Tissue specificity of mitochondrial adaptations in rats after 4 weeks of normobaric hypoxia.

PURPOSE: Exposure to hypoxia has been suggested to activate multiple adaptive pathways so that muscles are better able to maintain cellular energy homeostasis. However, there is limited research regarding the tissue specificity of this response. The aim of this study was to investigate the influence of tissue specificity on mitochondrial adaptations of rat skeletal and heart muscles after 4 weeks of normobaric hypoxia (FiO2: 0.10). METHODS: Twenty male Wistar rats were randomly assigned to either normobaric hypoxia or normoxia. Mitochondrial respiration was determined in permeabilised muscle fibres from left and right ventricles, soleus and extensorum digitorum longus (EDL). Citrate synthase activity and the relative abundance of proteins associated with mitochondrial biogenesis were also analysed. RESULTS: After hypoxia exposure, only the soleus and left ventricle (both predominantly oxidative) presented a greater maximal mass-specific respiration (+48 and +25%, p < 0.05) and mitochondrial-specific respiration (+75 and +28%, p < 0.05). Citrate synthase activity was higher in the EDL (0.63 ± 0.08 vs 0.41 ± 0.10 µmol min- 1 µg- 1) and lower in the soleus (0.65 ± 0.17 vs 0.87 ± 0.20 µmol min- 1 µg- 1) in hypoxia with respect to normoxia. There was a lower relative protein abundance of PGC-1α (-25%, p < 0.05) in the right ventricle and a higher relative protein abundance of PGC-1ß (+43%, p < 0.05) in the left ventricle of rats exposed to hypoxia, with few differences for protein abundance in the other muscles. CONCLUSION: Our results show a muscle-specific response to 4 weeks of normobaric hypoxia. Depending on fibre type, and the presence of ventricular hypertrophy, muscles respond differently to the same degree of environmental hypoxia.

Hepcidin regulation in a mouse model of acute hypoxia.

OBJECTIVE: During hypoxia, hepcidin expression is inhibited to allow iron mobilization to sustain erythropoietic expansion. We analyzed molecular mechanisms underlying hypoxia-induced hepcidin inhibition in an in vivo model of acute hypoxia. METHODS: Mice were kept under normal or hypoxic conditions for 6 hours and 15 hours and treated with α-PDGF-BB antibody or PDGF-BB receptor inhibitor. Blood, liver, spleen, and bone marrow were collected to extract RNA and protein or to quantify EPO and PDGF-BB. mRNA and protein levels were assessed by RT-PCR and Western blot. RESULTS: Hepcidin was strongly inhibited at 15 hours, and this downregulation followed erythropoiesis activation and upregulation of several growth factors. PDGF-BB, erythroferrone, GDF15, and TWSG1 were upregulated by hypoxia in the bone marrow, but not in spleen or liver. Inactivation of PDGF-BB or its receptor suppressed the hypoxia-induced hepcidin inhibition. CONCLUSION: Spleen and liver are not involved in the early stages of hypoxia-induced hepcidin downregulation. Our data support the role of PDGF-BB and probably also of erythroferrone in the recruitment of iron for erythropoiesis in the hypoxia setting. The rapid normalization of all the erythroid factors against persistent hepcidin suppression suggests that other signals are involved that should be clarified in future studies.

miRNA-regulated gene expression differs in celiac disease patients according to the age of presentation.

Celiac disease is an intestinal disease which shows different symptoms and clinical manifestations among pediatric and adult patients. These variations could be imputable to age-related changes in gut architecture and intestinal immune system, which could be characterized by gene expression differences possibly regulated by miRNAs. We analyzed a panel of miRNAs and their target genes in duodenal biopsies of Marsh 3AB and 3C pediatric celiac patients, compared to controls. Moreover, to assess variation of expression in plasma samples, we evaluated circulating miRNA levels in controls and patients at diagnosis or on gluten-free diet. We detected a decreased miR-192-5p expression in celiac patients, but no variations in NOD2 and CXCL2, targets previously identified in adults. Conversely, we detected a significant increase in mRNA and protein levels of another target, MAD2L1, protein related to cell cycle control. miR-31-5p and miR-338-3p were down-regulated and their respective targets, FOXP3 and RUNX1, involved in Treg function, resulted up-regulated in celiac patients. Finally, we detected, in celiac patients, an increased expression of miR-21-5p, possibly caused by a regulatory loop with its putative target STAT3, which showed an increased activation in Marsh 3C patients. The analysis of plasma revealed a trend similar to that observed in biopsies, but in presence of gluten-free diet we could not detect circulating miRNAs values comparable to controls. miRNAs and their gene targets showed an altered expression in duodenal mucosa and plasma of celiac disease pediatric patients, and these alterations could be different from adult ones.

miRNAs affect the expression of innate and adaptive immunity proteins in celiac disease.

OBJECTIVES: microRNAs (miRNAs) are short RNAs that regulate gene expression in various processes, including immune response. Altered immune response is a pivotal event in the pathogenesis of celiac disease (CD), and miRNAs could have a role in modulating both innate and adaptive response to gluten in celiac patients. METHODS: We compared miRNA profiles in duodenal biopsies of controls and CD patients by miRNA array. Differentially expressed miRNAs were validated in controls, Marsh 3A-B, and Marsh 3C patients by quantitative PCR (qPCR). Target gene expression was assessed by qPCR, western blotting, and immunohistochemistry, and the effect of gliadin was evaluated by in vitro stimulation experiments on duodenal biopsies. RESULTS: Seven miRNAs were identified as significantly downregulated in the duodenum of adult CD patients as compared with controls. qPCR validated the decreased expression of miR-192-5p, miR-31-5p, miR-338-3p, and miR-197, in particular in patients with more severe histological lesions (Marsh 3C). In silico analysis of possible miRNA targets identified several genes involved in innate and adaptive immunity. Among these, chemokine C-X-C motif ligand 2 (CXCL2) and NOD2 showed significantly increased mRNA and protein level in Marsh 3C patients and a significant inverse correlation with the regulatory miR-192-5p. In addition, forkhead box P3 (FOXP3), Run-related transcription factor 1, and interleukin-18 (targets of miR-31-5p, miR-338-3p, and miR-197, respectively) showed upregulation in CD patients. Furthermore, alterations in CXCL2 and NOD2, FOXP3, miR-192-5p, and miR-31-5p expression were triggered by gliadin exposure in CD patients. CONCLUSIONS: miRNA expression is significantly altered in duodenal mucosa of CD patients, and this alteration can increase the expression of molecules involved in immune response.