PURPOSE OF THE STUDY. The presence of mesenchymal cells, the primary source of fibrotic cells, has been described in BAL fluid of LTR patients as predictive of BOS onset (Badri et al, 2011). The aim of this study was (1) to analyse and characterize the cells isolated from BAL of BOS patients to verify the phenotype during the different phases of the disease and (2) to design a new therapeutic approach based on engineered nanoparticles (NP), which could inhibit proliferation and induce apoptosis of cells causing BOS (Cova et al, 2014). STATEMENTS OF THE METHODS. We collected BAL samples from 10 lung transplant patients at different stage of BOS (stable patients, BOS 0p, BOS 1 and BOS 3). Cells were isolated and cultured in high glucose DMEM medium with 10% fetal calf serum (FCS), 100 U mL–1 penicillin/streptomycin solution. We analyzed cell phenotype with flow-cytometry (CD44-FITC, CD105-FITC, CD90-PE, CD34-ECD, and CD45-APC750). We tried to differentiate cells toward mesenchymal lineage (bone and fat) and we performed qRT-PCR and Western blot analyses on genes and proteins associated with fibroblast and bone phenotype (alpha-SMA, RUNX-2 and Fibronectin). In addition, fluorescent-labeled NP functionalized with an anti-CD44 antibody and loaded with everolimus (CD44NPE) were used to assess cell uptake by confocal microscopy, cell apoptosis/death and proliferation by flow cytometry. RESULTS. Cells isolated from BAL exhibit a transition from a mesenchymal to a fibroblast phenotype along with BOS progression, evaluated by the response of cells to the differentiation toward mesenchymal lineage. All cells were positive for CD90 and CD105 (between 64 to 90%) and negative for CD34 and CD45 surface markers. Nevertheless, the expression of RUNX-2 (a marker of osteogenic phenotype) decreases from stable patients to BOS 3 patients (p<0.05) and, at the same time, the presence of alpha-SMA protein and the expression of Fibronectin gene increase (p<0.05), demonstrating a fibroblast transition of the cells associated with a severe BOS condition. In addition, the results obtained by treatment with NP showed that CD44NPE entered into the cells inhibiting proliferation and inducing apoptosis in all cells without significant differences between BOS grades. More specifically, cell proliferation was significantly reduced (p<0.05) at 24, 72 and 120 h after incubation while apoptosis was significantly higher (p<0.05) at 8, 24 and 48 h after treatment. CONCLUSIONS. We demonstrated that cells isolated from BAL of BOS patients present a transition from mesenchymal to fibroblast phenotype in association with BOS degree progression. The effectiveness of our nanoparticles in inhibiting mesenchymal cells and their characterization open new perspectives for therapeutic and diagnostic purposes of CLAD management.
An innovative cellular approach to BOS: targeted antibody-engineered gold nanoparticles and characterization of cells for new therapeutic treatment
CECCARELLI, GABRIELE;COVA, EMANUELA;BENEDETTI, LAURA;CUSELLA DE ANGELIS, MARIA GABRIELLA;MELONI, FEDERICA
2014-01-01
Abstract
PURPOSE OF THE STUDY. The presence of mesenchymal cells, the primary source of fibrotic cells, has been described in BAL fluid of LTR patients as predictive of BOS onset (Badri et al, 2011). The aim of this study was (1) to analyse and characterize the cells isolated from BAL of BOS patients to verify the phenotype during the different phases of the disease and (2) to design a new therapeutic approach based on engineered nanoparticles (NP), which could inhibit proliferation and induce apoptosis of cells causing BOS (Cova et al, 2014). STATEMENTS OF THE METHODS. We collected BAL samples from 10 lung transplant patients at different stage of BOS (stable patients, BOS 0p, BOS 1 and BOS 3). Cells were isolated and cultured in high glucose DMEM medium with 10% fetal calf serum (FCS), 100 U mL–1 penicillin/streptomycin solution. We analyzed cell phenotype with flow-cytometry (CD44-FITC, CD105-FITC, CD90-PE, CD34-ECD, and CD45-APC750). We tried to differentiate cells toward mesenchymal lineage (bone and fat) and we performed qRT-PCR and Western blot analyses on genes and proteins associated with fibroblast and bone phenotype (alpha-SMA, RUNX-2 and Fibronectin). In addition, fluorescent-labeled NP functionalized with an anti-CD44 antibody and loaded with everolimus (CD44NPE) were used to assess cell uptake by confocal microscopy, cell apoptosis/death and proliferation by flow cytometry. RESULTS. Cells isolated from BAL exhibit a transition from a mesenchymal to a fibroblast phenotype along with BOS progression, evaluated by the response of cells to the differentiation toward mesenchymal lineage. All cells were positive for CD90 and CD105 (between 64 to 90%) and negative for CD34 and CD45 surface markers. Nevertheless, the expression of RUNX-2 (a marker of osteogenic phenotype) decreases from stable patients to BOS 3 patients (p<0.05) and, at the same time, the presence of alpha-SMA protein and the expression of Fibronectin gene increase (p<0.05), demonstrating a fibroblast transition of the cells associated with a severe BOS condition. In addition, the results obtained by treatment with NP showed that CD44NPE entered into the cells inhibiting proliferation and inducing apoptosis in all cells without significant differences between BOS grades. More specifically, cell proliferation was significantly reduced (p<0.05) at 24, 72 and 120 h after incubation while apoptosis was significantly higher (p<0.05) at 8, 24 and 48 h after treatment. CONCLUSIONS. We demonstrated that cells isolated from BAL of BOS patients present a transition from mesenchymal to fibroblast phenotype in association with BOS degree progression. The effectiveness of our nanoparticles in inhibiting mesenchymal cells and their characterization open new perspectives for therapeutic and diagnostic purposes of CLAD management.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
