HSP70 increases extracellular matrix production by human vascular smooth muscle through TGF-β1 up-regulation. HSP70 increases extracellular matrix production by human vascular smooth muscle through TGF-β1 up-regulation.

Marta González-Ramos1,4,5 , Laura Calleros1,4,5, Susana López-Ongil3,4,5, Viviana Raoch3,4,5, Mercedes Griera3,4,5, Manuel Rodríguez-Puyol1,4,5, Sergio de Frutos1,4,5, Diego Rodríguez-Puyol2,3,4,5.

Link to Pubmed.

The circulating levels of heat shock proteins (HSP) are increased in cardiovascular diseases; however, the implication of this for the fibrotic process typical of such diseases remains unclear. HSP70 can interact with the vascular smooth muscle cells (SMC), the major producer of extracellular matrix (ECM) proteins, through the Toll-like receptors 4 (TLR4). The transforming growth factor type-β1 (TGF-β1) is a well known vascular pro-fibrotic cytokine that is regulated in part by AP-1-dependent transcriptional mechanisms. We hypothesized that extracellular HSP70 could interact with SMCs, inducing TGF-β1 synthesis and subsequent changes in the vascular ECM. We demonstrate that extracellular HSP70 binds to human aorta SMC TLR4, which up-regulates the AP-1-dependent transcriptional activity of the TGF-β1 promoter. This is achieved through the mitogen activated protein kinases JNK and ERK, as demonstrated by the use of specific blockers and the knockdown of TLR4 with specific small interfering RNAs. The TGF-β1 upregulation increase the expression of the ECM proteins type I collagen and fibronectin. This novel observation may elucidate the mechanisms by which HSP70 contributes in the inflammation and fibrosis present in atherosclerosis and other fibrosis-related diseases.


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

María del Nogal*,║,¶ Ph.D., Alicia Luengo*,║¶, Gemma Olmos*,¶,║ Ph.D., Marina Lasa§ Ph.D., Diego Rodriguez–Puyol†,‡,║,¶ M.D., Manuel Rodriguez–Puyol*,║¶ Ph.D., Laura Calleros*,║¶, # Ph.D.

Link to Pubmed.

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.


New losartan-hydrocaffeic acid hybrids as antihypertensive-antioxidant dual drugs: Ester, amide and amine linkersNew losartan-hydrocaffeic acid hybrids as antihypertensive-antioxidant dual drugs: Ester, amide and amine linkersNew losartan-hydrocaffeic acid hybrids as antihypertensive-antioxidant dual drugs: Ester, amide and amine linkersNew losartan-hydrocaffeic acid hybrids as antihypertensive-antioxidant dual drugs: Ester, amide and amine linkers

García G, Serrano I, Sánchez-Alonso P, Rodríguez-Puyol M, Alajarín R, Griera M, Vaquero JJ, Rodríguez-Puyol D, Alvarez-Builla J, Díez-Marqués ML.

Link to Pubmed

We report new examples of a series of losartan-hydrocaffeic hybrids that bear novel ester, amide and amine linkers. These compounds were made by linking hydrocaffeic acid to the side chain of losartan at the C-5 position of the imidazole ring through different strategies. Experiments performed in cultured cells demonstrate that these new hybrids retain the ability to block the angiotensin II effect and have increased antioxidant ability. Most of them reduced arterial pressure in rats better or as much as losartan.





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

González-Ramos M, Mora I, de Frutos S, Garesse R, Rodríguez-Puyol M, Olmos G, Rodríguez-Puyol D.

Link to Pubmed.

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.



Nitric oxide decreases the expression of endothelin-converting enzyme-1 through mRNA destabilization.

Raoch V, Rodríguez-Pascual F, López-Martínez V, Medrano-Andrés D, Rodríguez-Puyol M, Lamas S, Rodríguez-Puyol D, López-Ongil S.

Link to Pubmed.

OBJECTIVE: Endothelial function depends on the equilibrium in the synthesis of vasoactive endothelial factors. It is well known that endothelin and nitric oxide (NO) exhibit reciprocal regulation. We assessed the ability of NO to regulate endothelin-converting enzyme-1 (ECE-1) expression in vascular endothelial cells.

METHODS AND RESULTS: Bovine aortic endothelial cells were incubated with 2 different NO donors as well as with a cyclic-GMP analog, dibutyryl-cGMP (dB-cGMP). ECE-1 protein content and mRNA expression were evaluated by Western blot and Northern blot, respectively, promoter activity by transfection experiments, ECE-1 activity by ELISA, and cGMP production by radioimmunoassay. Both NO donors decreased ECE-1 protein content, mRNA expression, and ECE-1 activity. ODQ, an inhibitor of soluble guanylyl cyclase, blocked those effects. NO donors raised cGMP levels, and dB-cGMP mimicked their effects on ECE-1 expression, which were blocked by KT5823, a nonspecific PKG inhibitor. The changes on ECE-1 expression were due to a destabilization on 3'-untranslated region (3'-UTR) of this mRNA, because the activity of a luciferase reporter construct containing the 3'-UTR of the ECE-1 gene was reduced by dB-cGMP in a PKG-dependent manner. The biological relevance of this regulation was confirmed in bovine aortic endothelial cells coincubated with macrophages in the presence of lipopolysaccharide, in eNOS-deficient mice, and in Wistar rats treated with NO donors. In every case, an inverse relationship was observed between NO and ECE-1 protein content.

CONCLUSION:Our results support that NO regulates ECE-1 expression through a cGMP/PKG-dependent regulatory mechanism at the post-transcriptional level via the 3'-UTR of the ECE-1 gene.


Changes in extracellular matrix composition regulate cyclooxygenase-2 expression in human mesangial cells.

Alique M, Calleros L, Luengo A, Griera M, Iñiguez MÁ, Punzón C, Fresno M,Rodríguez-Puyol M, Rodríguez-Puyol D.

Link to Pubmed.

Glomerular diseases are characterized by a sustained synthesis and accumulation of abnormal extracellular matrix proteins, such as collagen type I. The extracellular matrix transmits information to cells through interactions with membrane components, which directly activate many intracellular signaling events. Moreover, accumulating evidence suggests that eicosanoids derived from cyclooxygenase (COX)-2 participate in a number of pathological processes in immune-mediated renal diseases, and it is known that protein kinase B (AKT) may act through different transcription factors in the regulation of the COX-2 promoter. The present results show that progressive accumulation of collagen I in the extracellular medium induces a significant increase of COX-2 expression in human mesangial cells, resulting in an enhancement in PGE(2) production. COX-2 overexpression is due to increased COX-2 mRNA levels. The study of the mechanism implicated in COX-2 upregulation by collagen I showed focal adhesion kinase (FAK) activation. Furthermore, we observed that the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway by collagen I and collagen I-induced COX-2 overexpression was abolished by PI3K and AKT inhibitors. Additionally, we showed that the cAMP response element (CRE) transcription factor is implicated. Finally, we studied COX-2 expression in an animal model, N(G)-nitro-l-arginine methyl ester hypertensive rats. In renal tissue and vascular walls, COX-2 and collagen type I content were upregulated. In summary, our results provide evidence that collagen type I increases COX-2 expression via the FAK/PI3K/AKT/cAMP response element binding protein signaling pathway.

Deletion of H-Ras decreases renal fibrosis and myofibroblast activation following ureteral obstruction in mice.

Grande MT, Fuentes-Calvo I, Arévalo M, Heredia F, Santos E, Martínez-Salgado C, Rodríguez-Puyol D, Nieto MA, López-Novoa JM.

Link to Pubmed.

Tubulointerstitial fibrosis is characterized by the presence of myofibroblasts that contribute to extracellular matrix accumulation. These cells may originate from resident fibroblasts, bone-marrow-derived cells, or renal epithelial cells converting to a mesenchymal phenotype. Ras GTPases are activated during renal fibrosis and play crucial roles in regulating both cell proliferation and TGF-beta-induced epithelial-mesenchymal transition. Here we set out to assess the contribution of Ras to experimental renal fibrosis using the well-established model of unilateral ureteral obstruction. Fifteen days after obstruction, both fibroblast proliferation and inducers of epithelial-mesenchymal transition were lower in obstructed kidneys of H-ras knockout mice and in fibroblast cell lines derived from these mice. Interestingly, fibronectin, collagen I accumulation, overall interstitial fibrosis, and the myofibroblast population were also lower in the knockout than in the wild-type mice. As expected, we found lower levels of activated Akt in the kidneys and cultured fibroblasts of the knockout. Whether Ras inhibition will turn out to prevent progression of renal fibrosis will require more direct studies.

Targeted genomic disruption of h-ras induces hypotension through a NO-cGMP-PKGpathway-dependent mechanism.

Chamorro-Jorganes A, Grande MT, Herranz B, Jerkic M, Griera M, Gonzalez-Nuñez M, Santos E, Rodriguez-Puyol D, Lopez-Novoa JM, Rodriguez-Puyol M.

Link to journal.

The aim of the present experiments was to evaluate the differences in arterial pressure between H-Ras lacking mice and control mice and to analyze the mechanisms involved in the genesis of the differences. H-Ras lacking mice and mouse embryonic fibroblasts from these animals were used. Blood pressure was measured using 3 different methods: direct intraarterial measurement in anesthetized animals, tail-cuff sphygmomanometer, and radiotelemetry. H-Ras lacking mice showed lower blood pressure than control animals. Moreover, the aorta protein content of endothelial nitric oxide synthase, soluble guanylyl cyclase, and cyclic guanosine monophosphate–dependent protein kinase was higher in H-Ras knockout mice than in control animals. The activity of these enzymes was increased, because urinary nitrite excretion, sodium nitroprusside–stimulated vascular cyclic guanosine monophosphate synthesis, and phosphorylated vasoactive-stimulated phosphoprotein in aortic tissue increased in these animals. Furthermore, mouse embryonic fibroblasts from H-Ras lacking mice showed higher cyclic guanosine monophosphate–dependent protein kinase promoter activity than control cells. These results strongly support the upregulation of the nitric oxide-cyclic guanosine monophosphate pathway in H-Ras–deficient mice. Moreover, they suggest that H-Ras pathway could be considered as a therapeutic target for hypertension treatment.

Fibronectin upregulates cGMP-dependent protein kinase type Iβ through C/EBP transcription factor activation in contractile cells.

Chamorro-Jorganes A, Calleros L, Griera M, Saura M, Luengo A, Rodriguez-Puyol D, Rodriguez-Puyol M.

Link to Pubmed.

The nitric oxide (NO)-soluble guanylate cyclase (sGC) pathway exerts most of its cellular actions through the activation of the cGMP-dependent protein kinase (PKG). Accumulation of extracellular matrix is one of the main structural changes in pathological conditions characterized by a decreased activity of this pathway, such as hypertension, diabetes, or aging, and it is a well-known fact that extracellular matrix proteins modulate cell phenotype through the interaction with membrane receptors such as integrins. The objectives of this study were 1) to evaluate whether extracellular matrix proteins, particularly fibronectin (FN), modulate PKG expression in contractile cells, 2) to analyze the mechanisms involved, and 3) to evaluate the functional consequences. FN increased type I PKG (PKG-I) protein content in human mesangial cells, an effect dependent on the interaction with β(1)-integrin. The FN upregulation of PKG-I protein content was due to increased mRNA expression, determined by augmented transcriptional activity of the PKG-I promoter region. Akt and the transcription factor CCAAT enhancer-binding protein (C/EBP) mediated the genesis of these changes. FN also increased PKG-I in another type of contractile cell, rat vascular smooth muscle cells (RVSMC). Tirofiban, a pharmacological analog of FN, increased PKG-I protein content in RVSMC and rat aortic walls and magnified the hypotensive effect of dibutyryl cGMP in conscious Wistar rats. The present results provide evidence of a mechanism able to increase PKG-I protein content in contractile cells. Elucidation of this novel mechanism provides a rationale for future pharmacotherapy in certain vascular diseases.



H2O2 regulation of vascular function through sGC mRNA stabilization by HuR. Arterioscler Thromb Vasc Biol.

Garrido A, González-Ramos M, Griera M, Guijarro B, Cannata-Andia J, Rodriguez-Puyol D, Rodriguez-Puyol M, Saura M.

Link to Pubmed.

OBJECTIVE: Hydrogen peroxide (H(2)O(2)) is an important mediator in the vasculature, but its role in the regulation of soluble guanylate cyclase (sGC) activity and expression is not completely understood. The aim of this study was to test the effect of H(2)O(2) on sGC expression and function and to explore the molecular mechanism involved.

METHODS AND RESULTS: H(2)O(2) increased sGCβ1 protein steady-state levels in rat aorta and aortic smooth muscle cells (RASMCs) in a time- and dose-dependent manner, and this effect was blocked by catalase. sGCα2 expression increased along with β1 subunit, whereas α1 subunit remained unchanged. Vascular relaxation to an NO donor (sodium nitroprusside) was enhanced by H(2)O(2), and it was prevented by ODQ (sGC inhibitor). cGMP production in both freshly isolated vessels and RASMCs exposed to H(2)O(2) was greatly increased after sodium nitroprusside treatment. The H(2)O(2)-dependent sGCβ1 upregulation was attributable to sGCβ1 mRNA stabilization, conditioned by the translocation of the mRNA-binding protein HuR from the nucleus to the cytosol, and the increased mRNA binding of HuR to the sGCβ1 3' untranslated region. HuR silencing reversed the effects of H(2)O(2) on sGCβ1 levels and cGMP synthesis.

CONCLUSIONS: Our results identify H(2)O(2) as an endogenous mediator contributing to the regulation of vascular tone and point to a key role of HuR in sGCβ1 mRNA stabilization.



Amadori Products Promote Cellular Senescence Activating Insulin-Like Growth Factor-1 Receptor And Down-Regulating The Antioxidant Enzyme Catalase.

María del Nogal-Ávila, Nuria Troyano-Suárez, Pablo Román-García, Jorge B. Cannata-Andía, Manuel Rodriguez-Puyol, Diego Rodriguez-Puyol, Makoto Kuro-O, María P. Ruiz-Torres

Link to Pubmed.

Activation of the insulin growth factor receptor-1 signaling pathways has been largely related to the aging process. Amadori products are produced in pathological conditions such as diabetes and aging, and are potentially involved in diabetic nephropathy or age-associated decline of renal function. We hypothesize that Amadori products induce senescence in primary human mesangial cells through the activation of IGF-1 receptor and investigate, in the present work, the intracellular mechanism involved after this activation. We treated cultured human mesangial cells with glycated albumin, one of the most abundant Amadori product, and senescence was assessed by determining the senescence associated β-galactosidase activity and the expression of the cell cycle regulators p53 and p21. We demonstrated that prolonged exposition (more than 24h) to glycated albumin induced senescence and, in parallel, incremented the release of IGF-1 and the activation of the IGF-1 receptor. Inhibition of the IGF-1 activation prevented the GA induced senescence. Activation of IGF-1R, after GA addition, promoted a reduction in the catalase content through the constitutive activation of Ras and erk1/2 proteins which were, in turn, responsible of the observed GA-induced senescence. In conclusion, we propose that the Amadori product, glycated albumin, promotes premature cell senescence in mesangial cells through the activation of the IGF-1 receptor and the subsequent reduction in the antioxidant enzyme catalase.


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

Gonzalez-Ramos M1, de Frutos S, Griera M, Luengo A, Olmos G, Rodriguez-Puyol D, Calleros L, Rodriguez-Puyol M.
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.

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