RESUMEN
Critical limb ischemia (CLI) represents the most severe form of peripheral arterial disease (PAD) and is the leading cause of non-traumatic amputations in western populations. In recent years, therapeutic angiogenesis has been considered to be a potential treatment option for CLI patients, however the molecular mechanism of ischemia-induced vascularization is still not fully understood. The identification of genetic factors underlying vascular responses to ischemia will improve our understanding of the biological causes of the disease and enhance personalized therapies in the future. In this work, we determined, for the first time, the expression profile of angiogenesis-related genes utilizing unique human material: the popliteal arteries retrieved during lower limb amputation from patients with CLI. Using custom-designed TaqMan Low-Density Array (TLDA) cards we investigated the mRNA level of 90 genes on CLI samples compared to healthy donors. We identified three significantly up-regulated genes in CLI group: matrix metalloproteinase 9 (MMP-9), VE-cadherin (CDH5) and integrin alpha 4 (ITGA4). However, among all investigated genes, only lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) was significantly reduced. In order to verify whether hypoxic conditions occur in popliteal arteries of CLI patients, we validated the transcription level of selected proangiogenic genes by real-time PCR on a larger number of samples. These results showed that the expression of key genes involved in angiogenesis, such as MMP9, HGF, HIF1A, VEGF-A and FLT1 were elevated in patients with CLI. Moreover, the study revealed that the expression of VEGF-A and FLT1 was associated with activation of HIF1A transcription. In conclusion, our data revealed the alteration in the mRNA level of genes involved in matrix remodelling, cell-cell adhesion as well as endothelial cell migration and proliferation in human popliteal arteries.
Asunto(s)
Isquemia/genética , Neovascularización Fisiológica/genética , Arteria Poplítea/metabolismo , Transcriptoma , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Extremidad Inferior/irrigación sanguínea , Masculino , Persona de Mediana Edad , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
BACKGROUND: MicroRNAs are small noncoding RNA molecules involved in the regulation of various physiological and pathological processes. Altered expression of different microRNAs has been observed in both solid tumours and haematological malignancies. OBJECTIVES: To investigate expression of several microRNAs in early and advanced mycosis fungoides (MF). METHODS: Biopsies were obtained from 43 patients with MF (18 early MF and 25 advanced MF) and 23 healthy volunteers. After microRNA isolation, reverse transcriptase reactions were performed, followed by cDNA amplification. The following microRNAs were analysed: miR-15a, miR-16, miR-155, let-7a, let-7d and let-7f. The relative amount of each microRNA was normalized according to the reference RNU48 level. RESULTS: Among the microRNAs studied, only MiR-155 was found to be slightly overexpressed in MF compared with healthy controls. Early MF showed a higher level of all analysed microRNAs after normalization against RNU48 level. Furthermore, metastatic MF demonstrated lower concentrations of let-7a, let-7d and let-7f when compared with MF limited to the skin. The univariate survival analysis and multivariate Cox's regression model revealed that the level of let-7a expression was an independent prognostic indicator. CONCLUSIONS: Altered expression of studied microRNAs and the differences between early and advanced MF may suggest that microRNAs play a significant role in MF pathogenesis. It seems that microRNAs could serve as potential therapeutic targets in the future.