RESUMEN
Purpose: Mitochondrial DNA (mtDNA) abnormalities were previously found to be causative in the pathogenesis of various diseases. Here, comprehensive mitochondrial and nuclear sequence and transcript analyses, along with analyses of the methylation aspects of nuclear genes related to mitochondrial function, were performed in patients with keratoconus (KTCN) to evaluate their contribution to the KTCN pathogenesis. Methods: Blood mtDNA of 42 KTCN and 51 non-KTCN individuals was Sanger sequenced and analyzed along with the previously obtained corneal RNA-sequencing data of 20 KTCN and 21 non-KTCN individuals. In addition, the expression and methylation of mtDNA genes and 1223 mitochondria-related nuclear genes were evaluated. Results: The mtDNA sequence alterations detected in blood coincided with variants identified in transcripts of the matched corneal tissues. In KTCN corneas, 97 mitochondria-related genes were deregulated, including TGFB1, P4HB, and BCL2, which are involved in the extracellular matrix (ECM) organization, collagen formation, and focal adhesion pathways. No changes in the expression of mtDNA transcripts and no differentially methylated genes among the assessed mitochondrial-nuclear gene sets were found. Conclusions: The absence of corneal-specific mtDNA variants indicates that there is no direct relationship between mitochondrial sequence variability and KTCN phenotype in the studied individuals. However, the identified KTCN-specific transcriptomic alterations of the nuclear genes directly related to the mitochondria functioning point to their possible involvement in the ECM organization, collagen formation, and focal adhesion. Translational Relevance: The identification of abnormalities within nuclear genes regulating ECM formation, collagen synthesis, and/or focal adhesion may form the basis of future treatment strategies or predict the progression of corneal changes in KTCN.
Asunto(s)
Queratocono , Colágeno/genética , Matriz Extracelular/genética , Adhesiones Focales , Expresión Génica , Humanos , Queratocono/genética , Mitocondrias/genéticaRESUMEN
Local anesthetics (LAs) are capable of influencing cell viability in systemic immunity and may also modify metabolism of those present in umbilical cord blood (UCB) following obstetric neuraxial analgesia and anaesthesia. Data regarding UCB immature cells, important for the neonate and critical for putative UCB transplantations, are lacking. LAs are capable of stimulating intracellular nitric oxide (NO) in human neutrophils; no information is available concerning newly perpetuated cells and its potential association with viability. The study aimed at assessing the LAs influence on the cell viability and intracellular NO production by UCB CD34+CD133- and CD34+ CD133+ cell populations. Mononuclear cells separated from UCB samples (n = 19) were incubated with bupivacaine (0.0005, 0.005, 1 mM), lidocaine (0.002, 0.02, 4 mM), and ropivacaine (0.0007, 0.007, 1.4 mM) for 4 h. Flow cytometry was applied for the assessment of cell viability and intracellular NO generation in CD34+CD133- and CD34+CD133+ cell populations using annexinV/7-AAD and DAF-2DA stainings, respectively. CD34+CD133+ cells showed less pronounced late apoptosis and necrosis as compared to CD34+CD133-population. Intracellular NO generation was comparable between both cell populations studied. LAs neither influenced cell viability nor changed NO production in either population. LAs do not interfere with viability and intracellular NO generation in the UCB CD34+CD133- and CD34+CD133+ cell populations.
RESUMEN
Nitric oxide (NO) generation by systemic neonatal neutrophils is not clarified. It is also not known whether local anaesthetics (LAs) transferred to the fetal systemic circulation following maternal epidural blockade may affect this process. In the present study, NO generation was evaluated in neutrophils from cord blood (CB, n = 11) and adult blood (n = 10) following exposure to bupivacaine (0.0005, 0.005, 1 mM), lidocaine (0.002, 0.02, 4 mM) and ropivacaine (0.0007, 0.007, 1.4 mM) using flow cytometry, as well as indirectly by determining nitrite concentrations in cell incubation media. To determine the role of NO synthase (NOS) isoforms in NO generation following exposure to LAs, experiments were repeated in the presence of the NOS inhibitors, NG-nitro-L-arginine methyl ester and aminoguanidine; in addition, the expression of NOS isoforms was analysed. CB neutrophils produced less NO than adult neutrophils. LAs, especially ropivacaine and lidocaine, stimulated neutrophil NO generation, but in CB neutrophils this effect was negligible at clinically relevant drug concentrations. A mechanism involving NOS activity was responsible for the observed phenomena. In conclusion, LAs are able to upregulate neutrophil NO production, but in neonates this effect is likely to be clinically insignificant.