Estudio de la transformación fibroblasto-miofibroblasto, la senescencia y el metabolismo de fibroblastos aislados de pacientes con fibrosis pulmonar idiopática. Papel de la nicotinamida n-metil transferasa (nnmt)

Resumen

Idiopathic Pulmonary Fibrosis (IPF) is defined as a chronic fibrosing interstitial pneumonia of unknown, progressive and fatal cause that mainly affects adults over 60 years of age, limited to the lungs and associated with the histopathological pattern of usual interstitial pneumonia (UIP). The absence of specific manifestations of IPF is an obstacle to its early detection. It is a disease of late and challenging diagnosis, with a therapeutic approach of reduced efficacy. These characteristics raise the need to find new biomarkers associated with fibrosis and new therapeutic targets for an early diagnosis and increase patients' quality and life expectancy. The pathogenesis of the disease involves a loss of alveolar epithelial cells, a proliferation of fibroblasts and their transformation to myofibroblasts in an environment of remodeling of the extracellular matrix produced by an imbalance of secretory and soluble factors. This fibrotic microenvironment favors cellular senescence, the acquisition of the mesenchymal phenotype and/or associated with the myofibroblast, as well as changes in cellular metabolism. The myofibroblast phenotype, considered an effector of the fibrotic process, plays a role of enormous importance due to its capacity for proliferation, migration, contraction and synthesis of collagen and other proteins of the extracellular matrix. Previous results from our group identify the metabolic enzyme NNMT as a director of epithelial-mesenchymal transformation (EMT) in models of non-small cell lung cancer tumor epithelial cells resistant to therapy. Other studies describe its importance in similar processes associated with gastric cancer, prostate cancer, ovarian cancer and glioblastomas. NNMT catalyzes the methylation of nicotinamide (NAM) and similar compounds using S-adenosylmethionine (SAM) as a methyl donor to produce S-adenosylhomocysteine (SAH) and methyl nicotinamide (1-MNA), modifying metabolism and cellular methylation capacity. With this background, we hypothesized that NNMT could play a relevant role in the fibrotic processes associated with fibroblast-myofibroblast transformation (FMT) that could be reflected in the clinical development of patients with IPF. Therefore, our objective has been to study the involvement of NNMT in FMT, senescence and metabolism of primary fibroblasts isolated from patients with IPF compared to control fibroblasts. Using clinical histology samples, we also considered whether NNMT could be a biomarker for the disease. Primary fibroblast cultures were isolated and cultured from lung biopsies of IPF patients (N = 15) and compared with fibroblast populations obtained from pneumothorax patients as a control group (N = 30). Using TGF-β1 as a profibrotic factor and IL-1β as an antifibrotic and pro-inflammatory factor, these populations were characterized by analyzing parameters associated with the FMT, EMT and senescence process. Next, using lentivirus gene transduction techniques, NNMT was inhibited and overexpressed to evaluate its role in these processes and its impact on cell metabolism. Finally, the presence of NNMT was analyzed in histological sections from patients, comparing it with other fibrotic markers. The results show us that IPF fibroblasts exhibit overexpression of fibrotic markers such as α-SMA, COL1A1 and COL1A2, and senescence markers (p16, p21 and β-galactosidase activity) compared to control fibroblasts. Induction with TGF-β1 produced an increase in the expression of fibrotic markers such as α-SMA, FN1, COL1A1 and COL1A2 and mesenchymal markers such as n-cadherin and SNAI1, both in fibrotic fibroblasts and controls. In contrast, IL-1β caused a decrease in these markers in fibrotic fibroblasts but not in control fibroblasts. The enzymatic activity of NNMT was shown to be increased in IPF fibroblasts compared to controls. This result was corroborated by observing a significant increase in the protein and gene expression of this protein in fibrotic fibroblasts. The inhibition of NNMT in IPF fibroblasts caused a decrease in the expression of fibrotic markers (α-SMA), mesenchymal (SNAI1 and vimentin) and senescence (p16, p21 and β-galactosidase activity) in shNNMT fibroblasts compared to the control ones (shNT). In contrast, the overexpression of NNMT in IPF fibroblasts (NNMT) produced a significant increase in fibrotic, mesenchymal and senescence markers compared to control fibroblasts (Flag). Regarding the wound closure capacity of fibroblast cultures, the repression and overexpression of NNMT produced an increase and a decrease in the area closed by migration and/or proliferation, respectively, compared to control fibroblasts. Immunofluorescence studies in these fibroblasts with fluorescent specific antibodies for NNMT and α-SMA showed that both markers co-express, which could indicate that they would have a direct relationship in the acquisition of the myofibroblast phenotype and the development of fibrosis. Modulation of NNMT levels represented a metabolic change in fibrotic fibroblasts. Fibroblast populations with high levels of NNMT showed a decrease in 1-MNA and NAM and an increase in metabolites of the polyamine pathway (spermine and spermidine). However, the levels of NAD + did not show changes between the different groups. Finally, the immunohistochemical results obtained from paraffin tissue sections from patients with IPF showed us that NNMT, like α-SMA, is expressed in reactive fibrosis areas and not in healthy tissue. In conclusion, our results indicate that NNMT is expressed in clinical samples from patients with IPF and in fibroblasts obtained from them. Furthermore, control and fibrotic fibroblasts overexpress NNMT in profibrotic environments (TGF-β1), and this expression is decreased in antifibrotic environments (IL-1β). NNMT gene overexpression increases the fibrotic and senescent phenotype associated with fibroblasts, and its repression decreases it. All of this points to the fact that NNMT plays an essential role in the fibrotic, senescence and metabolic processes associated with fibrotic fibroblasts and IPF and presents us with this enzyme as a promising fibrosis marker that could constitute a therapeutic target for the treatment of this pathology.