Idiopathic pulmonary hypertension (IPAH) is a rare disease characterized by a progressive increase in pulmonary vascular resistance leading to heart failure. MicroRNAs (miRNAs) are small noncoding RNAs that control the expression of genes, including some involved in the progression of IPAH, as studied in animals and lung tissue. These molecules circulate freely in the blood and their expression is associated with the progression of different vascular pathologies. Here, we studied the expression profile of circulating miRNAs in 12 well-characterized IPAH patients using microarrays. We found significant changes in 61 miRNAs, of which the expression of miR23a was correlated with the patients' pulmonary function. We also studied the expression profile of circulating messenger RNA (mRNAs) and found that miR23a controlled 17% of the significantly changed mRNA, including PGC1α, which was recently associated with the progression of IPAH. Finally we found that silencing of miR23a resulted in an increase of the expression of PGC1α, as well as in its well-known regulated genes CYC, SOD, NRF2, and HO1. The results point to the utility of circulating miRNA expression as a biomarker of disease progression.
e15511 Background: Paclitaxel skin toxicity is a frequent side effect extensively evaluated in the clinical setting. However little is known about the preclinical mechanisms that lead to this toxicity. The endpoint of this study was to analyse the cutaneous mechanisms that drive paclitaxel toxicity in a preclinical model. Methods: Primary human keratinocytes were co-cultured with human dermal fibroblast in collagen gel under air-liquid interface conditions to generate a multilayered 3D epidermis. Paclitaxel was added to 3D epidermis at 0.3 µM, 3 µM and 30 µM and total RNA and protein was extracted after 24h of incubation. Markers of cell senescence (p21 and p53), anti-apoptotic mediators (Bcl-2), skin elasticity (tropoelastin ELN, collagen type I and fibronectin), hidratation (aquaporin 3 AQP3), oxidative stress (NOX4), antioxidant (SOD1, Nrf2), and angiogenic markers (VEGFR, eNOS) were evaluated by RT-PCR and western blot. NfKB phosphorylation was measured by western blot and inflammatory citokines IL1α, IL-6 and IL-8 were measured in cultured supernatants by ELISA. Human primary melanocytes were cultured and stimulated with paclitaxel to measure melanogenesis through the expression of of TYR, TYRP1 and DCT genes. The effect of paclitaxel on skin endothelial cell angiogenesis was measured by endothelial tube formation. Results: In human 3D keratinocytesPaclitaxel inhibited the expression of Bcl-2, and increased the expression of p53 and p21. The angiogenic markers VEGF and eNOS were decreased. The expression of oxidative stress marker NOX4 was increased and the anti-oxidant mediators Nrf2 and SOD1 were decreased. Markers of elasticity, collagen type I, fibronectin and FN1 were decreased as occurs with AQP3 hidratation marker. Paclitaxel increased the phosphorylation of NfkB and elevated the secretion of IL1α, IL-6 and IL-8 cytokines. In skin endothelial cells, paclitaxel reduced the endothelial tube formation. In melanocytes, paclitaxel increased skin pigmentation represented by the increase of RNA expression of TYR, TYRP1 and DCT genes. Conclusions: This preclinical 3D model showed that paclitaxel impacts on the expression of proteins related with angiogenics, elasticity and senescence in human kerantinocytes. Moreover higher doses of paclitaxel increased inflammation parameters and confirmed phototoxic and anti-angiogenenic effects. This preclinical model could be a valuable tool to assess skin toxicity of new antineoplastic agents.
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