Artículos (Fisiología Médica y Biofísica)

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Olive leaves and mill waste exert anti-tumoral properties in hepatoma cell line
(Omics, 2014-05-07) Ranchal, Isidora; Luque de Castro, María Dolores; Muntané Relat, Jordi; Universidad de Sevilla. Departamento de Fisiología Médica y Biofísica; Universidad de Sevilla. CTS1098: Mecanismos Moleculares del Hepatocarcinoma y Sus Estrategias Terapéuticas
Different phenolic compounds such as oleuropein and hydroxytyrosol have demonstrated antitumoral activity in cancer cell lines. Olive derivatives have demonstrated beneficial properties in cardiovascular diseases. The study was addressed to show the potential beneficial properties of olive leaves and olive oil mill waste extracts in hepatoma cell lines. Aflatoxin B1 (AFB1) was administered to hepatoma cell line (HepG2). Different parameters related to cell death, cell proliferation and DNA damage were determined. The expression of p53 and c-Src (activated and inhibitory phosphorylated state) was assessed by Western blot analysis in HepG2. The olive leaves and olive oil mill waste extracts contained oleuropein and sydroxytyrosol, respectively. AFB1 induced cell proliferation and death, associated to a rise on p53 and c-Src expression in HepG2 cells. Oleuropein and hydroxytyrosol reduced cell necrosis and DNA damage in HepG2 cells. However, the administration of olive leaves and olive oil mill waste extracts increased cell necrosis, DNA damage. These effects of natural extracts were associated with a reduction of activated c-Src expression and cell proliferation in AFB1-treated HepG2 cells. These results support that olive leaves extract and olive oil mill waste extracts, but not oleuropein and hydroxytyrosol, may exert antitumoral effect against a hepatoma cancer cell line.
Impaired autophagic flux is associated with increased endoplasmic reticulum stress during the development of NAFLD
(Nature Publishing Group; Springernature, 2014-04-17) González-Rodríguez, A.; Mayoral, R.; Agra, N.; Valdés Pardo, V. G.; Valdecantos, M. P.; Miquilena-Colina, M. E.; Muntané Relat, Jordi; Valverde, A. M.; Universidad de Sevilla. Departamento de Fisiología Médica y Biofísica; Comunidad de Madrid; Ministerio de Economia, Industria y Competitividad (MINECO). España; Centro de Investigacion Biomedica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM, ISCIII, Barcelona, Spain); Centro de Investigacion Biomedica en Red de Enfermedades Hepaticas y Digestivas (CIBEREHD, ISCIII); Universidad de Sevilla. CTS1098: Mecanismos Moleculares del Hepatocarcinoma y Sus Estrategias Terapéuticas
The pathogenic mechanisms underlying the progression of non-alcoholic fatty liver disease (NAFLD) are not fully understood. In this study, we aimed to assess the relationship between endoplasmic reticulum (ER) stress and autophagy in human and mouse hepatocytes during NAFLD. ER stress and autophagy markers were analyzed in livers from patients with biopsy-proven non alcoholic steatosis (NAS) or non-alcoholic steatohepatitis (NASH) compared with livers from subjects with histologically normal liver, in livers from mice fed with chow diet (CHD) compared with mice fed withhigh fat diet (HFD) or methionine-choline-deficient (MCD) diet and in primary and Huh7 humanhepatocytesloadedwith palmitic acid (PA). In NASH patients, significant increases in hepatic messenger RNA levels of markers of ER stress (activating transcription factor 4 (ATF4), glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP)) and autophagy (BCN1) were found compared with NAS patients. Likewise, protein levels of GRP78, CHOP and p62/SQSTM1 (p62) autophagic substrate were significantly elevated in NASH compared with NAS patients. In livers from mice fed with HFD or MCD, ER stress-mediated signaling was parallel to the blockade of the autophagic flux assessed by increases in p62, microtubule-associated protein 2 light chain 3 (LC3-II)/LC3-I ratio and accumulation of autophagosomes compared with CHD fed mice. In Huh7 hepatic cells, treatment with PA for 8h triggered activation of both unfolding protein response and the autophagic flux. Conversely, prolonged treatment with PA (24h) induced ERstress and cell death together with a blockade of the autophagic flux. Under these conditions, cotreatment with rapamycin or CHOP silencing ameliorated these effects and decreased apoptosis. Our results demonstrated that the autophagic flux is impaired in the liver from both NAFLD patients and murine models of NAFLD, as well as in lipid-overloaded human hepatocytes, and it could be due to elevated ER stress leading to apoptosis. Consequently, therapies aimed to restore the autophagic flux might attenuate or prevent the progression of NAFLD.
Apicobasal polarity controls lymphocyte adhesion to hepatic epithelial cells
(Cell Press, 2015-09-25) Reglero-Real, Natalia; Álvarez-Varela, Adrián; Cernuda-Morollón, Eva; Feito, Jorge; Marcos-Ramiro, Beatriz; Muntané Relat, Jordi; Fernández-Martín, Laura; Millán, Jaime; Universidad de Sevilla. Departamento de Fisiología Médica y Biofísica; Comunidad de Madrid; Gobierno de España; Universidad de Sevilla. CTS1098: Mecanismos Moleculares del Hepatocarcinoma y Sus Estrategias Terapéuticas
Loss of apicobasal polarity is a hallmark of epithelial pathologies. Leukocyte infiltration and crosstalk with dysfunctional epithelial barriers are crucial for the inflammatory response. Here, we show that apicobasal architecture regulates the adhesion between hepatic epithelial cells and lymphocytes. Polarized hepatocytes and epithelium from bile ducts segregate the intercellular adhesion molecule 1 (ICAM-1) adhesion receptor onto their apical, microvilli-rich membranes, which are less accessible by circulating immune cells. Upon cell depolarization, hepatic ICAM-1 becomes exposed and increases lymphocyte binding. Polarized hepatic cells prevent ICAM-1 exposure to lymphocytes by redirecting basolateral ICAM-1 to apical domains. Loss of ICAM-1 polarity occurs in human inflammatory liver diseases and can be induced by the inflammatory cytokine tumor necrosis factor alpha (TNF-α). We propose that adhesion receptor polarization is a parenchymal immune checkpoint that allows functional epithelium to hamper leukocyte binding. This contributes to the haptotactic guidance of leukocytes toward neighboring damaged or chronically inflamed epithelial cells that expose their adhesion machinery.
Antisense therapeutics in oncology: current status
(Dove Medical Press Ltd, 2014-11-03) Farooqi, Ammad Ahmad; Rehman, Zia ur; Muntané Relat, Jordi; Universidad de Sevilla. Departamento de Fisiología Médica y Biofísica; Universidad de Sevilla. CTS1098: Mecanismos Moleculares del Hepatocarcinoma y Sus Estrategias Terapéuticas
There is increasing progress in translational oncology and tremendous breakthroughs have been made as evidenced by preclinical and clinical trials. Data obtained from high-throughput technologies are deepening our understanding about the molecular and gene network in cancer cells and rapidly emerging in vitro and in vivo evidence is highlighting the role of antisense agents as specific inhibitors of the expression of target genes, thus modulating the response of cancer cells to different therapeutic strategies. Much information is continuously being added into various facets of molecular oncology and it is now understood that overexpression of antiapoptotic proteins, oncogenes, oncogenic microRNAs (miRNA), and fusion proteins make cancer cells difficult to target. Delivery of antisense oligonucleotides has remained a challenge and technological developments have helped in overcoming hurdles by improving the ability to penetrate cells, effective and targeted binding to gene sequences, and downregulation of target gene function. Different delivery systems, including stable nucleic acid lipid particles, have shown potential in enhancing the delivery of cargo to the target site. In this review, we attempt to summarize the current progress in the development of antisense therapeutics and their potential in medical research. We partition this multicomponent review into introductory aspects about recent breakthroughs in antisense therapeutics. We also discuss how antisense therapeutics have shown potential in resensitizing resistant cancer cells to apoptosis by targeted inhibition of antiapoptotic proteins, oncogenic miRNAs, and BCR-ABL.
Redox regulation of metabolic and signaling pathways by thioredoxin and glutaredoxin in NOS-3 overexpressing hepatoblastoma cells
(Elsevier Science Bv, Elsevier, 2015-07-17) González, Raúl; López-Grueso, M. José; Muntané Relat, Jordi; Bárcena, J. A.; Padilla, C. Alicia; Universidad de Sevilla. Departamento de Fisiología Médica y Biofísica; Junta de Andalucía; Gobierno de España; Universidad de Sevilla. CTS1098: Mecanismos Moleculares del Hepatocarcinoma y Sus Estrategias Terapéuticas
Nitric oxide (NO) plays relevant roles in signal transduction in physiopathology and its effects are de pendent on several environmental factors. NO has both pro-apoptotic and anti-apoptotic functions but the molecular mechanisms responsible for these opposite effects are not fully understood. The action of NO occurs mainly through redox changes in target proteins, particularly by S-nitrosylation of reactive cysteine residues. Thioredoxin (Trx) and glutaredoxin (Grx) systems are the main cellular controllers of the thiolic redox state of proteins exerting controversial effects on apoptosis with consequences for the resistance to or the development of cancer. The aim of this study was to ascertain whether Trx and/or Grx systems mediate the antiproliferative effect of NO on hepatoblastoma cells by modulating the redox-state of key proteins. Proliferation decreased and apoptosis increased in HepG2 cells overexpressing Nitric Oxide Synthase 3 (NOS-3) as a result of multilevel cellular responses to the oxidative environment generated by NO. Enzyme levels and cysteine redox state at several metabolic checkpoints were consistent with promi nence of the pentose phosphate pathway to direct the metabolic flux toward NADPH for antioxidant defense and lowering of nucleotide biosynthesis and hence proliferation. Proteins involved in cell sur vival pathways, proteins of the redoxin systems and phosphorylation of MAPK were all significantly increased accompanied by a shift of the thiolic redox state of Akt1, Trx1 and Grx1 to more oxidized. Silencing of Trx1 and Grx1 neutralized the increases in CD95, Akt1 and pAkt levels induced by NO and produced a marked increase in caspase-3 and-8 activities in both control and NOS-3 overexpressing cells concomitant with a decrease in the number of cells. These results demonstrate that the antiproliferative effect of NO is actually hampered by Trx1 and Grx1 and support the strategy of weakening the thiolic antioxidant defenses when designing new an titumoral therapies.
Modulation of autophagy by sorafenib: effects on treatment response
(Frontiers Media SA, 2016-06-08) Prieto-Domínguez, Nestor; Ordóñez, Raquel; Fernández, Anna; García-Palomo, Andres; Muntané Relat, Jordi; González-Gallego, Javier; Mauriz, José L.; Universidad de Sevilla. Departamento de Fisiología Médica y Biofísica; Gobierno de España; Universidad de Sevilla. CTS1098: Mecanismos Moleculares del Hepatocarcinoma y Sus Estrategias Terapéuticas
The multikinase inhibitor sorafenib is, at present, the only drug approved for the treatment of hepatocellular carcinoma (HCC), one of the most lethal types of cancer worldwide. However, the increase in the number of sorafenib tumor resistant cells reduces efficiency. A better knowledge of the intracellular mechanism of the drug leading to reduced cell survival could help to improve the benefits of sorafenib therapy. Autophagy is a bulk cellular degradation process activated in a broad range of stress situations, which allows cells to degrade misfolded proteins or dysfunctional organelles. This cellular route can induce survival or death, depending on cell status and media signals. Sorafenib, alone or in combination with other drugs is able to induce autophagy, but cell response to the drug depends on the complex integrative crosstalk of different intracellular signals. In cancerous cells, autophagy can be regulated by different cellular pathways (Akt-related mammalian target of rapamycin (mTOR) inhibition, 5′ AMP-activated protein kinase (AMPK) induction, dissociation of B-cell lymphoma 2 (Bcl-2) family proteins from Beclin-1), or effects of some miRNAs. Inhibition of mTOR signaling by sorafenib and diminished interaction between Beclin-1 and myeloid cell leukemia 1 (Mcl-1) have been related to induction of autophagy in HCC. Furthermore, changes in some miRNAs, such as miR-30α, are able to modulate autophagy and modify sensitivity in sorafenib-resistant cells. However, although AMPK phosphorylation by sorafenib seems to play a role in the antiproliferative action of the drug, it does not relate with modulation of autophagy. In this review, we present an updated overview of the effects of sorafenib on autophagy and its related activation pathways, analyzing in detail the involvement of autophagy on sorafenib sensitivity and resistance.
Role of p63 and p73 isoforms on the cell death in patients with hepatocellular carcinoma submitted to orthotopic liver transplantation
(Public Library Science, 2017-03-28) González, Raúl; Rosa, Ángel J. de la; Rufini, Alessandro; Rodríguez-Hernández, María A.; Navarro-Villarán, Elena; Marchal, Trinidad; Pascasio Acevedo, Juan Manuel; Gómez Bravo, Miguel Ángel; Muntané Relat, Jordi; Universidad de Sevilla. Departamento de Cirugía; Universidad de Sevilla. Departamento de Fisiología médica y biofísica; Junta de Andalucía; Instituto de Salud Carlos III; Universidad de Sevilla. CTS664: Cirugía Avanzada y Trasplantes. Terapia Celular y Bioingeniería Aplicada a la Cirugía; Universidad de Sevilla. CTS1098: Mecanismos Moleculares del Hepatocarcinoma y Sus Estrategias Terapéuticas
Background & Aims Patients with hepatocellular carcinoma (HCC) submitted to orthotopic liver transplantation (OLT) have a variable 5-year survival rate limited mostly by tumor recurrence. The etiology, age, sex, alcohol, Child-Pugh, and the immunesuppressor have been associated with tumour recurrence. The expression of ΔNp73 is related to the reduced survival of patients with HCC. The study evaluated the expression of p63 and p73 isoforms and cell death receptors, and their relation to tumour recurrence and survival. The results were in vitro validated in HCC cell lines. Methods HCC sections from patients submitted to OLT were used. The in vitro study was done in differentiated hepatitis B virus (HBV)-expressing Hep3B and control HepG2 cells. The expression of cell death receptors and cFLIPS/L, caspase-8 and -3 activities, and cell proliferation were determined in control and p63 and p73 overexpressing HCC cells. Results The reduced tumor expression of cell death receptors and TAp63 and TAp73, and increased ΔNp63 and ΔNp73 expression were associated with tumor recurrence and reduced survival. The in vitro study demonstrated that HBV-expressing Hep3B vs HepG2 cells showed reduced expression of p63 and p73, cell death receptors and caspase activation, and increased cFLIPL/cFLIPS ratio. The overexpression of TAp63 and TAp73 exerted a more potent pro-apoptotic and anti-proliferative effects in Hep3B than HepG2-transfected cells which was related to cFLIPL upregulation. Conclusions The reduction of TAp63 and TAp73 isoforms, rather than alteration of ΔN isoform expression, exerted a significant functional repercussion on cell death and proliferation in HBV-expressing HepB cells.
Thioredoxin and glutaredoxin regulate metabolism through different multiplex thiol switches
(Elsevier Science Bv; Elsevier, 2019-02) López-Grueso, M. J.; González-Ojeda, R; Requejo-Aguilar, R.; McDonagh, B.; Fuentes-Almagro, C. A.; Muntané Relat, Jordi; Bárcena, J. A.; Padilla, C. A.; Universidad de Sevilla. Departamento de Fisiología Médica y Biofísica; Universidad de Sevilla. CTS1098: Mecanismos Moleculares del Hepatocarcinoma y Sus Estrategias Terapéuticas
The aim of the present study was to define the role of Trx and Grx on metabolic thiol redox regulation and identify their protein and metabolite targets. The hepatocarcinoma-derived HepG2 cell line under both normal and oxidative/nitrosative conditions by overexpression of NO synthase (NOS3) was used as experimental model. Grx1 or Trx1 silencing caused conspicuous changes in the redox proteome reflected by significant changes in the reduced/oxidized ratios of specific Cys's including several glycolytic enzymes. Cys91 of peroxiredoxin-6 (PRDX6) and Cys153 of phosphoglycerate mutase-1 (PGAM1), that are known to be involved in progression of tumor growth, are reported here for the first time as specific targets of Grx1. A group of proteins increased their CysRED/ CysOX ratio upon Trx1 and/or Grx1 silencing, including caspase-3 Cys163, glyceraldehyde-3-phosphate dehy drogenase (GAPDH) Cys247 and triose-phosphate isomerase (TPI) Cys255 likely by enhancement of NOS3 auto oxidation. The activities of several glycolytic enzymes were also significantly affected. Glycolysis metabolic flux increased upon Trx1 silencing, whereas silencing of Grx1 had the opposite effect. Diversion of metabolic fluxes toward synthesis of fatty acids and phospholipids was observed in siRNA-Grx1 treated cells, while siRNA-Trx1 treated cells showed elevated levels of various sphingomyelins and ceramides and signs of increased protein degradation. Glutathione synthesis was stimulated by both treatments. These data indicate that Trx and Grx have both, common and specific protein Cys redox targets and that down regulation of either redoxin has markedly different metabolic outcomes. They reflect the delicate sensitivity of redox equilibrium to changes in any of the elements involved and the difficulty of forecasting metabolic responses to redox environmental changes.
Zinc-based nanoparticles, but not silicon-based nanoparticles, accumulate in mitochondria and promote cell death in liver cancer cells
(Dove Medical Press Ltd, 2024-04-19) Pieretti, Joana C.; Muntané Relat, Jordi; Horne, Thaissa; García-Villasante, Natalia; Seabra, Amedea B.; Universidad de Sevilla. Departamento de Fisiología Médica y Biofísica; Universidad de Sevilla. CTS1098: Mecanismos Moleculares del Hepatocarcinoma y Sus Estrategias Terapéuticas
Introduction: Hepatocellular carcinoma (HCC) is the main hepatic primary malignancy. Patients with advanced HCC receiving the recommended therapies have a poor outcome. In different settings, nanotechnology has gained attraction as a potential alternative strategy for improving therapeutic effectiveness. Among several nanoparticles (NPs), inorganic NPs, such as zinc and silicon oxides (ZnO and SiO2), are mainly chosen as drug nanocarriers, as both present great adsorption properties and biocompatibility. Aim: The objective is to identify the molecular mechanisms underlying the proapoptotic effects of ZnO and SiO2 NPs in differentiated hepatoblastoma cells (HepG2) and mesenchymal liver cancer cells (SNU449). Methods: Dose-dependent induction of cell cytotoxicity by ZnO and SiO2 NPs (5 to 50 µg/mL) was determined in HepG2 and SNU449 cells. NPs intracellular localization was assessed using transmission electron microscopy (TEM). Cell death was determined by trypan blue staining and caspase-3 and −8 activities. Cell respiration was determined using MitroStress assay (Seahorse, Agilent). Results: ZnO NPs, but not SiO2 NPs, reduced cell viability in HepG2 and SNU449. Interestingly, SNU449 appeared to be more susceptible than HepG2 to ZnO NPs (IC50 of 27.4 ± 1.4 µg/mL and 41.8 ± 0.4 µg/mL, respectively). SiO2 NPs tended to be localized in lysosomes in both cell lines, while ZnO NPs demonstrated a random distribution with a high presence in mitochondria and related structures. As expected, SiO2 NPs did not reduce cell survival and cell respiration, while ZnO NPs promoted cell death and decreased oxygen consumption rate. ZnO NPs mitochondrial accumulation was associated with increased apoptosis in HepG2, while necroa poptosis was mainly involved in ZnO-induced cell death in SNU449. Conclusion: SiO2 demonstrated no cytotoxic profile against liver cancer cells. ZnO NPs demonstrated to accumulate in mitochondria impacting cell respiration and cell death in liver cancer cells. ZnO induced apoptosis and necroptosis in HepG2 and SNU449, respectively.
Involvement of mTOR signaling in physiopathology and its potential therapeutic interventions
(Bioscientífica Ltd., 2024-01-16) Martin-Bocanegra, Irene; Horne, Thaissa; Maqueda-Hernández, Belén; Navarro-Vilarán, Elena; Muntané Relat, Jordi; Universidad de Sevilla. Departamento de Fisiología Médica y Biofísica; Junta de Andalucía; Instituto de Salud Carlos III; European Commission (EC). Fondo Europeo de Desarrollo Regional (FEDER); Universidad de Sevilla. CTS1098: Mecanismos Moleculares del Hepatocarcinoma y Sus Estrategias Terapéuticas
The mammalian target of rapamycin (mTOR) is a master regulator of the cell metabolism which impacts numerous signaling involved in cell proliferation and death and recycling cell constituents to readapt to new physiological or pathological environments. mTOR is constituted by two structural and functional different complexes, mTORC1 and mTORC2. Both can be independently regulated, which has a great impact on the effectiveness of therapeutic interventions in different clinical and experimental situations. Furthermore, mTORC1 interacts with specific chaperones or immunophilins which are intracellular receptors of the immunosuppressive drugs. Low and high molecular weights of immunophilins have different intracellular functions. The present review updates the molecular structure and signaling of mTOR as well as their regulation by immunophilins and upstream and downstream signaling events, highlighting the potential therapeutic intervention of mTOR in cancer, metabolic disturbances, and aging.
In silico analysis of lncRNA-miRNA-mRNA signatures related to Sorafenib effectiveness in liver cancer cells
(Baishideng publishing group Inc., 2025-01-21) Cruz Ojeda, Patricia de la; Parras-Martínez, Ester; Rey-Pérez, Raquel; Muntané Relat, Jordi; Universidad de Sevilla. Departamento de Fisiología Médica y Biofísica; Junta de Andalucía; European Commission (EC). Fondo Europeo de Desarrollo Regional (FEDER); Instituto de Salud Carlos III; Universidad de Sevilla. CTS1098: Mecanismos Moleculares del Hepatocarcinoma y Sus Estrategias Terapéuticas
BACKGROUND Hepatocellular carcinoma (HCC) is the most common subtype of primary liver cancer with varied incidence and epidemiology worldwide. Sorafenib is still a recommended treatment for a large proportion of patients with advanced HCC. Different patterns of treatment responsiveness have been identified in differen tiated hepatoblastoma HepG2 cells and metastatic HCC SNU449 cells. AIM To define the long non-codingRNA-microRNA-mRNA (lncRNA-miRNA-mRNA) predicted signatures related to selected hallmarks of cancer (apoptosis, auto phagy, cell stress, cell dedifferentiation and invasiveness) in RNAseq studies using Sorafenib-treated HepG2 and SNU449 cells. Various available software analyses allowed us to establish the lncRNA-miRNA-mRNA regulatory axes following treatment in HepG2 and SNU449 cells. METHODS HepG2 and SNU449 cells were treated with Sorafenib (10 μmol/L) for 24 hours. Total RNA, including small and long RNA, was extracted with a commercial miRNeasy kit. RNAseq was carried out for the identification of changes in lncRNA-miRNA-mRNA regulatory axes. RESULTS MALAT, THAP9-AS1 and SNGH17 appeared to coordinately regulate miR-374b-3p and miR-769-5p that led to upregulation of SMAD7, TIRARP, TFAP4 and FAXDC2 in HepG2 cells. SNHG12, EPB41 L4A-AS1, LINC01578, SNHG12 and GAS5 interacted with let-7b-3p, miR-195-5p and VEGFA in SNU449 cells. The axes MALAT1/hsa mir-374b-3p/SMAD7 and MALAT1/hsa-mir-769-5p/TFAP4 were of high relevance for Sorafenib response in HepG2 cells, whereas PVT1/hsa-miR-195-5p/VEGFA was responsible for the differential response of SNU449 cells to Sorafenib treatment. CONCLUSION Critical lncRNAs acting as sponges of miRNA were identified that regulated mRNA expression, whose proteins mainly increased the antitumor effectiveness of the treatment (SMAD7, TIRARP, TFAP4, FAXDC2 and ADRB2). However, the broad regulatory axis leading to increased VEGFA expression may be related to the side effect of Sorafenib in SNU449 cells.
Acute oxygen sensing by vascular smooth muscle cells
(Frontiers Media, 2023-03-03) Moreno Domínguez, Alejandro; Colinas Miranda, Olalla; Smani Hajami, Tarik; Ureña López, Juan; López Barneo, José; Universidad de Sevilla. Departamento de Fisiología Médica y Biofísica; Junta de Andalucía; European Commission (EC). Fondo Europeo de Desarrollo Regional (FEDER); Gobierno de España
An adequate supply of oxygen (O2) is essential for most life forms on earth, making the delivery of appropriate levels of O2 to tissues a fundamental physiological challenge. When O2 levels in the alveoli and/or blood are low, compensatory adaptive reflexes are produced that increase the uptake of O2 and its distribution to tissues within a few seconds. This paper analyzes the most important acute vasomotor responses to lack of O2 (hypoxia): hypoxic pulmonary vasoconstriction (HPV) and hypoxic vasodilation (HVD). HPV affects distal pulmonary (resistance) arteries, with its homeostatic role being to divert blood to well ventilated alveoli to thereby optimize the ventilation/perfusion ratio. HVD is produced in most systemic arteries, in particular in the skeletal muscle, coronary, and cerebral circulations, to increase blood supply to poorly oxygenated tissues. Although vasomotor responses to hypoxia are modulated by endothelial factors and autonomic innervation, it is well established that arterial smooth muscle cells contain an acute O2 sensing system capable of detecting changes in O2 tension and to signal membrane ion channels, which in turn regulate cytosolic Ca2+ levels and myocyte contraction. Here, we summarize current knowledge on the nature of O2 sensing and signaling systems underlying acute vasomotor responses to hypoxia. We also discuss similarities and differences existing in O2 sensors and effectors in the various arterial territories.
Cytoskeletal Reorganization Evoked by Rho-associated kinase- and Protein Kinase C-catalyzed Phosphorylation of Cofilin and Heat Shock Protein 27, Respectively, Contributes to Myogenic Constriction of Rat Cerebral Arteries
(Elsevier, 2014-06-09) Moreno Domínguez, Alejandro; El-Yazbi, Ahmed F.; Zhu, Hai Lei; Colinas Miranda, Olalla; Zhong, X. Zoë; Walsh, Emma J.; Cole, Dylan M.; Kargacin, Gary J.; Walsh, Michael P.; Cole, William C.; Universidad de Sevilla. Departamento de Fisiología Médica y Biofísica; Alberta Heritage Foundation for Medical Research/Canadian Institutes of Health Research; Alberta Innovates-Health Solutions; Canadian Institutes of Health Research; Kertland Family Fellowship Fund & Andrew Family Professorship; Gobierno de España
Our understanding of the molecular events contributing to myogenic control of diameter in cerebral resistance arteries in response to changes in intravascular pressure, a fundamental mechanism regulating blood flow to the brain, is incomplete. Myosin light chain kinase and phosphatase activities are known to be increased and decreased, respectively, to augment phosphorylation of the 20-kDa regulatory light chain subunits (LC20) of myosin II, which permits cross-bridge cycling and force development. Here, we assessed the contribution of dynamic reorganization of the actin cytoskeleton and thin filament regulation to the myogenic response and serotonin-evoked constriction of pressurized rat middle cerebral arteries. Arterial diameter and the levels of phosphorylated LC20, calponin, caldesmon, cofilin, and HSP27, as well as G-actin content, were determined. A decline in G-actin content was observed following pressurization from 10 mm Hg to between 40 and 120 mm Hg and in three conditions in which myogenic or agonist-evoked constriction occurred in the absence of a detectable change in LC20 phosphorylation. No changes in thin filament protein phosphorylation were evident. Pressurization reduced G-actin content and elevated the levels of cofilin and HSP27 phosphorylation. Inhibitors of Rho-associated kinase and PKC prevented the decline in G-actin; reduced cofilin and HSP27 phosphoprotein content, respectively; and blocked the myogenic response. Furthermore, phosphorylation modulators of HSP27 and cofilin induced significant changes in arterial diameter and G-actin content of myogenically active arteries. Taken together, our findings suggest that dynamic reorganization of the cytoskeleton involving increased actin polymerization in response to Rho-associated kinase and PKC signaling contributes significantly to force generation in myogenic constriction of cerebral resistance arteries.
Hif1α-dependent mitochondrial acute O2 sensing and signaling to myocyte Ca2+ channels mediate arterial hypoxic vasodilation
(Nature Research, 2024-08-05) Moreno Domínguez, Alejandro; Colinas Miranda, Olalla; Arias Mayenco, Ignacio; Cabeza, José M.; López Ogayar, Juan L.; Chandel, Navdeep S.; Weissmann, Norbert; Sommer, Natascha; Pascual, Alberto; López Barneo, José; Universidad de Sevilla. Departamento de Fisiología Médica y Biofísica; European Research Council (ERC); Gobierno de España
Vasodilation in response to low oxygen (O2) tension (hypoxic vasodilation) is an essential homeostatic response of systemic arteries that facilitates O2 supply to tissues according to demand. However, how blood vessels react to O2 deficiency is not well understood. A common belief is that arterial myocytes are O2-sensitive. Supporting this concept, it has been shown that the activity of myocyte L-type Ca2+channels, the main ion channels responsible for vascular contractility, is reversibly inhibited by hypoxia, although the underlying molecular mechanisms have remained elusive. Here, we show that genetic or pharmacological disruption of mitochondrial electron transport selectively abolishes O2 modulation of Ca2+ channels and hypoxic vasodilation. Mitochondria function as O2 sensors and effectors that signal myocyte Ca2+ channels due to constitutive Hif1α-mediated expression of specific electron transport subunit isoforms. These findings reveal the acute O2-sensing mechanisms of vascular cells and may guide new developments in vascular pharmacology.
Hypotension Due to Kir6.1 Gain‐of‐Function in Vascular Smooth Muscle
(Wiley, 2013-08-23) Li, Anlong; Knutsen, Russell H.; Zhang, Haixia; Osei-Owusu, Patrick; Moreno Domínguez, Alejandro; Harter, Theresa M.; Nichols, Colin G.; Universidad de Sevilla. Departamento de Fisiología Médica y Biofísica; American Heart Association; Barnes-Jewish/Christian Foundation; National Institutes of Health (NIH)
Background KATP channels, assembled from pore‐forming (Kir6.1 or Kir6.2) and regulatory (SUR1 or SUR2) subunits, link metabolism to excitability. Loss of Kir6.2 results in hypoglycemia and hyperinsulinemia, whereas loss of Kir6.1 causes Prinzmetal angina–like symptoms in mice. Conversely, overactivity of Kir6.2 induces neonatal diabetes in mice and humans, but consequences of Kir6.1 overactivity are unknown. Methods and Results We generated transgenic mice expressing wild‐type (WT), ATP‐insensitive Kir6.1 [Gly343Asp] (GD), and ATP‐insensitive Kir6.1 [Gly343Asp,Gln53Arg] (GD‐QR) subunits, under Cre‐recombinase control. Expression was induced in smooth muscle cells by crossing with smooth muscle myosin heavy chain promoter–driven tamoxifen‐inducible Cre‐recombinase (SMMHC‐Cre‐ER) mice. Three weeks after tamoxifen induction, we assessed blood pressure in anesthetized and conscious animals, as well as contractility of mesenteric artery smooth muscle and KATP currents in isolated mesenteric artery myocytes. Both systolic and diastolic blood pressures were significantly reduced in GD and GD‐QR mice but normal in mice expressing the WT transgene and elevated in Kir6.1 knockout mice as well as in mice expressing dominant‐negative Kir6.1 [AAA] in smooth muscle. Contractile response of isolated GD‐QR mesenteric arteries was blunted relative to WT controls, but nitroprusside relaxation was unaffected. Basal KATP conductance and pinacidil‐activated conductance were elevated in GD but not in WT myocytes. Conclusions KATP overactivity in vascular muscle can lead directly to reduced vascular contractility and lower blood pressure. We predict that gain of vascular KATP function in humans would lead to a chronic vasodilatory phenotype, as indeed has recently been demonstrated in Cantu syndrome.
Sos1 ablation alters focal adhesion dynamics and increases Mmp2/9-dependent gelatinase activity in primary mouse embryonic fibroblasts
(Biomed central LTD, 2025-03-03) Liceras-Boillos, Pilar; Garcia-Navas, Rósula; Llorente-González, Clara; Lorenzo-Martin, L. Francisco; Luna-Ramírez, Luis; Fuentes-Mateos, Rocío; Calvo Baltanás, Fernando; Universidad de Sevilla. Departamento de Fisiología Médica y Biofísica; Instituto de Biomedicina de Sevilla (IBIS); Universidad de Sevilla. CTS007: Fisiopatología de células madre neurales
Background Sos1 and Sos2 are guanine-nucleotide exchange factors for Ras and Rac small GTPases, which are involved in a wide range of cellular responses including proliferation and migration. We have previously shown that Sos1 and Sos2 have different effects on cell migration, but the underlying mechanisms are not clear. Methods Using a 4-hydroxytamoxifen-inducible conditional Sos1KO mutation, here we evaluated the functional specificity or redundancy of Sos1 and Sos2 regarding the control of cell migration and dynamics of focal adhesions (FAs) in primary mouse embryonic fibroblasts (MEFs). Results Functional analysis of the transcriptome of primary Sos1/2WT, Sos1KO, Sos2KO and Sos1/2DKO-MEFs revealed a specific, dominant role of Sos1 over Sos2 in transcriptional regulation. Sos1KO MEFs had an increased number and stability of focal adhesions (FAs) and curbed protrusion and spreading. Conversely, Sos2KO MEFs displayed unstable FAs with increased protrusion. Interestingly, Sos1, but not Sos2, ablation reduced the levels of GTP-bound Rac at the leading edge. In 3D, however, only Sos1/2KO MEFs showed increased invasion and matrix degradative capacity, which correlated with increased expression of the Mmp2 and Mmp9 gelatinases. Moreover, increased matrix degradation in Sos1/2KO MEFs was abrogated by treatment with Mmp2/9 inhibitors. Conclusions Our data demonstrate that Sos1 and Sos2 have different functions in FAs distribution and dynamics in 2D whereas in 3D they act together to regulate invasion and unveil a previously undescribed mechanistic connection between Sos1/2 and the regulation of Mmp2/9 expression in primary MEFs.
Regulation of ER stress-induced apoptotic and inflammatory responses via YAP/TAZ-mediated control of the TRAIL-R2/DR5 signaling pathway
(Springer Nature, 2025-02-04) El Yousfi, Younes; Fernández-Farrán, FJ; Oliver, FJ; López-Rivas, Abelardo; Yerbes, Rosario; Universidad de Sevilla. Departamento de Fisiología Médica y Biofísica; Junta de Andalucía; Gobierno de España; Universidad de Sevilla. CTS007: Fisiopatología de Células Madre Neurales
In tumors, cancer cells are frequently exposed to adverse environmental conditions that result in endoplasmic reticulum (ER) stress. Mechanical signals emerging from extracellular matrix (ECM) rigidity and cell shape regulate the activity of transcriptional co activators Yes-associated protein (YAP) and its paralog Transcriptional Coactivator with PDZ-binding motif (TAZ). However, the role of ECM rigidity and YAP/TAZ in tumor cell fate decisions under ER stress remains relatively unexplored. Our results suggest that the YAP/TAZ system plays an important role in the control of ER stress-induced cell death by mechanical signaling arising from ECM stiffness in tumor cells. Mechanistically, YAP/TAZ regulates apoptosis induced by ER stress in tumor cells by controlling the activation of the TRAIL-R2/DR5-mediated extrinsic apoptotic pathway through a dual mechanism. On the one hand, the YAP/TAZ system prevents intracellular TRAIL-R2/DR5 clustering in tumor cells. On the other hand, it inhibits cFLIP down-regulation in tumor cells experiencing ER stress. In addition, YAP/TAZ controls the expression of pro-inflammatory interleukin-8 (IL-8/CXCL8) in tumor cells undergoing ER stress by a TRAIL-R2/DR5/caspase-8-dependent mechanism. Although other mechanisms may also be involved in controlling cell death and inflammation in tumor cells facing environmental stress, our results support a model in which regulation of the subcellular localization and activity of the YAP/TAZ transcriptional co-activators could contribute to the microenvironmental control of cell fate decisions in tumor cells undergoing ER stress.
An immunoassay that distinguishes real neuromyelitis optica signals from a labeling detected in patients receiving natalizumab
(BioMed Central, 2014-07-01) Sánchez Gomar, Ismael; Díaz Sánchez, María; Uclés Sánchez, Antonio José; Casado Chocán, José Luis; Ramírez Lorca, Reposo; Serna, Ana; Villadiego Luque, Francisco Javier; Toledo Aral, Juan José; Echevarría Irusta, Miriam; Universidad de Sevilla. Departamento de Fisiología Médica y Biofísica; Junta de Andalucía; Instituto de Salud Carlos III; Gobierno de España; Genzyme Foundation
Background Cell-based assays for neuromyelitis optica (NMO) diagnosis are the most sensitive and specific methods to detect anti-aquaporin 4 (AQP4) antibodies in serum, but some improvements in their quantitative and specificity capacities would be desirable. Thus the aim of the present work was to develop a sensitive quantitative method for detection of anti-AQP4 antibodies that allows clear diagnosis of NMO and distinction of false labeling produced by natalizumab treatment. Methods Sera from 167 individuals, patients diagnosed with NMO (16), multiple sclerosis (85), optic neuritis (24), idiopathic myelitis (21), or other neurological disorders (13) and healthy controls (8), were used as the primary antibody in an immunofluorescence assay on HEK cells transfected with the M23 isoform of human AQP4 fused with enhanced green fluorescent protein. Cells used were freshly transfected or stored frozen and then thawed just before adding the serum. Results Microscopic observation and fluorescence quantification produced similar results in fresh and frozen samples. Serum samples from patients diagnosed with NMO were 100% positive for anti-AQP4 antibodies, while all the other sera were negative. Using serum from patients treated with natalizumab, a small and unspecific fluorescent signal was produced from all HEK cells, regardless of AQP4 expression. Conclusions Our cell-based double-label fluorescence immunoassay protocol significantly increases the signal specificity and reduces false diagnosis of NMO patients, especially in those receiving natalizumab treatment. Frozen pretreated cells allow faster detection of anti-AQP4 antibodies.
RIPK1/RIPK3 promotes vascular permeability to allow tumor cell extravasation independent of its necroptotic function
(Nature publishing group; Springer nature, 2017-02-02) Hanggi, Kay; Vasilikos, Lazaros; Valls, Aida Freire; Yerbes, Rosario; Knop, Janin; Spilgies, Lisanne M; Wong, WWei Lynn; Universidad de Sevilla. Departamento de Fisiología Médica y Biofísica; Universidad de Sevilla. CTS007: Fisiopatología de Células Madre Neurales
Necroptosis is an inflammatory form of programmed cell death requiring receptor-interacting protein kinase 1, 3 (RIPK1, RIPK3) and mixedlineage kinase domain-like protein (MLKL). The kinase of RIPK3 phosphorylates MLKL causing MLKLtoformapore-like structure, allowing intracellular contents to release and cell death to occur. Alternatively, RIPK1 and RIPK3 have been shown to regulate cytokine production directly influencing inflammatory immune infiltrates. Recent data suggest that necroptosis may contribute to the malignant transformation of tumor cells in vivo and we asked whether necroptosis may have a role in the tumor microenvironment altering the ability of the tumor to grow or metastasize. To determine if necroptosis in the tumor microenvironment could promote inflammation alone or by initiating necroptosis and thereby influencing growth or metastasis of tumors, we utilized a syngeneic tumor model of metastasis. Loss of RIPK3 in the tumor microenvironment reduced the number of tumor nodules in the lung by46%. Loss of the kinase activity in RIPK1, a memberof the necrosome also reduced tumor nodules in the lung by 38%. However, the loss of kinase activity in RIPK3 or the loss of MLKL only marginally altered the ability of tumor cells to form in the lung. Using bone marrow chimeras, the decrease in tumor nodules in the Ripk3−/− appeared to be due to the stromal compartment rather than the hematopoietic compartment. Transmigration assays showed decreased ability of tumor cells to transmigrate through the vascular endothelial layer, which correlated with decreased permeability in the Ripk3−/− mice after tumor injection. In response to permeability factors, such as vascular endothelial growth factor, RIPK3 null endothelial cells showed decreased p38/HSP27 activation. Taken together, our results suggest an alternative function for RIPK1/RIPK3 in vascular permeability leading to decreased number of metastasis.
Role of the yap/taz-tead transcriptional complex in the metabolic control of trail sensitivity by the mevalonate pathway in cancer cells
(Mdpi Ag, 2023-09-27) El Yousfi, Younes; Mora-Molina, Rocío; López-Rivas, Abelardo; Yerbes, Rosario; Universidad de Sevilla. Departamento de Fisiología Médica y Biofísica; Instituto de Biomedicina de Sevilla (IBIS); Ministerio de Ciencia e Innovación (MICIN). España; Junta de Andalucía; European Commission (EC). Fondo Europeo de Desarrollo Regional (FEDER); Universidad de Sevilla. CTS007: Fisiopatología de Células Madre Neurales
Different studies have reported that inhibiting the mevalonate pathway with statins may increase the sensitivity of cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), although the signaling mechanism leading to this sensitization remains largely unknown. Weinvestigated the role of the YAP (Yes-associated protein)/TAZ (transcriptional co-activator with PDZ-binding motif)-TEAD (TEA/ATTS domain) transcriptional complex in the metabolic control of TRAIL sensitivity by the mevalonate pathway. We show that depleting nuclear YAP/TAZ in tumor cells, either via treatment with statins or by silencing YAP/TAZ expression with siRNAs, facilitates the activation of apoptosis by TRAIL. Furthermore, the blockage of TEAD transcriptional activity either pharmacologically or through the ectopic expression of a disruptor of the YAP/TAZ interaction with TEADtranscription factors, overcomes the resistance of tumor cells to the induction of apoptosis by TRAIL. Our results show that the mevalonate pathway controls cellular the FLICE-inhibitory protein (cFLIP) expression in tumor cells. Importantly, inhibiting the YAP/TAZ-TEAD signaling pathwayinducescFLIPdown-regulation, leading to a marked sensitization of tumor cells to apoptosis induction by TRAIL. Our data suggest that a combined strategy of targeting TEAD activity and selectively activating apoptosis signaling by agonists of apoptotic TRAIL receptors could be explored as a potential therapeutic approach in cancer treatment.

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