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1.
Am J Respir Crit Care Med ; 204(4): 431-444, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-33882264

RESUMO

Rationale: Lymphangioleiomyomatosis (LAM) is a multisystem disease that causes lung cysts and respiratory failure. Loss of TSC (tuberous sclerosis complex) gene function results in a clone of "LAM cells" with dysregulated mTOR (mechanistic target of rapamycin) activity. LAM cells and fibroblasts form lung nodules that also contain mast cells, although their significance is unknown. Objectives: To understand the mechanism of mast-cell accumulation and the role of mast cells in the pathogenesis of LAM. Methods: Gene expression was examined using transcriptional profiling and qRT-PCR. Mast cell/LAM nodule interactions were examined in vitro using spheroid TSC2-null cell/fibroblast cocultures and in vivo using an immunocompetent Tsc2-null murine homograft model. Measurements and Main Results: LAM-derived cell/fibroblast cocultures induced multiple CXC chemokines in fibroblasts. LAM lungs had increased tryptase-positive mast cells expressing CXCRs (CXC chemokine receptors) (P < 0.05). Mast cells located around the periphery of LAM nodules were positively associated with the rate of lung function loss (P = 0.016). LAM spheroids attracted mast cells, and this process was inhibited by pharmacologic and CRISPR/cas9 inhibition of CXCR1 and CXCR2. LAM spheroids caused mast-cell tryptase release, which induced fibroblast proliferation and increased LAM-spheroid size (1.36 ± 0.24-fold; P = 0.0019). The tryptase inhibitor APC366 and sodium cromoglycate (SCG) inhibited mast cell-induced spheroid growth. In vivo, SCG reduced mast-cell activation and Tsc2-null lung tumor burden (vehicle: 32.5.3% ± 23.6%; SCG: 5.5% ± 4.3%; P = 0.0035). Conclusions: LAM-cell/fibroblast interactions attract mast cells where tryptase release contributes to disease progression. Repurposing SCG for use in LAM should be studied as an alternative or adjunct to mTOR inhibitor therapy.


Assuntos
Biomarcadores Tumorais/metabolismo , Fibroblastos/metabolismo , Neoplasias Pulmonares/metabolismo , Linfangioleiomiomatose/metabolismo , Mastócitos/metabolismo , Triptases/metabolismo , Adulto , Animais , Biomarcadores Tumorais/genética , Quimiocinas/metabolismo , Progressão da Doença , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Linfangioleiomiomatose/genética , Linfangioleiomiomatose/patologia , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Esferoides Celulares , Células Tumorais Cultivadas
2.
Am J Pathol ; 187(8): 1750-1762, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28623674

RESUMO

Lymphangioleiomyomatosis (LAM) is a rare disease in which LAM cells and fibroblasts form lung nodules and it is hypothesized that LAM nodule-derived proteases cause cyst formation and tissue damage. On protease gene expression profiling in whole lung tissue, cathepsin K gene expression was 40-fold overexpressed in LAM compared with control lung tissue (P ≤ 0.0001). Immunohistochemistry confirmed cathepsin K protein was expressed in LAM but not control lungs. Cathepsin K gene expression and protein and protease activity were detected in LAM-associated fibroblasts but not the LAM cell line 621-101. In lung nodules, cathepsin K immunoreactivity predominantly co-localized with LAM-associated fibroblasts. In vitro, fibroblast extracellular cathepsin K activity was minimal at pH 7.5 but significantly enhanced at pH 7 and 6. 621-101 cells reduced extracellular pH with acidification dependent on 621-101 mechanistic target of rapamycin activity and net hydrogen ion exporters, particularly sodium bicarbonate co-transporters and carbonic anhydrases, which were also expressed in LAM lung tissue. In LAM cell-fibroblast co-cultures, acidification paralleled cathepsin K activity, and both were reduced by sodium bicarbonate co-transporter (P ≤ 0.0001) and carbonic anhydrase inhibitors (P = 0.0021). Our findings suggest that cathepsin K activity is dependent on LAM cell-fibroblast interactions, and inhibitors of extracellular acidification may be potential therapies for LAM.


Assuntos
Catepsina K/metabolismo , Fibroblastos/metabolismo , Neoplasias Pulmonares/metabolismo , Pulmão/metabolismo , Linfangioleiomiomatose/metabolismo , Peptídeo Hidrolases/metabolismo , Adulto , Comunicação Celular , Linhagem Celular , Técnicas de Cocultura , Feminino , Fibroblastos/patologia , Humanos , Pulmão/patologia , Neoplasias Pulmonares/patologia , Linfangioleiomiomatose/patologia
3.
Lab Invest ; 92(4): 600-5, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22330339

RESUMO

Aneurysmal bone cyst (ABC) is a benign osteolytic bone lesion in which there are blood-filled spaces separated by fibrous septa containing giant cells. The nature of the giant cells in this lesion and the mechanism of bone destruction in ABC is not certain. In this study, we have analysed several characteristics of mononuclear and multinucleated cells in the ABC and examined the cellular and molecular mechanisms of ABC osteolysis. The antigenic and functional phenotype of giant cells in ABC was determined by histochemistry/immunohistochemistry using antibodies to macrophage and osteoclast markers. Giant cells and CD14+ and CD14- mononuclear cells were isolated from ABC specimens and cultured on dentine slices and coverslips with receptor activator of nuclear factor κB ligand (RANKL)+/- macrophage-colony stimulating factor (M-CSF) and functional and cytochemical evidence of osteoclast differentiation sought. Giant cells in ABC expressed an osteoclast-like phenotype (CD51+, CD14-, cathepsin K+, TRAP+) and were capable of lacunar resorption, which was inhibited by zoledronate, calcitonin and osteoprotegerin (OPG). When cultured with RANKL±M-CSF, CD14+, but not CD14-, mononuclear cells differentiated into TRAP+ multinucleated cells that were capable of lacunar resorption. M-CSF was not necessary for osteoclast formation from CD14+ cell cultures. CD14- cells variably expressed RANKL, OPG and M-CSF but supported osteoclast differentiation. Our findings show that the giant cells in ABC express an osteoclast-like phenotype and are formed from CD14+ macrophage precursors. CD14- mononuclear stromal cells express osteoclastogenic factors and most likely interact with CD14+ cells to form osteoclast-like giant cells by a RANKL-dependent mechanism.


Assuntos
Cistos Ósseos Aneurismáticos/patologia , Diferenciação Celular , Células Gigantes/patologia , Monócitos/fisiologia , Osteoclastos/patologia , Adolescente , Adulto , Técnicas de Cultura de Células , Criança , Pré-Escolar , Feminino , Células Gigantes/metabolismo , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Osteoprotegerina/metabolismo , Fenótipo , Ligante RANK/metabolismo , Adulto Jovem
4.
EBioMedicine ; 58: 102904, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32763828

RESUMO

BACKGROUND: Chronic myelomonocytic leukaemia (CMML) is a clinically heterogeneous stem cell malignancy with overlapping features of myelodysplasia and myeloproliferation. Over 90% of patients carry mutations in epigenetic and/or splicing genes, typically detectable in the Lin-CD34+CD38- immunophenotypic stem cell compartment in which the leukaemia-initiating cells reside. Transcriptional dysregulation at the stem cell level is likely fundamental to disease onset and progression. METHODS: We performed single-cell RNA sequencing on 6826 Lin-CD34+CD38-stem cells from CMML patients and healthy controls using the droplet-based, ultra-high-throughput 10x platform. FINDINGS: We found substantial inter- and intra-patient heterogeneity, with CMML stem cells displaying distinctive transcriptional programs. Compared with normal controls, CMML stem cells exhibited transcriptomes characterized by increased expression of myeloid-lineage and cell cycle genes, and lower expression of genes selectively expressed by normal haematopoietic stem cells. Neutrophil-primed progenitor genes and a MYC transcription factor regulome were prominent in stem cells from CMML-1 patients, whereas CMML-2 stem cells exhibited strong expression of interferon-regulatory factor regulomes, including those associated with IRF1, IRF7 and IRF8. CMML-1 and CMML-2 stem cells (stages distinguished by proportion of downstream blasts and promonocytes) differed substantially in both transcriptome and pseudotime, indicating fundamentally different biology underpinning these disease states. Gene expression and pathway analyses highlighted potentially tractable therapeutic vulnerabilities for downstream investigation. Importantly, CMML patients harboured variably-sized subpopulations of transcriptionally normal stem cells, indicating a potential reservoir to restore functional haematopoiesis. INTERPRETATION: Our findings provide novel insights into the CMML stem cell compartment, revealing an unexpected degree of heterogeneity and demonstrating that CMML stem cell transcriptomes anticipate disease morphology, and therefore outcome. FUNDING: Project funding was supported by Oglesby Charitable Trust, Cancer Research UK, Blood Cancer UK, and UK Medical Research Council.


Assuntos
Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Leucemia Mielomonocítica Crônica/genética , Células-Tronco Neoplásicas/imunologia , Adulto , Estudos de Casos e Controles , Feminino , Regulação Neoplásica da Expressão Gênica , Heterogeneidade Genética , Humanos , Imunofenotipagem , Leucemia Mielomonocítica Crônica/imunologia , Masculino , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/química , Análise de Sequência de RNA , Análise de Célula Única
5.
PLoS One ; 10(5): e0126025, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25978616

RESUMO

Lymphangioleiomyomatosis (LAM) is a rare disease leading to lungs cysts and progressive respiratory failure. Cells of unknown origin accumulate in the lungs forming nodules and eventually resulting in lung cysts. These LAM cells are described as clonal with bi-allelic mutations in TSC-2 resulting in constitutive mTOR activation. However LAM nodules are heterogeneous structures containing cells of different phenotypes; we investigated whether recruited wild type cells were also present alongside mutation bearing cells. Cells were isolated from LAM lung tissue, cultured and characterised using microscopy, immunocytochemistry and western blotting. Fibroblast-like cells were identified in lung tissue using immunohistochemical markers. Fibroblast chemotaxis toward LAM cells was examined using migration assays and 3D cell culture. Fibroblast-like cells were obtained from LAM lungs: these cells had fibroblast-like morphology, actin stress fibres, full length tuberin protein and suppressible ribosomal protein S6 activity suggesting functional TSC-1/2 protein. Fibroblast Activation Protein, Fibroblast Specific Protein/S100A4 and Fibroblast Surface Protein all stained subsets of cells within LAM nodules from multiple donors. In a mouse model of LAM, tuberin positive host derived cells were also present within lung nodules of xenografted TSC-2 null cells. In vitro, LAM 621-101 cells and fibroblasts formed spontaneous aggregates over three days in 3D co-cultures. Fibroblast chemotaxis was enhanced two fold by LAM 621-101 conditioned medium (p=0.05), which was partially dependent upon LAM cell derived CXCL12. Further, LAM cell conditioned medium also halved fibroblast apoptosis under serum free conditions (p=0.03). Our findings suggest that LAM nodules contain a significant population of fibroblast-like cells. Analogous to cancer associated fibroblasts, these cells may provide a permissive environment for LAM cell growth and contribute to the lung pathology of LAM lung disease.


Assuntos
Pulmão/patologia , Linfangioleiomiomatose/patologia , Células-Tronco Mesenquimais/patologia , Animais , Apoptose , Western Blotting , Células Cultivadas , Modelos Animais de Doenças , Feminino , Fibroblastos/patologia , Imunofluorescência , Humanos , Pulmão/citologia , Camundongos
6.
PLoS One ; 9(2): e90565, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24587395

RESUMO

Airway remodelling describes the histopathological changes leading to fixed airway obstruction in patients with asthma and includes extra-cellular matrix (ECM) deposition. Matrix metalloproteinase-1 (MMP-1) is present in remodelled airways but its relationship with ECM proteins and the resulting functional consequences are unknown. We used airway smooth muscle cells (ASM) and bronchial biopsies from control donors and patients with asthma to examine the regulation of MMP-1 by ECM in ASM cells and the effect of MMP-1 on ASM contraction. Collagen-I and tenascin-C induced MMP-1 protein expression, which for tenascin-C, was greater in asthma derived ASM cells. Tenascin-C induced MMP-1 expression was dependent on ERK1/2, JNK and p38 MAPK activation and attenuated by function blocking antibodies against the ß1 and ß3 integrin subunits. Tenascin-C and MMP-1 were not expressed in normal airways but co-localised in the ASM bundles and reticular basement membrane of patients with asthma. Further, ECM from asthma derived ASM cells stimulated MMP-1 expression to a greater degree than ECM from normal ASM. Bradykinin induced contraction of ASM cells seeded in 3D collagen gels was reduced by the MMP inhibitor ilomastat and by siRNA knockdown of MMP-1. In summary, the induction of MMP-1 in ASM cells by tenascin-C occurs in part via integrin mediated MAPK signalling. MMP-1 and tenascin-C are co-localised in the smooth muscle bundles of patients with asthma where this interaction may contribute to enhanced airway contraction. Our findings suggest that ECM changes in airway remodelling via MMP-1 could contribute to an environment promoting greater airway narrowing in response to broncho-constrictor stimuli and worsening asthma symptoms.


Assuntos
Asma/fisiopatologia , Proteínas da Matriz Extracelular/farmacologia , Metaloproteinase 1 da Matriz/metabolismo , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Asma/genética , Asma/patologia , Bradicinina/farmacologia , Células Cultivadas , Colágeno Tipo I/farmacologia , Relação Dose-Resposta a Droga , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos , Immunoblotting , Indóis/farmacologia , Integrina beta1/metabolismo , Integrina beta3/metabolismo , Metaloproteinase 1 da Matriz/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso/fisiopatologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Interferência de RNA , Sistema Respiratório/efeitos dos fármacos , Sistema Respiratório/fisiopatologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tenascina/genética , Tenascina/metabolismo , Tenascina/farmacologia , Vasodilatadores/farmacologia
7.
Spine (Phila Pa 1976) ; 36(11): 899-904, 2011 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-21343852

RESUMO

STUDY DESIGN: Analysis of lymphatic vessels in childhood and adult normal and pathological vertebral bone and intervertebral disc tissue. OBJECTIVE: To determine whether lymphatic vessels are present in spinal vertebrae and intervertebral discs in normal children and adults (4-30 years) as well as in pathological lesions of the spine. SUMMARY OF BACKGROUND DATA: There is uncertainty regarding the presence or absence of lymphatic vessels in normal intervertebral discs and the role of lymphatics in the pathobiology of disc degeneration and infective, neoplastic, and other spinal pathology. METHODS: The presence of the specific lymphatic endothelial cell markers, podoplanin, and LYVE-1 was determined immuno-histochemically in normal cervical, thoracic, and lumbar disc and vertebral tissues of adults and children, as well as in a wide range of spinal disorders. RESULTS: Lymphatics were not found in intact normal intervertebral discs or within spinal vertebrae of children or adults. Lymphatics were present in the outer periosteum and paraspinal ligaments and surrounding connective tissue. Lymphatic vessels were seen in infected and displaced degenerate disc tissue. Lymphatic vessels in vertebral bone were seen only when neoplastic and non-neoplastic lesions of the spine were associated with vertebral destruction and the lesion extending through the bone cortex into surrounding connective tissue. CONCLUSION: Lymphatics are not found in intact normal spinal vertebrae or the intervertebral discs of children or adults. Lymphatics in vertebral bone are found in pathological lesions of the spine when these have extended beyond the normal anatomical confines of the vertebra or intervertebral disc; this most likely occurs by ingrowth of lymphatics from surrounding connective tissues. These findings strongly suggest that metastatic tumor spread to the spine does not occur by lymphatics and that lymph node involvement of primary malignant spinal tumors occurs only after extraosseous spread.


Assuntos
Degeneração do Disco Intervertebral/patologia , Disco Intervertebral/patologia , Vasos Linfáticos/patologia , Doenças da Coluna Vertebral/patologia , Coluna Vertebral/patologia , Adolescente , Adulto , Vértebras Cervicais/patologia , Vértebras Cervicais/fisiologia , Criança , Pré-Escolar , Feminino , Humanos , Disco Intervertebral/fisiologia , Degeneração do Disco Intervertebral/complicações , Degeneração do Disco Intervertebral/fisiopatologia , Vértebras Lombares/patologia , Vértebras Lombares/fisiologia , Vasos Linfáticos/fisiologia , Masculino , Doenças da Coluna Vertebral/complicações , Doenças da Coluna Vertebral/fisiopatologia , Neoplasias da Coluna Vertebral/patologia , Neoplasias da Coluna Vertebral/fisiopatologia , Coluna Vertebral/fisiologia , Vértebras Torácicas/patologia , Vértebras Torácicas/fisiologia , Adulto Jovem
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