Background: Adenosine (ADO) is an endogenous nucleoside ubiquitously expressed in the organism. It mediates its functions interacting with specific G-protein coupled membrane receptors: A 1 R, A 2a R, A 2b R and A 3 R. During stress or injury conditions, ADO is produced and released in excess: ATP is released to extracellular level, dephosphorylated to ADO, by two extracellular nucleotidases, CD39 and CD73. Unfortunately it is difficult to measure ADO concentrations since this nucleoside is rapidly degraded into inosine by adenosine deaminase (ADA). Fibrotic lung diseases represent a broad spectrum of pulmonary pathologies characterized by different degrees of inflammation and fibrosis. A possible link between the adenosinergic system and lung fibrosis -more specifically idiopathic pulmonary fibrosis- can be supported by the role of ADO and its receptors in lung diseases with an extensive fibrotic component. Experimental studies on animal models use bleomycin, a chemotherapic agent, to induce pulmonary fibrosis and to assess the biological pathways involved in the development of fibrosis. Aim of the study was to evaluate the possible changes of the adenosinergic transcriptomic profile (receptors, CD39, CD73 and ADA) in fibroblasts with or without bleomycin treatment, isolated from mice lungs. Methods: To isolate lung fibroblasts, five lungs of C57bl/j mice were explanted. From lung fragments fibroblast cultures were isolated and experiments conducted at the third passage. A part of fibroblast cultures was treated with 2mg/ml of bleomycin. Total RNA was extracted from cells (about 400.000) treated with bleomycin (BLEO, n=5) and from untreated cells (C, n=5) by specific extraction assays. Real-time PCR was performed and optimized for each ARs primer, CD39, CD73 and ADA. The experimental data were normalized with the three most stably expressed genes (HPRT1, PPiA, RPL13a). Results: For each analyzed receptor higher, although not significantly, levels of mRNA expression were observed in fibroblasts untreated as compared to fibroblasts treated with bleomycin: A 1 R: C=0.48±0.11, BLEO=0.30±0.11, p=0.324; A 2a R: C=0.23±0.09, BLEO=0.11±0.03, p=0.220; A 2b R: C=0.41±0.18, BLEO=0.32±0.14, p=0.682; CD73:C=0.07±0.02, BLEO=0.05±0.02, p=0.511. In contrast CD39 and ADA analysis showed a higher trend towards levels of mRNA expression in BLEO as compared to C (CD39: C=0.90±0.08, BLEO=1.02±0.16 p=0.55; ADA: C=0.44±0.25, BLEO=0.63±0.28, p=0.624). Discussion: The higher level of expression of CD39 and ADA compared to ARs and CD73 mRNA expression could represent a condition of desensitization and internalization of ARs in response to high concentrations ofADO released by lung fibroblasts after bleomycin induced damage.
Adenosinergic transcriptomic profile in murine lung fibroblasts treated or not with bleomycin.
Della Latta V;Cabiati M;Morales MA;Del Ry S
2014
Abstract
Background: Adenosine (ADO) is an endogenous nucleoside ubiquitously expressed in the organism. It mediates its functions interacting with specific G-protein coupled membrane receptors: A 1 R, A 2a R, A 2b R and A 3 R. During stress or injury conditions, ADO is produced and released in excess: ATP is released to extracellular level, dephosphorylated to ADO, by two extracellular nucleotidases, CD39 and CD73. Unfortunately it is difficult to measure ADO concentrations since this nucleoside is rapidly degraded into inosine by adenosine deaminase (ADA). Fibrotic lung diseases represent a broad spectrum of pulmonary pathologies characterized by different degrees of inflammation and fibrosis. A possible link between the adenosinergic system and lung fibrosis -more specifically idiopathic pulmonary fibrosis- can be supported by the role of ADO and its receptors in lung diseases with an extensive fibrotic component. Experimental studies on animal models use bleomycin, a chemotherapic agent, to induce pulmonary fibrosis and to assess the biological pathways involved in the development of fibrosis. Aim of the study was to evaluate the possible changes of the adenosinergic transcriptomic profile (receptors, CD39, CD73 and ADA) in fibroblasts with or without bleomycin treatment, isolated from mice lungs. Methods: To isolate lung fibroblasts, five lungs of C57bl/j mice were explanted. From lung fragments fibroblast cultures were isolated and experiments conducted at the third passage. A part of fibroblast cultures was treated with 2mg/ml of bleomycin. Total RNA was extracted from cells (about 400.000) treated with bleomycin (BLEO, n=5) and from untreated cells (C, n=5) by specific extraction assays. Real-time PCR was performed and optimized for each ARs primer, CD39, CD73 and ADA. The experimental data were normalized with the three most stably expressed genes (HPRT1, PPiA, RPL13a). Results: For each analyzed receptor higher, although not significantly, levels of mRNA expression were observed in fibroblasts untreated as compared to fibroblasts treated with bleomycin: A 1 R: C=0.48±0.11, BLEO=0.30±0.11, p=0.324; A 2a R: C=0.23±0.09, BLEO=0.11±0.03, p=0.220; A 2b R: C=0.41±0.18, BLEO=0.32±0.14, p=0.682; CD73:C=0.07±0.02, BLEO=0.05±0.02, p=0.511. In contrast CD39 and ADA analysis showed a higher trend towards levels of mRNA expression in BLEO as compared to C (CD39: C=0.90±0.08, BLEO=1.02±0.16 p=0.55; ADA: C=0.44±0.25, BLEO=0.63±0.28, p=0.624). Discussion: The higher level of expression of CD39 and ADA compared to ARs and CD73 mRNA expression could represent a condition of desensitization and internalization of ARs in response to high concentrations ofADO released by lung fibroblasts after bleomycin induced damage.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
