Gemma Olmos

Delivery to macrophages and toxic action of etoposide carried in mouse red blood cells

Erythrocytes could be used as physiological carriers of active compounds. Several substances can be loaded into erythrocytes by hypotonic dialysis methods. Furthermore, carrier erythrocyte membrane can be chemically modified in order to promote increased arrival of the loaded compound to macrophages. In this work, we have prepared erythrocytes loaded with etoposide. We found conditions to obtain high etoposide encapsulation yields with minor alteration of some cell parameters of these carrier erythrocytes. Etoposide loaded into erythrocytes is mainly localised in the cytoplasmic compartment. Membrane modification of etoposide-loaded erythrocytes with band 3 crosslinkers produces an increased incorporation of the drug into macrophages mainly by phagocytosis process. The toxic effect of etoposide conveyed in these carrier erythrocytes determined as DNA fragmentation in macrophages was higher than that shown by free etoposide added at the same concentration in the culture medium to macrophages. These results seem to indicate the usefulness of this model to deliver this anti-tumour compound to macrophages, which might be useful in therapy.

Intracellular redox equilibrium is essential for the constitutive expression of AP-1 dependent genes in resting cells: studies on TGF-β1 regulation.

The mechanisms involved in the continuous expression of constitutive genes are unclear. We hypothesize that steady state intracellular reactive oxygen species (ROS), which their levels are tightly maintained, could be regulating the expression of these constitutive genes in resting cells. We analyzed the regulation of an important constitutive gene, TGF-β1, after decreasing intracellular ROS concentration in human mesangial cells. Decreased intracellular hydrogen peroxide by catalase addition reduced TGF-β1 protein, mRNA expression and promoter activity. Furthermore, catalase decreased the basal activity of Activated Protein-1 (AP-1) that regulates TGF-β1 promoter activity. This effect disappeared when AP-1 binding site was removed. Similar results were observed with another protein containing AP-1 binding sites in its promoter, such as eNOS, but it was not the case in other constitutive genes without any AP-1 binding site, as COX1 or PKG1. The pharmacological inhibition of the different ROS synthesis sources by blocking NADPH oxidase, the mitochondrial respiratory chain or xanthine oxidase, or the use of human fibroblasts with genetically deficient mitochondrial activity, induced a similar, significant reduction of steady state ROS concentration as the one observed with catalase. Moreover, there was decreased TGF-β1 expression in all the cases excepting the xanthine oxidase blockade. These findings suggest a novel role for the steady state intracellular ROS concentration, where the compartmentalized, different systems involved in the intracellular ROS production, could be essential for the expression of constitutive AP1-dependent genes, as TGF-β1.

15-Deoxy-Δ12,14-prostaglandin-J2 reveals a new pVHL-independent, lysosomal-dependent mechanism of HIF-1α degradation

Hypoxia-inducible factor-1α (HIF-1α) protein is degraded under normoxia by its association to von Hippel-Lindau protein (pVHL) and further proteasomal digestion. However, human renal cells HK-2 treated with 15-deoxy-Δ12,14-prostaglandin-J2 (15d-PGJ2) accumulate HIF-1α in normoxic conditions. Thus, we aimed to investigate the mechanism involved in this accumulation. We found that 15d-PGJ2 induced an over-accumulation of HIF-1α in RCC4 cells, which lack pVHL and in HK-2 cells treated with inhibitors of the pVHL-proteasome pathway. These results indicated that pVHL-proteasome-independent mechanisms are involved, and therefore we aimed to ascertain them. We have identified a new lysosomal-dependent mechanism of HIF-1α degradation as a target for 15d-PGJ2 based on: (1) HIF-1α colocalized with the specific lysosomal marker Lamp-2a, (2) 15d-PGJ2 inhibited the activity of cathepsin B, a lysosomal protease, and (3) inhibition of lysosomal activity did not result in over-accumulation of HIF-1α in 15d-PGJ2-treated cells. Therefore, expression of HIF-1α is also modulated by lysosomal degradation.

Balance between apoptosis or survival induced by changes in extracellular-matrix composition in human mesangial cells: a key role for ILK-NFκB pathway

Renal fibrosis is the final outcome of many clinical conditions that lead to chronic renal failure, characterized by a progressive substitution of cellular elements by extracellular-matrix proteins, in particular collagen type I. The aim of this study was to identify the mechanisms responsible for human mesangial cell survival, conditioned by changes in extracellular-matrix composition. Our results indicate that collagen I induces apoptosis in cells but only after inactivation of the pro-survival factor NFκB by either the super-repressor IκBα or the PDTC inhibitor. Collagen I activates a death pathway, through ILK/GSK-3β-dependent Bim expression. Moreover, collagen I significantly increases NFκB-dependent transcription, IκBα degradation and p65/NFκB translocation to the nucleus; it activates β1 integrin and this is accompanied by increased activity of ILK which leads to AKT activation. Knockdown of ILK or AKT with small interfering RNA suppresses the increase in NFκB activity. NFκB mediates cell survival through the antiapoptotic protein Bcl-xL. Our data suggest that human mesangial cells exposed to abnormal collagen I are protected against apoptosis by a complex mechanism involving integrin β1/ILK/AKT-dependent NFκB activation with consequent Bcl-xL overexpression, that opposes a simultaneously activated ILK/GSK-3β-dependent Bim expression and this dual mechanism may play a role in the progression of glomerular dysfunction.

Integrin-linked kinase mediates the hydrogen peroxide-dependent transforming growth factor-β1 up-regulation.

Transforming growth factor type-β1 (TGF-β1) has been recognized as a central mediator in many pathological events related to extracellular matrix (ECM) proteins accumulation, where their locally increased expression has been implicated in the fibrosis process of numerous organs, including glomerular fibrosis in the kidney. We and others have reported the TGF-β1 synthesis regulation by reactive oxygen species (ROS), and moreover we also described the implication of integrin-linked kinase (ILK) in the AP-1-dependent TGF-β1 up-regulation. Thus, we propose here that hydrogen peroxide (H2O2)-dependent TGF-β1 regulation may be mediated by ILK activation. First we confirmed the increase in TGF-β1 expression in human mesangial cells (HMC) after treatment with H2O2 or with an alternative H2O2-generating system such as the glucose-oxidase enzyme (GOX). By using immunoblotting, immunofluorescence, and ELISA techniques, we demonstrate that extracellular H2O2 up-regulates TGF-β1 transcription, as well as increases TGF-β1 promoter activity. Furthermore, catalase-decreased intracellular H2O2 abolished TGF-β1 up-regulation. The use of pharmacological inhibitors as well as knockdown of ILK with small interfering RNA (siRNA) demonstrated the implication of a PI3K/ILK/AKT/ERK MAPK signaling pathway axis in the H2O2-induced TGF-β1 overexpression. Finally, we explored the physiological relevance of these findings by treating HMC with angiotensin II, a known stimuli of H2O2 synthesis. Our results confirm the relevance of previous findings after a more physiological stimulus. In summary, our results provide evidence that ILK activity changes may act as a mechanism in response to different stimuli such as H2O2 in the induced TGF-β1 up-regulation in pathological or even physiological conditions.

Delivery to macrophages of interleukin 3 loaded in mouse erythrocytes.

Mouse carrier erythrocytes containing 125I-interleukin 3 have been prepared and treated with band 3 crosslinking reagents. The incorporation of interleukin 3 by hypotonic treatment into mouse erythrocytes reached levels of about 15% of the interleukin 3 added to the medium being predominantly present in the cytosolic fraction (73%). Uptake fell to about 7.4% when using the same conditions but omitting hypotonic shock. The interaction of band 3 crosslinked interleukin 3 loaded erythrocytes with macrophages was also studied. A high level of incorporation of interleukin 3 into macrophages was observed either from band 3 crosslinked, interleukin 3-loaded erythrocytes or from interleukin 3 loaded erythrocytes. The observations encourage the view that the system may be able to deliver and target cytokines and other growth factors to macrophages.

Relevant role of PKG in the progression of fibrosis induced by TNF-like weak inducer of apoptosis

TNF-like weak inducer of apoptosis (TWEAK) is an inflammatory cytokine that activates the FGF-inducible 14 receptor. Both TWEAK and the FGF-inducible 14 receptor are constitutively expressed in the kidney. TWEAK has been shown to modulate several biological responses, such as inflammation, proliferation, differentiation, and apoptosis, that contribute to kidney injury. However, the role of TWEAK in fibrosis and TWEAK-activated intracellular signaling pathways remain poorly understood. We tested the hypothesis that TWEAK can be a potent inducer of renal fibrosis by increasing transforming growth factor (TGF)-β1 expression (a well-known switch in the fibrosis process) through PKG-I downregulation. We showed that in human mesangial cells, TWEAK increased TGF-β1 expression and activity, leading to higher levels of the extracellular matrix protein fibronectin and decreased PKG-I expression and activity via the Ras pathway. PKG-I activation with 8-bromo-cGMP, Ras inactivation with dominant negative Ras, or Ras pathway inhibition with the ERK1/2 inhibitor PD-98059 resulted in the prevention of TWEAK-induced TGF-β1 upregulation. In vivo, exogenous administration of TWEAK to wild-type mice downregulated kidney PKG-I and increased kidney TGF-β1 expression. These effects were blunted in H-Ras knockout mice. Together, these data demonstrate, for the first time, the key role of PKG-I in TGF-β1 induction by TWEAK in kidney cells.

Differential In vitro and In vivo Behavior of Mouse Ascorbate/Fe3+ and Diamide Oxidized Erythrocytes

Chemical oxidation of mouse erythrocytes has been carried out using two different oxidizing systems namely: Diamide and Ascorbate/Fe3+ together with different concentrations of the oxidant. These oxidation treatments produced different extents of modification in membrane proteins as was observed by electrophoretic analyses that showed a possible formation of high molecular weight aggregates. Lipid peroxidation was also observed as the result of these chemical treatments. The action of these two oxidation treatments produced different extents of lipid peroxidation in which the effect Ascorbate/Fe3+ reached higher values than that shown by diamide treatments. To study the resulting in vitro behavior of such oxidized erythrocytes, we have evaluated the recognition of oxidized erythrocytes by peritoneal macrophages. In the conditions used, diamide oxidized erythrocytes were more highly recognized by macrophages than Ascorbate/Fe3+ treated erythrocytes. However, in both cases an influence of serum factors in the recognition process can be inferred. Additionally, we have correlated on one side the action of different oxidation systems on mouse erythrocytes with different in vivo behavior and organ uptake of the oxidized erythrocytes. On the other side, differential targeting of oxidized erythrocytes to a liver or spleen was observed on dependence of the oxidant used.

Hyperphosphatemia induces cellular senescence in human aorta smooth muscle cells through integrin linked kinase (ILK) up-regulation.

Aging is conditioned by genetic and environmental factors. Hyperphosphatemia is related to some pathologies, affecting to vascular cells behavior. This work analyze whether high concentration of extracellular phosphate induces vascular smooth muscle cells senescence, exploring the intracellular mechanisms and highlighting the in vivo relevance of this phenomenon. Human aortic smooth muscle cells treated with β-Glycerophosphate (BGP, 10mM) suffered cellular senescence by increasing p53, p21 and p16 expression and the senescence associated β-galactosidase activity. In parallel, BGP induced ILK overexpression, dependent on the IGF-1 receptor activation, and oxidative stress. Down-regulating ILK expression prevented BGP-induced senescence and oxidative stress. Aortic rings from young rats treated with 10mM BGP for 48h, showed increased p53, p16 and ILK expression and SA-β-gal activity. Seven/eight nephrectomized rats feeding a hyperphosphatemic diet and fifteenth- month old mice showed hyperphosphatemia and aortic ILK, p53 and p16 expression. In conclusion, we demonstrated that high extracellular concentration of phosphate induced senescence in cultured smooth muscle through the activation of IGF-1 receptor and ILK overexpression and provided solid evidences for the in vivo relevance of these results since aged animals showed high levels of serum phosphate linked to increased expression of ILK and senescence genes.

Hyperphosphatemia induces senescence in human endothelial cells by increasing endothelin‐1 production

Hyperphosphatemia is related to some pathologies, affecting vascular cell behavior. This work analyzes whether high concentration of extracellular phosphate induces endothelial senescence through up‐regulation of endothelin‐1 (ET‐1), exploring the mechanisms involved. The phosphate donor β‐glycerophosphate (BGP) in human endothelial cells increased ET‐1 production, endothelin‐converting enzyme‐1 (ECE‐1) protein, and mRNA expression, which depend on the AP‐1 activation through ROS production. In parallel, BGP also induced endothelial senescence by increasing p16 expression and the senescence‐associated β‐galactosidase (SA‐ß‐GAL) activity. ET‐1 itself was able to induce endothelial senescence, increasing p16 expression and SA‐ß‐GAL activity. In addition, senescence induced by BGP was blocked when different ET‐1 system antagonists were used. BGP increased ROS production at short times, and the presence of antioxidants prevented the effect of BGP on AP1 activation, ECE‐1 expression, and endothelial senescence. These findings were confirmed in vivo with two animal models in which phosphate serum levels were increased: seven/eight nephrectomized rats as chronic kidney disease models fed on a high phosphate diet and aged mice. Both models showed hyperphosphatemia, higher levels of ET‐1, and up‐regulation in aortic ECE‐1, suggesting a direct relationship between hyperphosphatemia and ET‐1. Present results point to a new and relevant role of hyperphosphatemia on the regulation of ET‐1 system and senescence induction at endothelial level, both in endothelial cells and aorta from two animal models. The mechanism involved showed a higher ROS production, which probably activates AP‐1 transcription factor and, as a result, ECE‐1 expression, increasing ET‐1 synthesis, which in consequence induces endothelial senescence.