Abstract
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.
Highlights
Lymphangioleiomyomatosis (LAM) is a rare and progressive multi-system disease affecting women, which leads to respiratory failure over a variable period of time[1]
We examined LAM tissue for the presence of wild type, nonLAM cells by a range of methods, and we show that LAM nodules contain wild type fibroblast-like cells which are attracted to LAM cells, in part by production of the chemokine CXCL12, and that LAM cell/fibroblast aggregates protect LAM cells from stress-induced apoptosis
We found that sub-populations of cells within LAM nodules reacted with antibodies against Fibroblast Activation Protein (FAP), S100A4 and Fibroblast Surface Protein (FSP)
Summary
Lymphangioleiomyomatosis (LAM) is a rare and progressive multi-system disease affecting women, which leads to respiratory failure over a variable period of time[1]. Wild Type Mesenchymal Cells in Lymphangioleiomyomatosis sporadically, but is common in patients with tuberous sclerosis complex (TSC). Histological examination shows that a heterogeneous population of mesenchymal cells, termed LAM cells, infiltrate the lungs and lymphatics of these patients. Women with LAM may develop lymphatic masses, chylous collections and the tumour angiomyolipoma, the main morbidity is caused by the lung disease [2]. LAM cells form nodular aggregates and, probably due to the production of proteolytic enzymes [3, 4], damage lung tissue to form cysts, which gradually increase in number
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