Verónica Miguel

Antioxidant responses and cellular adjustments to oxidative stress

Redox biological reactions are now accepted to bear the Janus faceted feature of promoting both physiological signaling responses and pathophysiological cues. Endogenous antioxidant molecules participate in both scenarios. This review focuses on the role of crucial cellular nucleophiles, such as glutathione, and their capacity to interact with oxidants and to establish networks with other critical enzymes such as peroxiredoxins. We discuss the importance of the Nrf2-Keap1 pathway as an example of a transcriptional antioxidant response and we summarize transcriptional routes related to redox activation. As examples of pathophysiological cellular and tissular settings where antioxidant responses are major players we highlight endoplasmic reticulum stress and ischemia reperfusion. Topologically confined redox-mediated post-translational modifications of thiols are considered important molecular mechanisms mediating many antioxidant responses, whereas redox-sensitive microRNAs have emerged as key players in the posttranscriptional regulation of redox-mediated gene expression. Understanding such mechanisms may provide the basis for antioxidant-based therapeutic interventions in redox-related diseases.

Polyelectrolyte Multilayers Containing Triblock Copolymers of Different Charge Ratio

Multilayers formed by the sodium salt of poly(4-styrenesulfonate), PSS, and triblock copolymers of the form PDMAEMA-PCL-PDMAEMA (PDMAEMA corresponding to poly[2-(N,N-dimethylamino)ethyl methacrylate), and PCL to poly(epsilon-caprolactone) have been built by layer-by-layer self-assembly from the aqueous polyelectrolyte solutions. Two types of block copolymers have been used which differ on the type of the amino groups, either hydrochloride or quaternized. This leads to changes in the charge density of the chains for the same content of amino groups. The growth of the multilayers has been followed using dissipative quartz crystal microbalance and ellipsometry techniques. The results show that, independently of the conditions used in the assembling, the film thickness grows linearly with the number of layers. The comparison of the thickness values obtained from D-QCM and ellipsometry has allowed us to calculate the water content of the polymer film. The analysis of the D-QCM data also provides the shear modulus, whose values are typical of a rubber-like polymer system. The analysis of the mass adsorbed calculated by the ellipsometric measurements indicated that the nature of the charge compensation mechanism is extrinsic for all the studied systems, although the degree of extrinsic compensation is strongly dependent on the copolymer used and the concentration in solution. Finally, it was found that the adsorption kinetic of the layers is bimodal for all the films built. Even though the characteristic adsorption times depend on the specific copolymer used, no dependence on the number of layers has been found for a given multilayer.

NADPH Oxidase 4 (Nox4) Suppresses Mitochondrial Biogenesis and Bioenergetics in Lung Fibroblasts via a Nuclear Factor Erythroid-Derived 2-like 2 (Nrf2)-Dependent Pathway

Mitochondrial bioenergetics are critical for cellular homeostasis and stress responses. The reactive oxygen species-generating enzyme, NADPH oxidase 4 (Nox4), regulates a number of physiological and pathological processes, including cellular differentiation, host defense, and tissue fibrosis. In this study we explored the role of constitutive Nox4 activity in regulating mitochondrial function. An increase in mitochondrial oxygen consumption and reserve capacity was observed in murine and human lung fibroblasts with genetic deficiency (or silencing) of Nox4. Inhibition of Nox4 expression/activity by genetic or pharmacological approaches resulted in stimulation of mitochondrial biogenesis, as evidenced by elevated mitochondrial-to-nuclear DNA ratio and increased expression of the mitochondrial markers transcription factor A (TFAM), citrate synthase, voltage-dependent anion channel (VDAC), and cytochrome c oxidase subunit 4 (COX IV). Induction of mitochondrial biogenesis was dependent on TFAM up-regulation but was independent of the activation of the peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α). The enhancement of mitochondrial bioenergetics as well as the increase in mitochondrial proteins in Nox4-deficient lung fibroblasts is inhibited by silencing of nuclear factor erythroid-derived 2-like 2 (Nrf2), supporting a key role for Nrf2 in control of mitochondrial biogenesis. Together, these results indicate a critical role for both Nox4 and Nrf2 in counter-regulation of mitochondrial biogenesis and metabolism.

Role of redoximiRs in fibrogenesis

Fibrosis can be defined as an excessive accumulation of extracellular matrix (ECM) components, ultimately leading to stiffness, scarring and devitalized tissue. MicroRNAs (miRNAs) are short, 19–25 nucleotides (nt), non-coding RNAs involved in the post-transcriptional regulation of gene expression. Recently, miRNAs have also emerged as powerful regulators of fibrotic processes and have been termed “fibromiRs”. Oxidative stress represents a self-perpetuating mechanism in fibrogenesis. MiRNAs can also influence the expression of genes responsible for the generation of reactive oxygen species (ROS) and antioxidant defence and are termed “redoximiRs”. Here, we review the current knowledge of mechanisms by which “redoximiRs” regulate fibrogenesis. This new set of miRNAs may be called “redoxifibromiRs”.

Protective role for miR-9-5p in the fibrogenic transformation of human dermal fibroblasts

BackgroundExcessive accumulation of extracellular matrix (ECM) proteins is the hallmark of fibrotic diseases, including skin fibrosis. This response relies on the activation of dermal fibroblasts that evolve into a pro-fibrogenic phenotype. One of the major players in this process is the cytokine transforming growth factor-β (TGF-β). MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression affecting a wide range of pathophysiological events including fibrogenesis. MicroRNA-9-5p (miR-9-5p) has been shown to exert a protective role in lung and peritoneal fibrosis. This study aimed to evaluate the role of miR-9-5p in skin fibrosis.ResultsmiR-9-5p is up-regulated in TGF-β1-treated human dermal fibroblasts (HDFs). In silico identification of miR-9-5p targets spotted the type II TGF-β receptor (TGFBR2) as a potential TGF-β signaling-related effector for this miRNA. Consistently, over-expression of miR-9-5p in HDFs down-regulated TGFBR2 at both the mRNA and protein levels and reduced the phosphorylation of Smad2 and the translocation of Smad2/3 to the nucleus. In keeping, over-expression of miR-9-5p significantly delayed TGF-β1-dependent transformation of dermal fibroblasts, decreasing the expression of ECM protein collagen, type I, alpha 1 (Col1α1), and fibronectin (FN), the amount of secreted collagen proteins, and the expression of the archetypal myofibroblast marker alpha-smooth muscle actin (α-SMA). By contrast, specific inhibition of miR-9-5p resulted in enhanced presence of fibrosis markers. The expression of miR-9-5p was also detected in the skin and plasma in the mouse model of bleomycin-induced dermal fibrosis. Using lentiviral constructs, we demonstrated that miR-9-5p over-expression was also capable of deterring fibrogenesis in this same model.ConclusionsmiR-9-5p significantly prevents fibrogenesis in skin fibrosis. This is mediated by an abrogation of TGF-β-mediated signaling through the down-regulation of TGFBR2 expression in HDFs. These results may pave the way for future diagnostic or therapeutic developments for skin fibrosis based on miR-9-5p.

Role of glutathione biosynthesis in endothelial dysfunction and fibrosis

Glutathione (GSH) biosynthesis is essential for cellular redox homeostasis and antioxidant defense. The rate-limiting step requires glutamate-cysteine ligase (GCL), which is composed of the catalytic (GCLc) and the modulatory (GCLm) subunits. To evaluate the contribution of GCLc to endothelial function we generated an endothelial-specific Gclc haplo-insufficient mouse model (Gclc e/+ mice). In murine lung endothelial cells (MLEC) derived from these mice we observed a 50% reduction in GCLc levels compared to lung fibroblasts from the same mice. MLEC obtained from haplo-insufficient mice showed significant reduction in GSH levels as well as increased basal and stimulated ROS levels, reduced phosphorylation of eNOS (Ser 1177) and increased eNOS S-glutathionylation, compared to MLEC from wild type (WT) mice. Studies in mesenteric arteries demonstrated impaired endothelium-dependent vasodilation in Gclc(e/+) male mice, which was corrected by pre-incubation with GSH-ethyl-ester and BH4. To study the contribution of endothelial GSH synthesis to renal fibrosis we employed the unilateral ureteral obstruction model in WT and Gclc(e/+) mice. We observed that obstructed kidneys from Gclc(e/+) mice exhibited increased deposition of fibrotic markers and reduced Nrf2 levels. We conclude that the preservation of endothelial GSH biosynthesis is not only critical for endothelial function but also in anti-fibrotic responses.