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Biomedical subjects

Santiago Lamas

Publications and source records attributed to Santiago Lamas.

At least 19 recordsLinked to original sources
PubMed2007

Mitochondrial dysfunction in human pathologies.

The integrity of mitochondrial function is fundamental to cell life. The cell demands for mitochondria and their complex integration into cell biology, extends far beyond the provision of ATP. It follows that disturbances of mitochondrial function lead to disruption of cell function, expressed as disease or even death. Mitochondria are major producers of free radical species and also possibly of nitric oxide, and are, at the same time, major targets for oxidative damage. In this review we consider recent developments in our knowledge of how the mitochondrial production of reactive oxygen species (ROS) plays a critical role in several major human pathologies. We will also consider recent advances in our understanding of the molecular mechanisms involved in mitochondrial ROS detoxification.

Aging↗
PubMed2007

Proteomic identification of S-nitrosylated proteins in endothelial cells.

Nitric oxide (NO) produced in endothelial cells exerts important roles in the vascular system. In recent years, posttranslational modifications induced by NO have been increasingly studied and, among them, cysteine modification by S-nitrosylation (also called S-nitrosation) has been hypothesized to represent a relevant mechanism for cell signaling. Thus, knowledge of the proteins that can be S-nitrosylated in endothelial cells will help to better understand the possible role of this modification. We describe a protocol to identify the S-nitrosylome or S-nitrosoproteome of endothelial cells, based on the specific derivatization of the S-nitrosylation, substituting it by a biotinylation, and the purification of the biotinylated proteins.

Cysteine↗
PubMed2006

Viral protease cleavage of inhibitor of kappaBalpha triggers host cell apoptosis.

Apoptosis is an innate immune response to viral infection that limits viral replication. However, the mechanisms by which cells detect viral infection and activate apoptosis are not completely understood. We now show that during Coxsackievirus infection, the viral protease 3C(pro) cleaves inhibitor of kappaBalpha (IkappaBalpha). A proteolytic fragment of IkappaBalpha then forms a stable complex with NF-kappaB, translocates to the nucleus, and inhibits NF-kappaB transactivation, increasing apoptosis and decreasing viral replication. In contrast, cells with reduced IkappaBalpha expression are more susceptible to viral infection, with less apoptosis and more viral replication. IkappaBalpha thus acts as a sensor of viral infection. Cleavage of host proteins by pathogen proteases is a novel mechanism by which the host recognizes and responds to viral infection.

3C Viral Proteases↗
PubMed2006

Role of peroxynitrite in endothelial damage mediated by Cyclosporine A.

Although Cyclosporine A (CsA) is an effective therapy for immunosuppression, its use encompasses serious side effects that have been associated with oxidative stress. We previously reported the intracellular formation of both peroxynitrite and 3-nitrotyrosine in cultured bovine aortic endothelial cells (BAEC) when exposed to CsA. Here we show that re-addition of CsA to BAEC increases peroxynitrite formation in a concentration-dependent manner. This effect is inhibited by the glutathione donor and antioxidant, N-acetylcysteine (NAC). BAEC exposed to CsA showed impaired integrity of plasma membranes and increased cytolysis, a phenomenon prevented by NAC. When CsA was administered to mice, the increased presence of 3-nitrotyrosine was detected in the aortic endothelium, an effect also abrogated by the concomitant administration of NAC. An increase in nitrated MnSOD was detected in BAEC treated with CsA and the peroxynitrite donor SIN-1 and recapitulated in recombinant MnSOD, exposed to the conditioned media from BAEC. We propose that CsA promotes nitration of specific molecular targets, such as MnSOD, within vascular endothelial cells. This may represent a pathogenetic mechanism of vascular injury. Inhibition of this process by clinically applicable antioxidants, such as NAC, lends a basis for the exploration of therapeutic alternatives in patients treated with CsA.

Acetylcysteine↗
PubMed2006

Nitric oxide regulates mitochondrial oxidative stress protection via the transcriptional coactivator PGC-1alpha.

Nitric oxide (NO) has both prooxidant and antioxidant activities in the endothelium; however, the molecular mechanisms involved are still a matter of controversy. PGC-1alpha [peroxisome proliferators-activated receptor (PPAR) gamma coactivator 1-alpha] induces the expression of several members of the mitochondrial reactive oxygen species (ROS) detoxification system. Here, we show that NO regulates this system through the modulation of PGC-1alpha expression. Short-term (<12 h) treatment of endothelial cells with NO donors down-regulates PGC-1alpha expression, whereas long-term (>24 h) treatment up-regulates it. Treatment with the NOS inhibitor l-NAME has the opposite effect. Down-regulation of PGC-1alpha by NO is mediated by protein kinase G (PKG). It is blocked by the soluble guanylate cyclase (sGC) inhibitor ODQ and the PKG inhibitor KT5823, and mimicked by the cGMP analog 8-Br-cGMP. Changes in PGC-1alpha expression are in all cases paralleled by corresponding variations in the mitochondrial ROS detoxification system. Cells that transiently overexpress PGC-1alpha from the cytomeglovirus (CMV) promoter respond poorly to NO donors. Analysis of tissues from eNOS(-/-) mice showed reduced levels of PGC-1alpha and the mitochondrial ROS detoxification system. These data suggest that NO can regulate the mitochondrial ROS detoxification system both positively and negatively through PGC-1alpha.

Animals↗
PubMed2006

Cbfa-1 mediates nitric oxide regulation of MMP-13 in osteoblasts.

During bone development, osteoblast differentiation requires remodeling of the extracellular matrix. Although underlying mechanisms have not been elucidated, evidence points to the participation of the nitric oxide (NO) and cyclic guanosine 3',5'-monophosphate (cGMP) system. Here, we detected increased matrix metalloproteinase (MMP)-13 mRNA, protein and activity, as well as increased inducible NO synthase (iNOS) and NO production during the differentiation of MC3T3-E1 osteoblasts. Transcriptional activity of the MMP-13 promoter was augmented by NO, 8-bromo-cGMP (8-Br-cGMP), and by a dominant-positive form of protein kinase G (PKG1-alpha). The stimulatory effect on the MMP-13 promoter was partially inhibited by mutation of the osteoblast-specific element 2 (OSE-2) binding site. Core binding factor-1 (Cbfa-1) expression peaked at 7 days of differentiation, and was phosphorylated by PKG in vitro. Cbfa-1 was localized to cell nuclei, and its translocation was inhibited by the iNOS inhibitor 1400W. Immunohistological examination revealed that MMP-13 and Cbfa-1 expression levels are both reduced in 17-day-old embryos of iNOS-deficient mice. Silencing of Cbfa-1 mRNA blocked MMP-13 expression without interfering with endogenous NO production, confirming its role in NO-induced MMP-13 expression by MC3T3-E1 cells. The results described here suggest a mechanism by which NO regulates osteogenesis.

3T3 Cells↗
PubMed2006

NMDA induces post-transcriptional regulation of alpha2-guanylyl-cyclase-subunit expression in cerebellar granule cells.

Activation of N-methyl-D-aspartate (NMDA) glutamate receptors commonly affects gene expression in different neurons. We reported previously that chronic treatment of rat cerebellar granule cells with NMDA (24 hours) upregulates the expression of mRNA encoding the alpha2 subunit of the nitric-oxide-sensitive guanylyl cyclase. However, the molecular mechanisms involved in this process remained to be elucidated. Here, we have performed mRNA-decay experiments using the transcriptional inhibitor actinomycin D, providing evidence that the half-life of alpha2 mRNA is significantly prolonged in cells exposed to NMDA. The role of the 3' untranslated region of the alpha2 transcripts in NMDA-induced mRNA stabilisation was examined and an association between the RNA-binding proteins AUF1 and ELAV-like protein 1 (HuR/HuA), and endogenous alpha2 mRNA was demonstrated in vivo, as revealed by coimmunoprecipitation experiments with specific antibodies against AUF1 and HuR. Further studies indicated that stimulation of the NMDA receptor induces a downregulation in AUF1 levels stabilising the alpha2 mRNA transcripts. These events are triggered through a mechanism that depends on formation of nitric oxide, and on the subsequent activation of guanylyl cyclase and cGMP dependent protein kinases.

3' Untranslated Regions↗
PubMed2006

Constitutive ALK5-independent c-Jun N-terminal kinase activation contributes to endothelin-1 overexpression in pulmonary fibrosis: evidence of an autocrine endothelin loop operating through the endothelin A and B receptors.

The signal transduction mechanisms generating pathological fibrosis are almost wholly unknown. Endothelin-1 (ET-1), which is up-regulated during tissue repair and fibrosis, induces lung fibroblasts to produce and contract extracellular matrix. Lung fibroblasts isolated from scleroderma patients with chronic pulmonary fibrosis produce elevated levels of ET-1, which contribute to the persistent fibrotic phenotype of these cells. Transforming growth factor beta (TGF-beta) induces fibroblasts to produce and contract matrix. In this report, we show that TGF-beta induces ET-1 in normal and fibrotic lung fibroblasts in a Smad-independent ALK5/c-Jun N-terminal kinase (JNK)/Ap-1-dependent fashion. ET-1 induces JNK through TAK1. Fibrotic lung fibroblasts display constitutive JNK activation, which was reduced by the dual ETA/ETB receptor inhibitor, bosentan, providing evidence of an autocrine endothelin loop. Thus, ET-1 and TGF-beta are likely to cooperate in the pathogenesis of pulmonary fibrosis. As elevated JNK activation in fibrotic lung fibroblasts contributes to the persistence of the myofibroblast phenotype in pulmonary fibrosis by promoting an autocrine ET-1 loop, targeting the ETA and ETB receptors or constitutive JNK activation by fibrotic lung fibroblasts is likely to be of benefit in combating chronic pulmonary fibrosis.

Actins↗
PubMed2006

ACE clock scoring: a comparison with eight standard correction methods in a population of low educational level.

The authors compared the accuracy of Addenbrooke's Cognitive Examination Clock Drawing Test scoring method with 8 standard Clock Drawing Test scoring methods in a Spanish speaking population of low educational level. A clinical group composed of 70 patients affected by dementia and 25 patients with memory complaints without dementia was compared with 72 controls matched for gender, age, and educational level. The clinical group was studied with standard neuropsychological instruments and neuroimaging. Clock Drawing Tests extracted from Addenbrooke's Cognitive Examination were randomly distributed and scored by 2 independent raters. There were statistically significant differences between the different scoring methods. Addenbrooke's Cognitive Examination Clock Drawing Test scoring method obtained and optimal areas under the curve and interrater reliability, showing no statistical differences with previously standardized systems. In our population, Addenbrooke's Cognitive Examination and the methods of Cahn, Mendez, Rouleau, Shulman, and Sunderland were of optimal and equivalent usefulness regarding dementia detection.

Aged↗
PubMed2006

Transforming growth factor-beta receptor requirements for the induction of the endothelin-1 gene.

Expression of the endothelin (ET)-1 gene is subject to complex regulation by numerous factors, among which the cytokine transforming growth factor-beta (TGF-beta) is one of the most important. TGF-beta action is based on the activation of the Smad signaling pathway. Smad proteins activate transcription of the gene by cooperation with activator protein-1 (AP-1) at specific sites on the ET-1 promoter. Smad signaling pathway is initiated by binding of the cytokine to a heteromeric complex of type I and type II receptors. Signal is then propagated to the nucleus by specific members of the Smad family. Most cell types contain a type I receptor known as ALK5. However, endothelial cells are unique because they coexpress an additional type I receptor named ALK1. These forms do not constitute redundant receptors with the same function, but they actually activate different Smad-mediated expression programs that lead to specific endothelial phenotypes. TGF-beta/ALK5/Smad3 pathway is associated to a mature endothelium because it leads to inhibition of cell migration/proliferation. Conversely, TGF-beta/ALK1/Smad5 activates both processes and is more related to the angiogenic state. We have analyzed the TGF-beta receptor subtype requirements for the activation of the ET-1 gene. For that purpose, we have overexpressed type I receptor and Smad isoforms in endothelial cells and analyzed the effect on ET-1 expression. Our experiments indicate that TGF-beta induces ET-1 expression preferentially through the activation of the ALK5/Smad3 pathway and, therefore, the expression of the vaso-constrictor may be associated to a quiescent and mature endothelial phenotype.

Activin Receptors, Type I↗
PubMed2005

Direct interaction between the reductase domain of endothelial nitric oxide synthase and the ryanodine receptor.

We have performed the recombinant expression and purification of the reductase domain of endothelial nitric oxide synthase (eNOS) and used it as a bait in search for interacting proteins present in endothelial cells. Using mass spectrometry of the bound proteins run in a PAGE-SDS gel, we were able to identify the ryanodine receptor (RyR) as a novel eNOS-binding partner. This interaction was confirmed through immunoprecipitation of both RyR and eNOS from endothelial cells and cardiac myocytes. Immunofluorescence data indicated that a subpopulation of eNOS associates with RyR in perinuclear regions of the cell, where eNOS might be responsible for the known nitrosylation of RyR.

Animals↗
PubMed2005

S-nitrosylation of Hsp90 promotes the inhibition of its ATPase and endothelial nitric oxide synthase regulatory activities.

Nitric oxide is implicated in a variety of signaling pathways in different systems, notably in endothelial cells. Some of its effects can be exerted through covalent modifications of proteins and, among these modifications, increasing attention is being paid to S-nitrosylation as a signaling mechanism. In this work, we show by a variety of methods (ozone chemiluminescence, biotin switch, and mass spectrometry) that the molecular chaperone Hsp90 is a target of S-nitrosylation and identify a susceptible cysteine residue in the region of the C-terminal domain that interacts with endothelial nitric oxide synthase (eNOS). We also show that the modification occurs in endothelial cells when they are treated with S-nitroso-l-cysteine and when they are exposed to eNOS activators. Hsp90 ATPase activity and its positive effect on eNOS activity are both inhibited by S-nitrosylation. Together, these data suggest that S-nitrosylation may functionally regulate the general activities of Hsp90 and provide a feedback mechanism for limiting eNOS activation.

Adenosine Triphosphatases↗
PubMed2005

Endothelin-1 expression is strongly repressed by AU-rich elements in the 3'-untranslated region of the gene.

The regulation of the synthesis of the endothelial-derived vasoconstrictor ET-1 (endothelin-1) is a complex process that occurs mainly at the mRNA level. Transcription of the gene accounts for an important part of the regulation of expression, as already described for different modulators such as the cytokine TGF-beta (transforming growth factor-beta). However, very little is known about mechanisms governing ET-1 expression at the post-transcriptional level. The aim of the present study was to investigate the regulation of the ET-1 expression at this level. Since the 3'-UTR (3'-untranslated region) of mRNAs commonly contains genetic determinants for the post-transcriptional control of gene expression, we focused on the potential role of the 3'-UTR of ET-1 mRNA. Experiments performed with luciferase reporter constructs containing the 3'-UTR showed that this region exerts a potent destabilizing effect. Deletional analyses allowed us to locate this activity within a region at positions 924-1127. Some (but not all) of the AREs (AU-rich elements) present in this region were found to be essential for this mRNA-destabilizing activity. We also present evidence that cytosolic proteins from endothelial cells interact specifically with these RNA elements, and that a close correlation exists between the ability of the AREs to destabilize ET-1 mRNA and the binding of proteins to these elements. Our results are compatible with the existence of a strong repressional control of ET-1 expression mediated by destabilization of the mRNA exerted through the interaction of specific cytosolic proteins with AREs present in the 3'-UTR of the gene.

3' Untranslated Regions↗
PubMed2005

PGC-1alpha regulates the mitochondrial antioxidant defense system in vascular endothelial cells.

OBJECTIVE: Mitochondrial production of oxidants contributes to a variety of pathological conditions including the vascular complications of diabetes, neurodegenerative diseases, and cellular senescence. We postulated that a transcriptional coactivator, peroxisome proliferator activated receptor-gamma coactivator 1alpha (PGC-1alpha), a major regulator of oxidative metabolism and mitochondrial biogenesis, could be involved in the transcriptional regulation of the mitochondrial antioxidant defense system in vascular endothelial cells. METHODS AND RESULTS: We show that PGC-1alpha is present in human, bovine, and mouse endothelial cells and positively modulates the expression of the mitochondrial detoxification system. Endothelial cells that overexpress PGC-1alpha show reduced accumulation of reactive oxygen species (ROS), increased mitochondrial membrane potential, and reduced apoptotic cell death both in basal and oxidative stress conditions. Downregulation of PGC-1alpha levels by siRNA reduces the expression of mitochondrial detoxification proteins. CONCLUSIONS: These results unveil a novel regulatory pathway that links mitochondrial activity and mitochondrial oxidative stress protective systems. In addition, they suggest that PGC-1alpha could play a crucial protective role in vascular complications of diabetes, where the mitochondrial metabolism of glucose has been shown to result in oxidative stress and vascular endothelial cell dysfunction.

Animals↗
PubMed2005

Matrix metalloproteinase 13 mediates nitric oxide activation of endothelial cell migration.

To explore the mechanisms by which NO elicits endothelial cell (EC) migration we used murine and bovine aortic ECs in an in vitro wound-healing model. We found that exogenous or endogenous NO stimulated EC migration. Moreover, migration was significantly delayed in ECs derived from endothelial NO synthase-deficient mice compared with WT murine aortic EC. To assess the contribution of matrix metalloproteinase (MMP)-13 to NO-mediated EC migration, we used RNA interference to silence MMP-13 expression in ECs. Migration was delayed in cells in which MMP-13 was silenced. In untreated cells MMP-13 was localized to caveolae, forming a complex with caveolin-1. Stimulation with NO disrupted this complex and significantly increased extracellular MMP-13 abundance, leading to collagen breakdown. Our findings show that MMP-13 is an important effector of NO-activated endothelial migration.

Animals↗
PubMed2005

Nitrosylation of thiols in vascular homeostasis and disease.

Post-translational modification of thiols to form a nitrosothiol (S-nitrosylation) is gaining attention as a mechanism by which nitric oxide can exert some of its effects in the cardiovascular system, as well as in other systems. It has been proposed that this modification would have a particularly important role in cell signaling. However, its study has been hampered by many technical difficulties. In this paper, we summarize current achievements in the field that may help to answer the questions that have been posed about the functional role of this modification. Some of these achievements have arisen from methodologic improvements, such as proteomic studies or approaches to identify subcellular localization and tissue distribution of the modification. New functional consequences of the modification of individual proteins are also summarized.

Animals↗