Ivonne Olmedo

University of Santiago, Chile, CiudadSantiago, Santiago, Chile

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Publications (15)47 Total impact

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    ABSTRACT: Cardiac fibroblast (CF) survival is important for the maintenance of the extracellular matrix homeostasis in the heart; providing a functional support to cardiomyocytes necessary for the correct myocardial function. Endoplasmic reticulum (ER) stress causes cellular dysfunction and cell death by apoptosis; and thapsigargin is a well-known ER stress inducer. On the other hand, the chemical chaperone, 4-phenylbutyric acid (4-PBA) had showed to prevent ER stress; however, in cardiac fibroblast both the ER stress induced by thapsigargin and prevention by 4-PBA, have not been studied in detail. Neonate rat CF were treated with thapsigargin in presence or absence of 4-PBA, and cell viability was evaluated by trypan blue exclusion and apoptosis by flow cytometry; whereas CHOP, BIP, PDI, ATF4 and procollagen protein levels were assessed by western blot. In CF, thapsigargin triggered the unfolded protein response detected by early increases in ATF4, CHOP, PDI and BIP protein levels as well as, the accumulation of intracellular procollagen. Thapsigargin also stimulated CF death in a time and concentration-dependent manner. ER stress, CF death and apoptosis induced by thapsigargin were prevented by 4-PBA. Conclusion our data suggest that 4-PBA prevent ER stress, intracellular procollagen accumulation, CF death and apoptosis induced by thapsigargin.
    Toxicology in Vitro 08/2014; 28(8). DOI:10.1016/j.tiv.2014.07.013 · 3.21 Impact Factor
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    ABSTRACT: Cardiac fibroblast (CF) survival is important for the maintenance of the extracellular matrix homeostasis in the heart; providing a functional support to cardiomyocytes necessary for the correct myocardial function. Endoplasmic reticulum (ER) stress causes cellular dysfunction and cell death by apoptosis; and thapsigargin is a well-known ER stress inducer. On the other hand, the chemical chaperone, 4-phenylbutyric acid (4-PBA) had showed to prevent ER stress; however, in cardiac fibroblast both the ER stress induced by thapsigargin and prevention by 4-PBA, have not been studied in detail. Neonate rat CF were treated with thapsigargin in presence or absence of 4-PBA, and cell viability was evaluated by trypan blue exclusion and apoptosis by flow cytometry; whereas CHOP, BIP, PDI, ATF4 and procollagen protein levels were assessed by western blot. In CF, thapsigargin triggered the unfolded protein response detected by early increases in ATF4, CHOP, PDI and BIP protein levels as well as, the accumulation of intracellular procollagen. Thapsigargin also stimulated CF death in a time and concentration-dependent manner. ER stress, CF death and apoptosis induced by thapsigargin were prevented by 4-PBA. Conclusion our data suggest that 4-PBA prevent ER stress, intracellular procollagen accumulation, CF death and apoptosis induced by thapsigargin.
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    ABSTRACT: In the heart, cardiac fibroblasts (CF) and cardiac myofibroblasts (CMF) are the main cells responsible for wound healing after cardiac insult. Exchange protein activated by cAMP (EPAC) is a downstream effector of cAMP, and it has been not completely studied on CF. Moreover, in CMF, which are the main cells responsible for cardiac healing, EPAC expression and function are unknown. We evaluated in both CF and CMF the effect of transforming growth factor β1 (TGF-β1) on EPAC-1 expression. We also studied the EPAC involvement on collagen synthesis, adhesion, migration and collagen gel contraction. Rat neonatal CF and CMF were treated with TGF-β1 at different times and concentration. EPAC-1 protein levels and Rap1 activation were measured by western blot and pull down assay respectively. EPAC cellular functions were determined by adhesion, migration and collagen gel contraction assay; collagen expression was determined by western blot. TGF-β1 through Smad and JNK significantly reduced EPAC-1 expression in CF, while in CMF this cytokine increased EPAC-1 expression through ERK1/2, JNK, p38, AKT and Smad3. EPAC activation was able to induce higher Rap1-GTP levels in CMF than in CF. EPAC and PKA, both cAMP effectors, promoted CF and CMF adhesion on fibronectin, as well as CF migration; however, this effect was not observed in CMF. EPAC but not PKA activation mediated collagen gel contraction in CF, while in CMF both PKA and EPAC mediated collagen gel contraction. Finally, the EPAC and PKA activation reduced collagen synthesis in CF and CMF. TGF-β1 differentially regulates the expression of EPAC in CF and CMF; and EPAC regulates differentially CF and CMF functions associated with cardiac remodeling.
    Toxicology and Applied Pharmacology 07/2013; 272(2). DOI:10.1016/j.taap.2013.06.022 · 3.63 Impact Factor
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    ABSTRACT: Ischemia/reperfusion injury is a major cause of myocardial death. In the heart, cardiac fibroblasts play a critical role in healing post myocardial infarction. TGF-β1 has shown cardioprotective effects in cardiac damage; however, if TGF-β1 can prevent cardiac fibroblast death triggered by ischemia/reperfusion is unknown. Therefore, we test this hypothesis, and whether the canonical and/or non-canonical TGF-β1 signaling pathways are involved in this protective effect. Cultured rat cardiac fibroblasts were subjected to simulated ischemia/reperfusion. Cell viability was analyzed by trypan blue exclusion and propidium iodide by flow cytometry. The processing of procaspases 8, 9 and 3 to their active forms was assessed by Western blot, whereas subG1 population was evaluated by flow cytometry. Levels of total and phosphorylated forms of ERK1/2, Akt and Smad2/3 were determined by Western blot. The role of these signaling pathways on the protective effect of TGF-β1 was studied using specific chemical inhibitors. Simulated ischemia over 8h triggers a significant cardiac fibroblast death, which increased by reperfusion, with apoptosis actively involved. These effects were only prevented by the addition of TGF-β1 during reperfusion. TGF-β1 pretreatment increased the levels of phosphorylated forms of ERK1/2, Akt and Smad2/3. The inhibition of ERK1/2, Akt and Smad3 also blocked the preventive effects of TGF-β1 on cardiac fibroblast apoptosis induced by simulated ischemia/reperfusion. Overall, our data suggest that TGF-β1 prevents cardiac fibroblast apoptosis induced by simulated ischemia-reperfusion through the canonical (Smad3) and non canonical (ERK1/2 and Akt) signaling pathways.
    Biochimica et Biophysica Acta 02/2013; 1832(6). DOI:10.1016/j.bbadis.2013.02.004 · 4.66 Impact Factor
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    ABSTRACT: Kinins mediate their cellular effects through B1 (B1R) and B2 (B2R) receptors, and the activation of B2R reduces collagen synthesis in cardiac fibroblasts (CF). However, the question of whether B1R and/or B2R have a role in cardiac myofibroblasts remains unanswered. CF were isolated from neonate rats and myofibroblasts were generated by an 84 h treatment with TGF-β1 (CMF). B1R was evaluated by western blot, immunocytochemistry and radioligand assay; B2R, inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and cyclooxygenases 1 and 2 (COX-1, and COX-2) were evaluated by western blot; intracellular Ca⁺² levels were evaluated with Fluo-4AM; collagen secretion was measured in the culture media using the picrosirius red assay kit. B2R, iNOS, COX-1 and low levels of B1R but not eNOS, were detected by western blot in CF. Also, B1R, B2R, and COX-2 but not iNOS, eNOS or COX-1, were detected by western blot in CMF. By immunocytochemistry, our results showed lower intracellular B1R levels in CF and higher B1R levels in CMF, mainly localized on the cell membrane. Additionally, we found B1R only in CMF cellular membrane through radioligand displacement assay. Bradykinin (BK) B2R agonist increased intracellular Ca²⁺ levels and reduced collagen secretion both in CF and CMF. These effects were blocked by HOE-140, and inhibited by L-NAME, 1400 W and indomethacin. Des-Arg-kallidin (DAKD) B1R agonist did not increase intracellular Ca²⁺ levels in CF; however, after preincubation for 1h with DAKD and re-stimulation with the same agonist, we found a low increase in intracellular Ca²⁺ levels. Finally, DAKD increased intracellular Ca²⁺ levels and decreased collagen secretion in CMF, being this effect blocked by the B1R antagonist des-Arg9-Leu8-kallidin and indomethacin, but not by L-NAME or 1400 W. B1R, B2R, iNOS and COX-1 were expressed differently between CF and CMF, and collagen secretion was regulated differentially by kinin receptor agonists in cultured CF and CMF.
    Toxicology and Applied Pharmacology 04/2012; 261(3):300-8. DOI:10.1016/j.taap.2012.04.013 · 3.63 Impact Factor
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    ABSTRACT: Cardiac fibroblast (CF) death by ischemia/reperfusion (I/R) has major implications for cardiac wound healing. Although IGF-1 has well-known cytoprotective effects, no study has been done on CF subjected to simulated I/R. Simulated ischemia of neonate rat CF was performed in a free oxygen chamber in an ischemic medium; reperfusion was done in normal culture conditions. Cell viability was evaluated by trypan blue assay, and apoptosis by a FACS flow cytometer; p-ERK-1/2 and p-Akt levels were determined by western blot. We showed that simulated I/R triggers CF death by necrosis and apoptosis. IGF-1 partially inhibits I/R-induced apoptosis. PD98059 and LY294002 neutralize the preventive effects of IGF-1. CONCLUSION: IGF-1 partially inhibits CF apoptosis induced by simulated I/R by PI3K/Akt- and MEK/ERK1/2-dependent signaling pathways.
    Experimental and Molecular Pathology 04/2012; 93(1):1-7. DOI:10.1016/j.yexmp.2012.01.010 · 2.88 Impact Factor
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    ABSTRACT: Statins reduce the isoprenoids farnesyl and geranylgeranyl pyrophosphate, essential intermediates, which control a diversity of cellular events such as cytoskeleton integrity, adhesion, migration and viability. Cardiac fibroblasts are the major non-myocyte cell constituent in the normal heart, and play a key role in the maintenance of extracellular matrix. The effects of simvastatin on cardiac fibroblast processes previously mentioned remain unknown. Our aims were to investigate the effects of simvastatin on cytoskeleton structure and focal adhesion complex assembly and their relationships with cell adhesion, migration and viability in cultured cardiac fibroblasts. To this end, cells were treated with simvastatin for 24 h and changes in actin cytoskeleton, levels of vimentin and paxillin as well as their subcellular localization were analyzed by Western blot and immunocytochemistry, respectively. Cell adhesion to plastic or collagen coated dishes, migration in Transwell chambers, and cell viability were analyzed after simvastatin treatment. Our results show that simvastatin disrupts actin cytoskeleton and focal adhesion complex evaluated by phalloidin stain and immunocytochemistry for paxillin and vinculin. All these effects occurred by a cholesterol synthesis-independent mechanism. Simvastatin decreased cell adhesion, migration and viability in a concentration-dependent manner. Finally, simvastatin decreased angiotensin II-induced phospho-paxillin levels and cell adhesion. We concluded that simvastatin disrupts cytoskeleton integrity and focal adhesion complex assembly in cultured cardiac fibroblasts by a cholesterol-independent mechanism and consequently decreases cell migration, adhesion and viability.
    Toxicology 03/2012; 294(1):42-9. DOI:10.1016/j.tox.2012.01.011 · 3.75 Impact Factor
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    ABSTRACT: Several clinical trials have shown the beneficial effects of statins in the prevention of coronary heart disease. Additionally, statins promote apoptosis in vascular smooth muscle cells, in renal tubular epithelial cells and also in a variety of cell lines; yet, the effects of statins on cardiac fibroblast and myofibroblast, primarily responsible for cardiac tissue healing are almost unknown. Here, we investigated the effects of simvastatin on cardiac fibroblast and myofibroblast viability and studied the molecular cell death mechanism triggered by simvastatin in both cell types. Rat neonatal cardiac fibroblasts and myofibroblasts were treated with simvastatin (0.1-10μM) up to 72h. Cell viability and apoptosis were evaluated by trypan blue exclusion method and by flow cytometry, respectively. Caspase-3 activation and Rho protein levels and activity were also determined by Western blot and pull-down assay, respectively. Simvastatin induces caspase-dependent apoptosis of cardiac fibroblasts and myofibroblasts in a concentration- and time-dependent manner, with greater effects on fibroblasts than myofibroblasts. These effects were prevented by mevalonate, farnesylpyrophosphate and geranylgeranylpyrophosphate, but not squalene. These last results suggest that apoptosis was dependent on small GTPases of the Rho family rather than Ras. Simvastatin triggered apoptosis of cardiac fibroblasts and myofibroblasts by a mechanism independent of cholesterol synthesis, but dependent of isoprenilation of Rho protein. Additionally, cardiac fibroblasts were more susceptible to simvastatin-induced apoptosis than cardiac myofibroblasts. Thus simvastatin could avoid adverse cardiac remodeling leading to a less fibrotic repair of the damaged tissues.
    Toxicology and Applied Pharmacology 05/2011; 255(1):57-64. DOI:10.1016/j.taap.2011.05.016 · 3.63 Impact Factor
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    ABSTRACT: Functionalization of gold nanoparticles (AuNPs) with both a targeting peptide (an analogue of the peptide Bombesin) and a drug peptide ligand (an analogue of the RAF peptide) with the aim of improving selectivity in the delivery of the conjugates as well as the antitumor activity is described. Studies on the internalization mechanism of peptide-AuNP conjugates and viability of cells were carried out. An enhancement of the activity and selectivity of the peptide multifunctionalized conjugates was observed.
    Bioconjugate Chemistry 06/2010; 21(6):1070-8. DOI:10.1021/bc1000164 · 4.82 Impact Factor
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    ABSTRACT: Gold nanoparticles (GNPs) offer a great promise in biomedicine. Currently, there is no data available regarding the accumulation of nanoparticles in vivo after repeated administration. The purpose of the present study was to evaluate the bioaccumulation and toxic effects of different doses (40, 200, and 400 microg/kg/day) of 12.5 nm GNPs upon intraperitoneal administration in mice every day for 8 days. The gold levels in blood did not increase with the dose administered, whereas in all the organs examined there was a proportional increase on gold, indicating efficient tissue uptake. Although brain was the organ containing the lowest quantity of injected GNPs, our data suggest that GNPs are able to cross the blood-brain barrier and accumulate in the neural tissue. Importantly, no evidence of toxicity was observed in any of the diverse studies performed, including survival, behavior, animal weight, organ morphology, blood biochemistry and tissue histology. The results indicate that tissue accumulation pattern of GNPs depend on the doses administered and the accumulation of the particles does not produce sub-acute physiological damage.
    Biochemical and Biophysical Research Communications 02/2010; 393(4):649-55. DOI:10.1016/j.bbrc.2010.02.046 · 2.28 Impact Factor
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    The Power of Functional Resins in Organic Synthesis, 1st edited by Judit Tulla-Puche, Fernando Albericio, 02/2009: chapter Nanoparticles Functionalized with Bioactive Molecules: Biomedical Applications: pages 45-80; John Wiley & Sons., ISBN: 9783527319367
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    ABSTRACT: Peptide-Gold nanoparticles selectively attached to beta-amyloid protein (A beta) amyloidogenic aggregates were irradiated with microwave. This treatment produces dramatic effects on the A beta aggregates, inhibiting both the amyloidogenesis and the restoration of the amyloidogenic potential. This novel approach offers a new strategy to inhibit, locally and remotely, the amyloidogenic process, which could have application in Alzheimer's disease therapy. We have studied the irradiation effect on the amyloidogenic process in the presence of conjugates peptide-nanoparticle by transmission electronic microscopy observations and by Thioflavine T assays to quantify the amount of fibrils in suspension. The amyloidogenic aggregates rather than the amyloid fibrils seem to be better targets for the treatment of the disease. Our results could contribute to the development of a new therapeutic strategy to inhibit the amyloidogenic process in Alzheimer's disease. This work was supported by FONDECYT 1061142, FONDAP 11980002 (17 07 0002), and AECI A/010967/07.
    Nanoscale Research Letters 11/2008; DOI:10.1007/s11671-008-9178-5 · 2.52 Impact Factor
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    ABSTRACT: A new synthesis and stabilization method was developed for paramagnetic nanoparticles composed of nickel and nickel oxides. Nickel/nickel oxides nanoparticles were synthesized by a method based on ligand displacement of bis(1,5-cyclooctadiene)-nickel(0), zerovalent organometallic precursor and simultaneous formation of a thiourea inclusion compound. Nickel/nickel oxides nanoparticles were stabilized with the amphipathic peptide H2N-Cys-Leu-Pro-Phe-Phe-Asp-NH2 having H2N-Leu-Pro-Phe-Phe-Asp-NH2 a peptide with potential properties for Alzheimer's disease therapy. The inclusion compound formed after displacement was characterized by X-ray powder diffraction, and nickel/nickel oxides nanoparticles were characterized using transmission electron microscopy, atomic force microscopy, UV-Visible spectroscopy, X-ray photoelectron spectroscopy, and superconducting quantum interference device magnetometry. In addition, a cell viability assay in primary rat hippocampal neurons was carried out.
    Journal of Nanoscience and Nanotechnology 09/2008; 8(8):3820-7. DOI:10.1166/jnn.2008.199 · 1.34 Impact Factor
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    ABSTRACT: In a previous work, we studied the interaction of beta-amyloid fibrils (Abeta) with gold nanoparticles (AuNP) conjugated with the peptide CLPFFD-NH2. Here, we studied the effect of changing the residue sequence of the peptide CLPFFD-NH2 on the efficiency of conjugation to AuNP, the stability of the conjugates, and the affinity of the conjugates to the Abeta fibrils. We conjugated the AuNP with CLPFFD-NH 2 isomeric peptides (CDLPFF-NH2 and CLPDFF-NH2) and characterized the resulting conjugates with different techniques including UV-Vis, TEM, EELS, XPS, analysis of amino acids, agarose gel electrophoresis, and CD. In addition, we determined the proportion of AuNP bonded to the Abeta fibrils by ICP-MS. AuNP-CLPFFD-NH2 was the most stable of the conjugates and presented more affinity for Abeta fibrils with respect to the other conjugates and bare AuNP. These findings help to better understand the way peptide sequences affect conjugation and stability of AuNP and their interaction with Abeta fibrils. The peptide sequence, the steric effects, and the charge and disposition of hydrophilic and hydrophobic residues are crucial parameters when considering the design of AuNP peptide conjugates for biomedical applications.
    Bioconjugate Chemistry 07/2008; 19(6):1154-63. DOI:10.1021/bc800016y · 4.82 Impact Factor
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    ABSTRACT: In this review, we describe the contribution of peptides to the biomedical applications of metallic nanoparticles. We also discuss strategies for the preparation of peptide-nanoparticle conjugates and the synthesis of the peptides and metallic nanoparticles. An overview of the techniques used for the characterization of the conjugates is also provided. Mainly for biomedical purposes, metallic nanoparticles conjugated to peptides have been prepared from Au and iron oxide (magnetic nanoparticles). Peptides with the capacity to penetrate the plasma membrane are used to deliver nanoparticles to the cell. In addition, peptides that recognize specific cell receptors are used for targeting nanoparticles. The potential application of peptide-nanoparticle conjugates in cancer and Alzheimer's disease therapy is discussed. Several peptide-nanoparticle conjugates show biocompatibility and present a low degree of cytotoxicity. Furthermore, several peptide-metallic nanoparticle conjugates are used for in vitro diagnosis.
    Nanomedicine 07/2007; 2(3):287-306. DOI:10.2217/17435889.2.3.287 · 5.82 Impact Factor