mouse model to identify unique monocyte/macrophage populations by differential expression of Ly6C in CD11b(+) cells and to define the function of these cells in the pathophysiology of disease on the basis of microarray gene signatures and reduction strategies. Macrophage populations were isolated from kidney homogenates by fluorescence-activated cell sorting for whole genome microarray analysis. The CD11b(+)/Ly6C(high) population associated with the onset of renal injury and increase in proinflammatory cytokines, whereas the CD11b(+)/Ly6C(intermediate) population peaked during kidney repair. The CD11b(+)/Ly6C(low) population emerged with developing renal fibrosis. Principal component and hierarchical cluster analyses identified gene signatures unique to each population. The CD11b(+)/Ly6C(intermediate) population had a distinct phenotype of wound healing, confirmed by results of studies inhibiting the macrophage colony-stimulating factor 1 receptor,whereas the CD11b(+)/Ly6C(low) population had a profibrotic phenotype. All populations, including the CD11b(+)/Ly6C(high) population, carried differential inflammatory signatures. The expression of M2-specific markers was detected in both the CD11b(+)/Ly6C(intermediate) and CD11b(+)/Ly6C(low) populations, suggesting these in vivo populations do not fit into the traditional classifications defined by in vitro systems. Results of this study in a renal ischemia-reperfusion injury model allow phenotype and function to be assigned to CD11b(+)/Ly6C(+) monocyte/macrophage populations in the pathophysiology of disease after AKI.


Asunto(s)
Antígenos Ly/biosíntesis , Riñón/metabolismo , Macrófagos/clasificación , Daño por Reperfusión/metabolismo , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Fenotipo , Daño por Reperfusión/sangre
2.
Aging (Albany NY) ; 16(13): 10694-10723, 2024 Jun 29.
Artículo en Inglés | MEDLINE | ID: mdl-38976646

RESUMEN

Idiopathic pulmonary fibrosis (IPF) is an age-related disease with poor prognosis and limited therapeutic options. Activation of lung fibroblasts and differentiation to myofibroblasts are the principal effectors of disease pathology, but damage and senescence of alveolar epithelial cells, specifically type II (ATII) cells, has recently been identified as a potential trigger event for the progressive disease cycle. Targeting ATII senescence and the senescence-associated secretory phenotype (SASP) is an attractive therapeutic strategy; however, translatable primary human cell models that enable mechanistic studies and drug development are lacking. Here, we describe a novel system of conditioned medium (CM) transfer from bleomycin-induced senescent primary alveolar epithelial cells (AEC) onto normal human lung fibroblasts (NHLF) that demonstrates an enhanced fibrotic transcriptional and secretory phenotype compared to non-senescent AEC CM treatment or direct bleomycin damage of the NHLFs. In this system, the bleomycin-treated AECs exhibit classical hallmarks of cellular senescence, including SASP and a gene expression profile that resembles aberrant epithelial cells of the IPF lung. Fibroblast activation by CM transfer is attenuated by pre-treatment of senescent AECs with the senolytic Navitoclax and AD80, but not with the standard of care agent Nintedanib or senomorphic JAK-targeting drugs (e.g., ABT-317, ruxolitinib). This model provides a relevant human system for profiling novel senescence-targeting therapeutics for IPF drug development.


Asunto(s)
Células Epiteliales Alveolares , Bleomicina , Senescencia Celular , Fibroblastos , Fibrosis Pulmonar Idiopática , Humanos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Bleomicina/toxicidad , Bleomicina/farmacología , Senescencia Celular/efectos de los fármacos , Células Epiteliales Alveolares/efectos de los fármacos , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/patología , Fibrosis Pulmonar Idiopática/patología , Fibrosis Pulmonar Idiopática/metabolismo , Medios de Cultivo Condicionados/farmacología , Indoles/farmacología , Fenotipo Secretor Asociado a la Senescencia/efectos de los fármacos , Pulmón/patología , Pulmón/citología , Pulmón/efectos de los fármacos , Sulfonamidas/farmacología , Senoterapéuticos/farmacología , Células Cultivadas , Pirimidinas/farmacología , Pirazoles/farmacología , Nitrilos/farmacología , Compuestos de Anilina
3.
Front Immunol ; 15: 1293883, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38455057

RESUMEN

Fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF) and systemic scleroderma (SSc), are commonly associated with high morbidity and mortality, thereby representing a significant unmet medical need. Interleukin 11 (IL11)-mediated cell activation has been identified as a central mechanism for promoting fibrosis downstream of TGFß. IL11 signaling has recently been reported to promote fibroblast-to-myofibroblast transition, thus leading to various pro-fibrotic phenotypic changes. We confirmed increased mRNA expression of IL11 and IL11Rα in fibrotic diseases by OMICs approaches and in situ hybridization. However, the vital role of IL11 as a driver for fibrosis was not recapitulated. While induction of IL11 secretion was observed downstream of TGFß signaling in human lung fibroblasts and epithelial cells, the cellular responses induced by IL11 was quantitatively and qualitatively inferior to that of TGFß at the transcriptional and translational levels. IL11 blocking antibodies inhibited IL11Rα-proximal STAT3 activation but failed to block TGFß-induced profibrotic signals. In summary, our results challenge the concept of IL11 blockade as a strategy for providing transformative treatment for fibrosis.


Asunto(s)
Interleucina-11 , Factor de Crecimiento Transformador beta , Humanos , Factor de Crecimiento Transformador beta/metabolismo , Transducción de Señal , Fibrosis , Miofibroblastos/metabolismo
4.
PLoS One ; 16(11): e0248034, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34752458

RESUMEN

Retinoic acid receptor-related orphan nuclear receptor (ROR) γt is a member of the RORC nuclear hormone receptor family of transcription factors. RORγt functions as a critical regulator of thymopoiesis and immune responses. RORγt is expressed in multiple immune cell populations including Th17 cells, where its primary function is regulation of immune responses to bacteria and fungi through IL-17A production. However, excessive IL-17A production has been linked to numerous autoimmune diseases. Moreover, Th17 cells have been shown to elicit both pro- and anti-tumor effects. Thus, modulation of the RORγt/IL-17A axis may represent an attractive therapeutic target for the treatment of autoimmune disorders and some cancers. Herein we report the design, synthesis and characterization of three selective allosteric RORγt inhibitors in preclinical models of inflammation and tumor growth. We demonstrate that these compounds can inhibit Th17 differentiation and maintenance in vitro and Th17-dependent inflammation and associated gene expression in vivo, in a dose-dependent manner. Finally, RORγt inhibitors were assessed for efficacy against tumor formation. While, RORγt inhibitors were shown to inhibit tumor formation in pancreatic ductal adenocarcinoma (PDAC) organoids in vitro and modulate RORγt target genes in vivo, this activity was not sufficient to delay tumor volume in a KP/C human tumor mouse model of pancreatic cancer.


Asunto(s)
Expresión Génica/efectos de los fármacos , Inflamación/genética , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/antagonistas & inhibidores , Células Th17/efectos de los fármacos , Animales , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Inflamación/metabolismo , Interleucina-17/metabolismo , Ratones , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Células Th17/metabolismo
5.
PLoS One ; 8(8): e70464, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23940580

RESUMEN

Recent findings indicate that elderly patients with acute kidney injury (AKI) have an increased incidence of progression to chronic kidney disease (CKD) due to incomplete recovery from an acute insult. In the current study, a co-morbid model of AKI was developed to better mimic the patient population and to investigate whether age exacerbates the fibrosis and inflammation that develop in the sequelae of progressive kidney disease following acute injury. Young (8-10 weeks) and aged (46-49 weeks) C57BL/6 mice were subjected to 30 min bilateral renal ischemia-reperfusion (I/R) to induce AKI. The aged animals have greater mortality and prolonged elevation of plasma creatinine correlating with less tubular epithelial cell proliferation compared to the young. Six weeks post-reperfusion, interstitial fibrosis is greater in aged kidneys based on picrosirius red staining and immunolocalization of cellular fibronectin, collagen III and collagen IV. Aged kidneys 6 weeks post-reperfusion also express higher levels of p53 and p21 compared to the young, correlating with greater increases in senescence associated (SA) ß-galactosidase, a known marker of cellular senescence. A higher influx of F4/80(+) macrophages and CD4(+) T lymphocytes is measured and is accompanied by increases in mRNA of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α). Importantly, microvascular density is significantly less, correlating with an increase in nitro-tyrosine, a marker of oxidative stress. Collectively, these data demonstrate that prolonged acute injury in the aged animals results in an accelerated progression of kidney disease in a chronic state.


Asunto(s)
Lesión Renal Aguda/patología , Senescencia Celular/fisiología , Fibrosis/patología , Riñón/patología , Daño por Reperfusión/patología , Lesión Renal Aguda/metabolismo , Animales , Quimiocina CCL2/metabolismo , Fibrosis/metabolismo , Riñón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Daño por Reperfusión/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo
6.
Biochem Mol Biol Educ ; 38(6): 385-92, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21567867

RESUMEN

A key goal of molecular/cell biology/biotechnology is to identify essential genes in virtually every physiological process to uncover basic mechanisms of cell function and to establish potential targets of drug therapy combating human disease. This article describes a semester-long, project-oriented molecular/cellular/biotechnology laboratory providing students, within a framework of bone cell biology, with a modern approach to gene discovery. Students are introduced to the topics of bone cells, bone synthesis, bone resorption, and osteoporosis. They then review the theory of microchip gene arrays, and study microchip array data generated during the differentiation of bone-resorbing osteoclasts in vitro. The class selects genes whose expression increases during osteoclastogenesis, and researches them in small groups using web-based bioinformatics tools. Students then go to a biotechnology company website to find and order small inhibitory RNAs (siRNAs) designed to "knockdown" expression of the gene of interest. Students then learn to transfect these siRNAs into osteoclasts, stimulate the cells to differentiate, assay osteoclast differentiation in vitro, and measure specific gene expression using real-time PCR and immunoblotting. Specific siRNA knockdown resulting in a decrease in osteoclastogenesis is indicative of a gene's physiological relevance. The results are analyzed statistically and presented to the class in groups. In the past 2 years, students identified several genes essential for optimal osteoclast differentiation, including Myo1d. The students hypothesize that the myo1d protein functions in osteoclasts to deliver important proteins to the cell surface via vesicular transport along microfilaments. Student response to the new course was overwhelmingly positive.

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