Browsing by Author "Saez, Claudia G."

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    Clot lysis time in platelet-rich plasma: Method assessment, comparison with assays in platelet-free and platelet-poor plasmas, and response to tranexamic acid
    (TAYLOR & FRANCIS INC, 2012) Panes, Olga; Padilla, Oslando; Matus, Valeria; Saez, Claudia G.; Berkovits, Alejandro; Pereira, Jaime; Mezzano, Diego
    Fibrinolysis dysfunctions cause bleeding or predisposition to thrombosis. Platelets contain several factors of the fibrinolytic system, which could up or down regulate this process. However, the temporal relationship and relative contributions of plasma and platelet components in clot lysis are mostly unknown. We developed a clot lysis time (CLT) assay in platelet-rich plasma (PRP-CLT, with and without stimulation) and compared it to a similar one in platelet-free plasma (PFP) and to another previously reported test in platelet-poor plasma (PPP). We also studied the differential effects of a single dose of tranexamic acid (TXA) on these tests in healthy subjects. PFP- and PPP-CLT were significantly shorter than PRP-CLT, and the three assays were highly correlated (p < 0.0001). PFP- and PPP-, but more significantly PRP-CLT, were positively correlated with age and plasma PAI-1, von Willebrand factor, fibrinogen, LDL-cholesterol, and triglycerides (p < 0.001). All these CLT assays had no significant correlations with platelet aggregation/secretion, platelet counts, and pro-coagulant tests to explore factor X activation by platelets, PRP clotting time, and thrombin generation in PRP. Among all the studied variables, PFP-CLT was independently associated with plasma PAI-1, LDL-cholesterol, and triglycerides and, additionally, stimulated PRP-CLT was also independently associated with plasma fibrinogen. A single 1 g dose of TXA strikingly prolonged all three CLTs, but in contrast to the results without the drug, the lysis times were substantially shorter in non-stimulated or stimulated PRP than in PFP and PPP. This standardized PRP-CLT may become a useful tool to study the role of platelets in clot resistance and lysis. Our results suggest that initially, the platelets enmeshed in the clot slow down the fibrinolysis process. However, the increased clot resistance to lysis induced by TXA is overcome earlier in platelet-rich clots than in PFP or PPP clots. This is likely explained by the display of platelet pro-fibrinolytic effects. Focused research is needed to disclose the mechanisms for the relationship between CLT and plasma cholesterol and its potential pathophysiologic and clinical relevance.
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    Differences in lung glutathione metabolism may account for rodent susceptibility in elastase-induced emphysema development
    (AMER PHYSIOLOGICAL SOC, 2009) Borzone, Gisella R.; Liberona, Leonel F.; Bustamante, Andrea P.; Saez, Claudia G.; Olmos, Pablo R.; Vecchiola, Andrea; Villagran, Andrea; Serrano, Carolina; Reyes, Tatiana P.
    Borzone GR, Liberona LF, Bustamante AP, Saez CG, Olmos PR, Vecchiola A, Villagran A, Serrano C, Reyes TP. Differences in lung glutathione metabolism may account for rodent susceptibility in elastase-induced emphysema development. Am J Physiol Regul Integr Comp Physiol 296: R1113-R1123, 2009. First published January 14, 2009; doi: 10.1152/ajpregu.90361.2008.-Syrian Golden hamsters develop more severe emphysema than Sprague-Dawley rats after intratracheal instillation of the same dose of elastase/body weight. Although species variations in antielastase defenses may largely explain these results, other variables, such as differences in lung antioxidants, cannot be overlooked since oxidative stress modulates antiprotease activity. We propose that elastase instillation might affect lung glutathione (GSH) metabolism differently in these species. Our aim was to study in hamsters and rats, lung glutathione metabolism at different times, from the stage of diffuse alveolar damage to advanced emphysema. We measured total and oxidized glutathione content as well as activity and expression of enzymes related to GSH synthesis and redox cycling: gamma-glutamylcysteine synthetase, glutathione peroxidase, and glutathione reductase. Whereas rats showed no significant changes in these measurements, hamsters showed significant derangement in GSH metabolism early after elastase instillation: 25% fall in total GSH (P < 0.05) with no increase in oxidized glutathione associated with reduced enzyme activities 24 h after elastase [60% for gamma-glutamylcysteine synthetase (P < 0.01), 30% for glutathione peroxidase (P < 0.01), and 75% for glutathione reductase (P < 0.001)]. GSH homeostasis was restored at the end of the first week, involving transient increased expression of these enzymes. We conclude that elastase induces significant alterations in GSH metabolism of hamster lungs and no overall change in rat lungs. Although differences in disease severity may account for our findings, the hamster becomes vulnerable to functional inhibition of alpha(1)-antitrypsin by oxidants and thus, even more susceptible to injury than it would be, considering only its low alpha(1)-antitrypsin level.
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    Human platelets synthesize and express functional tissue factor
    (AMER SOC HEMATOLOGY, 2007) Panes, Olga; Matus, Valeria; Saez, Claudia G.; Quiroga, Teresa; Pereira, Jaime; Mezzano, Diego
    The source and significance of bloodborne tissue factor (TF) are controversial. TF mRNA, protein, and TF-dependent procoagulant activity (PCA) have been detected in human platelets, but direct evidence of TF synthesis is missing. Nonstimulated monocyte-free platelets from most patients expressed TF mRNA, which was enhanced or induced in all of them after platelet activation. Immunoprecipitation assays revealed TF protein (mainly of a molecular weight [Mr] of approximately 47 kDa, with other bands of approximately 35 and approximately 60 kDa) in nonstimulated platelet membranes, which also increased after activation. This enhancement was concomitant with TF translocation to the plasma membrane, as demonstrated by immunofluorescence-confocal microscopy and biotinylation of membrane proteins. Platelet PCA, assessed by factor Xa (FXa) generation, was induced after activation and was inhibited by 48% and 76% with anti-TF and anti-FVIIa, respectively, but not by intrinsic pathway inhibitors. Platelets incorporated [35S]-methionine into TF proteins with Mr of approximately 47 kDa, approximately 35 kDa, and approximately 60 kDa, more intensely after activation. Puromycin but not actinomycin D or DRB (5,6-dichloro-1 -beta-D-ribofuranosylbenzimidazole)inhibited TIF neosynthesis. Thus, human platelets not only assemble the clotting reactions on their membrane, but also supply their own TIF for thrombin generation in a timely and spatially circumscribed process. These observations simplify, unify, and provide a more coherent formulation of the current cellbased model of hemostasis.
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    Identification of Statin’s Action in a Small Cohort of Patients with Major Depression
    (2021) Thakkar, Ishani; Massardo, Teresa; Pereira, Jaime; Quintana, Juan Carlos; Risco, Luis; Saez, Claudia G.; Corral, Sebastián; Villa, Carolina; Spuler, Jane; Olivares, Nixa; Valenzuela, Guillermo; Castro, Gabriel; Riedel, Byron; Vicentini, Daniel; Muñoz, Diego; Lastra, Raúl; Rodriguez-Fernandez, Maria
    Statins are widely used as an effective therapy for ischemic vascular disorders and employed for primary and secondary prevention in cardiac and cerebrovascular diseases. Their hemostatic mechanism has also been shown to induce changes in cerebral blood flow that may result in neurocognitive improvement in subjects with Major Depressive Disorder. Behavioral data, various blood tests, and resting-state brain perfusion data were obtained at the start of this study and three months post-therapy from a small cohort of participants diagnosed with Major Depressive Disorder. Subjects received either rosuvastatin (10 mg) or placebo with their standard selective serotonin reuptake inhibitors therapy. At the end of the study, patients using rosuvastatin reported more positive mood changes than placebo users. However, standard statistical tests revealed no significant differences in any non-behavioral variables before and after the study. In contrast, feature selection techniques allowed identifying a small set of variables that may be affected by statin use and contribute to mood improvement. Classification models built to assess the distinguishability between the two groups showed an accuracy higher than 85% using only five selected features: two peripheral platelet activation markers, perfusion abnormality in the left inferior temporal gyrus, Attention Switching Task Reaction latency, and serum phosphorus levels. Thus, using machine learning tools, we could identify factors that may be causing self-reported mood improvement in patients due to statin use, possibly suggesting a regulatory role of statins in the pathogenesis of clinical depression.
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    Increased number of circulating endothelial cells and plasma markers of endothelial damage in chronic cocaine users
    (PERGAMON-ELSEVIER SCIENCE LTD, 2011) Saez, Claudia G.; Olivares, Paulina; Pallavicini, Julio; Panes, Olga; Moreno, Natalia; Massardo, Teresa; Mezzano, Diego; Pereira, Jaime
    Background: Cocaine use has been related with the development of accelerated atherosclerosis and with an increased risk of cardiac and cerebrovascular events, such as myocardial infarction, sudden cardiac death, and ischemic stroke. The underlying mechanisms leading to these complications are not fully understood, although thrombus formation and altered vascular function are prominent findings.
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    Maternal obesity and high leptin levels prime pro-inflammatory pathways in human cord blood leukocytes
    (2023) Krause, Bernardo J.; Vega-Tapia, Fabian A.; Soto-Carrasco, Gustavo; Lefever, Isidora; Letelier, Catalina; Saez, Claudia G.; Castro-Rodriguez, Jose A.
    Introduction: Maternal obesity alters the immune function in the offspring. We hypothesize that maternal obesity and pro-inflammatory pathways induce leptin-related genes in neonatal monocytes, whereby high leptin levels enhance their inflammatory response. Methods: Transcriptional profiles of cord blood leukocytes (CBL) in basal and pro-inflammatory conditions were studied to determine differentially expressed genes (DEG). The DNA methylation profile of CB monocytes (CBM) of neonates born to control BMI mothers and women with obesity was assayed to identify differentially methylated probes (DMP). CBM-derived macrophages were cultured with or without leptin (10-100 ng/ml) and then stimulated with lipopolysaccharide (LPS, 100 ng/ml) and interferon-gamma (20 ng/ml) to assess the induction of TNF-& alpha; and IL-10 transcripts. Results: CBL from pregnancies with obesity (CBL-Ob) showed 12,183 DEG, affecting 49 out of 78 from the leptin pathway. Control CBM exposed to LPS showed 45 leptin-related DEG, an effect prevented by the co-exposure to LPS and IL-10. Conversely, CBM-Ob showed 5279 DMP enriched in insulin- and leptin-related genes, and Lasso regression of leptin-related DMP showed high predictive value for plasma leptin levels (r2 = 0.9897) and maternal BMI categories (AUC = 1). Chronic exposure to leptin increased TNF-& alpha; and decreased IL-10 levels in control BMI samples but not in Ob-CBM. Enhanced TNF-& alpha; induction after proinflammatory stimulation was observed in leptin-treated control BMI samples. Discussion: Obesity in pregnancy is associated with a distinctive expression and DNA methylation profile of leptin-related genes in cord blood monocytes, meanwhile, leptin enhances the expression of pro-inflammatory cytokines upon stimulation with M1-skewing agents.
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    Platelet activation in chronic cocaine users: Effect of short term abstinence
    (TAYLOR & FRANCIS INC, 2011) Pereira, Jaime; Saez, Claudia G.; Pallavicini, Julio; Panes, Olga; Pereira Flores, Karla; Cabreras, Manuel J.; Massardo, Teresa; Mezzano, Diego
    Cocaine abuse increases the risk of cardiac and cerebrovascular events, such as myocardial infarction and ischemic stroke. The underlying mechanisms leading to these complications are not fully understood although intravascular thrombus formation has been observed. The aim of this study was to investigate the existence of platelet activation and the effect of short-term abstinence in chronic cocaine consumers. We studied 23 cocaine dependent individuals (aged 20-54 years) who met DSM-IV criteria for cocaine dependence and 20 controls. Samples were obtained at baseline, within 72 h of last drug exposure and after 4 weeks of controlled abstinence. Monocyte-platelet aggregates (MPA) were measured by flow cytometry. Plasma levels of soluble CD40L (sCD40L), Neutrophil-Activating Peptide-2 (NAP-2) and regulated on activation normal T cells expressed and secreted (RANTES) were determined by ELISA. Levels of MPA, sCD40L, NAP-2 and RANTES were significantly higher (all p < 0.05) in cocaine addicts compared to controls at baseline. All the parameters returned to values similar to the control group after 4-weeks' abstinence. Levels of sCD40L and RANTES were associated with an index of intensity of drug consumption (p < 0.02). Our results demonstrate that cocaine use induces platelet activation which is a prominent finding after recent consumption. The persistence over time of this condition may contribute not only to acute thrombotic complications but also to the development of early-onset atherosclerotic process observed in cocaine abusers.
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    Smoking habits do not affect biological responses induced by leucocyte and platelet-rich fibrin in periodontal ligament cells
    (2023) Rios, Susana; Alvarez, Simon; Smith, Patricio C.; Saez, Claudia G.; Andrade, Catherine; Pinto, Nelson; Martinez, Constanza E.
    Background and Objective: Leucocyte- and platelet-rich fibrin has been developed to stimulate wound healing response. However, it is currently unknown whether smoking affects the biological responses elicited by leucocyte- and platelet-rich fibrin on periodontal ligament-derived mesenchymal stromal cells. This study analyzes the kinetics of biomolecule release from leucocyte- and platelet-rich fibrin derived from smokers and nonsmokers and their effect on periodontal ligament cell proliferation and migration as essential biological activities during wound healing. Methods: Biomolecules present in leucocyte- and platelet-rich fibrin exudates and conditioned media collected from smokers and nonsmokers were analyzed by Luminex arrays. Periodontal ligament-derived mesenchymal stromal cell obtained from one nonsmoker were treated with leucocyte- and platelet-rich fibrin exudates or leucocyte- and platelet-rich fibrin conditioned media derived from both smokers and nonsmokers. The parameters evaluated included cell proliferation, determined by Ki67 immunostaining and migration assessed using transwell assays. Also, cells were treated with nicotine in the presence of fetal bovine serum 10% or leucocyte- and platelet-rich fibrin conditioned media. Results: A similar biomolecular profile was detected in leucocyte- and platelet-rich fibrin exudates and leucocyte- and platelet-rich fibrin conditioned media from smokers and nonsmokers, stimulating (periodontal ligament-derived mesenchymal stromal cell) proliferation, and migration to a comparable degree. Nicotine reduced cell proliferation and migration of periodontal cells; however, this effect was recovered in the presence of leucocyte- and platelet-rich fibrin conditioned media. Conclusion: Leucocyte- and platelet-rich fibrin derived from smokers could be an autologous source of biomolecules to stimulate cell biological activities involved in wound healing in smokers who have difficulties in ceasing this habit. Clinical trials are required to evaluate the impact of leucocyte- and platelet-rich fibrin on healing responses in smokers.
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    Transcriptional Profiling of Human Endothelial Cells Unveils PIEZO1 and Mechanosensitive Gene Regulation by Prooxidant and Inflammatory Inputs
    (2023) Arenas, German A.; Valenzuela, Jose G.; Penaloza, Estefania; Paz, Adolfo A.; Iturriaga, Rodrigo; Saez, Claudia G.; Krause, Bernardo J.
    PIEZO1 is a mechanosensitive cation channel implicated in shear stress-mediated endothelial-dependent vasorelaxation. Since altered shear stress patterns induce a pro-inflammatory endothelial environment, we analyzed transcriptional profiles of human endothelial cells to determine the effect of altered shear stress patterns and subsequent prooxidant and inflammatory conditions on PIEZO1 and mechanosensitive-related genes (MRG). In silico analyses were validated in vitro by assessing PIEZO1 transcript levels in both the umbilical artery (HUAEC) and vein (HUVEC) endothelium. Transcriptional profiling showed that PIEZO1 and some MRG associated with the inflammatory response were upregulated in response to high (15 dyn/cm(2)) and extremely high shear stress (30 dyn/cm(2)) in HUVEC. Changes in PIEZO1 and inflammatory MRG were paralleled by p65 but not KLF or YAP1 transcription factors. Similarly, PIEZO1 transcript levels were upregulated by TNF-alpha (TNF-alpha) in diverse endothelial cell types, and pre-treatment with agents that prevent p65 translocation to the nucleus abolished PIEZO1 induction. ChIP-seq analysis revealed that p65 bonded to the PIEZO1 promoter region, an effect increased by the stimulation with TNF-alpha. Altogether this data showed that NF-kappa B activation via p65 signaling regulates PIEZO1 expression, providing a new molecular link for prooxidant and inflammatory responses and mechanosensitive pathways in the endothelium.