No description available.

Purpose: The aim of this study was to determine whether the combination of 2 angiogenic growth factor, vascular endothelial growth factor 165(VEGF165) and angiopoietin-1 (Ang1), could increase angiogenesis and cardiomyocyte(CM) proliferation in an infarcted myocardium. Methods: Myocardial ischemia was induced in rats by ligation of the left anterior descending (LAD) coronary artery. Replication-deficient adenoviruses encoding VEGF165 (Ad-VEGF165), Ang1 (Ad-Ang1) or enhanced green fluorescence protein (Ad-EGFP) was injected into the ischemic myocardium immediately. Bromodexyuridine (BrdU) was administered intraperitoneally 1 week after ligation. One week later, the hearts were harvested and sectioned for hematoxylin-eosin (HE) and immunohistochemistry to evaluate densities of capillary, arteriole and double labelled BrdU(+) CM. M-mode echocardiography was used to evaluate the cardiac function. Results: Ang1 significantly increased collateral vessel formation. Both VEGF165 and Ang1 significantly increased densities of capillary and arteriole, as well as the number of double labelled BrdU(+) CM, and improved cardiac function. Conclusion: Our results suggest that the combination of VEGF165 and Ang1 can increase both myocardial angiogenesis and CM proliferation following myocardial ischemia in rats, leading to improved cardiac function.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Normal endothelial cell function is controlled in part by a tightly regulated balance between angiopoietin-1 and -2 (Ang-1 and Ang-2). Angiopoietin dysregulation (decreased Ang-1 and increased Ang-2) leads to an activated endothelium that is contractile, adhesive, and prothrombotic. Since an activated endothelial phenotype is seen in invasive group A streptococcal infection, E. coli O157:H7-induced hemolytic-uremic syndrome (HUS), and sepsis, we hypothesized that angiopoietin dysregulation might also be present in these syndromes, and to that end, measured angiopoietin levels in several well-characterized patient cohorts. Decreased Ang-1 and/or increased Ang-2 were found in all three syndromes, and were predictive of clinical outcome in HUS and sepsis. The prognostic utility of Ang-2 in sepsis was further enhanced by combination with biomarkers of inflammation. Angiopoietin dysregulation may therefore represent a shared final common pathway to endothelial activation as well as a clinically useful prognostic biomarker in streptococcal toxic shock, HUS, and sepsis.

Normal endothelial cell function is controlled in part by a tightly regulated balance between angiopoietin-1 and -2 (Ang-1 and Ang-2). Angiopoietin dysregulation (decreased Ang-1 and increased Ang-2) leads to an activated endothelium that is contractile, adhesive, and prothrombotic. Since an activated endothelial phenotype is seen in invasive group A streptococcal infection, E. coli O157:H7-induced hemolytic-uremic syndrome (HUS), and sepsis, we hypothesized that angiopoietin dysregulation might also be present in these syndromes, and to that end, measured angiopoietin levels in several well-characterized patient cohorts. Decreased Ang-1 and/or increased Ang-2 were found in all three syndromes, and were predictive of clinical outcome in HUS and sepsis. The prognostic utility of Ang-2 in sepsis was further enhanced by combination with biomarkers of inflammation. Angiopoietin dysregulation may therefore represent a shared final common pathway to endothelial activation as well as a clinically useful prognostic biomarker in streptococcal toxic shock, HUS, and sepsis.

Doxorubicin (Dox) is a major anticancer chemotherapeutic agent. However, it causes cardiomyopathy due to the side effect of cardiomyocyte apoptosis. We have previously reported that angiopoietin-1 significantly reduced myocardial infarction after ischemic injury and protected cardiomyocytes from oxidative stress-induced apoptosis. It is hypothesized that angiopoietin-1 may protect cardiomyocytes from Dox-induced apoptosis. Cardiomyocytes H9C2 were transfected with adenovirus expressing angiopoietin-1 (Ad5-Ang-1) 24 h before the cells were challenged with Dox at a concentration of 2 μmol/L. Ad5-GFP served as the vector control. Cardiomyocyte apoptosis was evaluated using Annexin V-FITC staining and caspase-3 and caspase-8 activity was determined by Western blotting. The results showed that Dox treatment significantly induced cardiomyocyte apoptosis as evidenced by the greater number of Annexin V-FITC stained cells and increases in caspase-3 and caspase-8 activity. In contrast, overexpression of angiopoietin-1 significantly prevented Dox-induced cardiomyocyte apoptosis. To elucidate the mechanisms by which angiopoietin-1 protected cells from Dox-induced apoptosis, we analyzed both extrinsic and intrinsic apoptotic signaling pathways. We observed that angiopoietin-1 prevented Dox-induced activation of both extrinsic and intrinsic apoptotic signaling pathways. Specifically, angiopoietin-1 prevented DOX-induced in-creases in FasL and Bax levels and cleaved caspase-3 and caspase-8 levels in H9C2 cells. In addition, overexpression of angiopoietin-1 also activated the pro-survival phosphoinositide-3 kinase (PI3K)/Akt signaling pathway and decreased Dox-induced nuclear factor-kappaB (NF-κB) activation. Our data suggest that promoting the expression of angiopoietin-1 could be a potential approach for reducing Dox-induced cardiomyocyte cytoxicity.

Sowohl der ANP und BNP bindende Guanylylzyklase-A-Rezeptor, als auch der CNP bindende Guanylylzyklase-B-Rezeptor auf den die Endothelzellen ummantelnden Perizyten sind für eine normale postnatale Gefäßentwicklung in der Netzhaut der Maus von entscheidender Bedeutung. Eine perizytenspezifische Deletion der Guanylylzyklase-Rezeptoren führt in Mäusen zu einer signifikanten Verminderung der postnatalen Ausdehnung sowie der Dichte des Gefäßnetzes. Dies ist nicht auf eine Verminderung der Bedeckung des Endothels durch Perizyten zurückzuführen. Weiterhin geht diese Rezeptordeletion mit einer geschlechterunabhängigen Erhöhung des systolischen Blutdrucks einher. Die intrazelluläre Weiterleitung, des durch die natriuretischen Peptide ausgelösten cGMP-Signals erfolgt über die cGMP-abhängige Proteinkinase Typ I (cGKI). / Both the ANP- and BNP-binding guanylyl cyclase A receptor and the CNP-binding guanylyl cyclase B receptor on pericytes lining endothelial cells are crucial for a normal postnatal vascular development in the mouse retina. A pericyte-specific deletion of the guanylyl cyclase receptors in mice leads to a significant reduction in the postnatal extent and density of the vascular plexus. This reduction is not caused by a reduced pericyte-coverage of the endothelium. Furthermore, the deletion of this receptor is associated with a gender-independent increase in systolic blood pressure. The intracellular transmission of the cGMP signal triggered by the natriuretic peptides takes place via the cGMP-dependent protein kinase type I (cGKI).

Angiopoietin-1 (Ang-1), the main ligand for the endothelial cell (EC)-selective Tie-2 receptors, promotes survival, proliferation, migration and differentiation of these cells. Despite its importance in various aspects of vascular biology, the mechanisms of action of the Ang-1/Tie-2 receptor pathway have not been fully explored. / To identify the downstream modulators of Ang-1, we evaluated changes in the transcriptome of human umbilical vein endothelial cells (HUVECs) treated with Ang-1 protein for four hours by employing the oligonucleotide rnicroarray technology. Eighty-six genes were significantly upregulated by this treatment and forty-nine genes were significantly downregulated. These genes are involved in the regulation of cell cycle, proliferation, apoptosis, transcription and differentiation. Furthermore, we found that the Erk1/2, PI3-Kinase and mTOR pathways are implicated in promoting gene expression in HUVECs in response to Ang-1. Analysis of the microarray data employing the Ingenuity Pathways analysis software to place the regulated genes in the context of biological networks revealed several highly connected nodes including the chemokine Interleukin-8 (IL-8) and the transcription factor Early growth response-1 (Egr-1). Due to the importance of these genes in promoting angiogenesis, we decided to evaluate their roles in Ang-1/Tie-2 receptor signaling and biological effects. / Ang-1 induced IL-8 expression in a time- and dose-dependent manner in ECs through both transcriptional and post-transcriptional mechanisms. To study the functional role of Ang-1-induced IL-8, we generated HUVECs that overexpress Ang-1. In these cells, neutralizing IL-8 significantly reduced EC proliferation and migration. IL-8 promoter activity experiments and gel shift assays revealed the involvement of the transcription factor AP-1 in Ang-1-induced IL-8. Ang-1 stimulated the phosphorylation of c-Jun through activation of Erk1/2, JNK and PI-3 kinase pathways. Similarly, Ang-1 provoked the expression and DNA binding of Egr-1 in HUVECs. Employing siRNA and DNAzyme to specifically knock-down Egr-1, we found that Ang-1-induced Egr-1 also promotes EC proliferation and migration. / We conclude that Ang-1 provokes a coordinated response intended to promote EC survival, proliferation, and angiogenesis and to inhibit EC apoptosis. Ang-1 induces EC proliferation and migration in part through the secretion of the soluble mediator Interleukin-8 and through induction of the transcription factor Egr-1.

No description available.

Orientador: Erich Vinicius de Paula / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-18T13:46:04Z (GMT). No. of bitstreams: 1 Alves_BrunnaEulalio_D.pdf: 4762678 bytes, checksum: 34eea414d90830a52ed9c9b044538888 (MD5) Previous issue date: 2011 / Resumo: Pacientes portadores de neoplasia hematológica e neutropenia febril representam um grupo de risco elevado de sepse e choque séptico. Nas últimas décadas, estratégias terapêuticas alvo-específicas para a sepse não modificaram de forma significativa a sobrevida dos pacientes e o tratamento permanece baseado em antibioticoterapia e cuidados de suporte, com altas taxas de mortalidade. A quebra da barreira endotelial é um evento fundamental na fisiopatologia do choque séptico e a compreensão dos mecanismos envolvidos neste evento tem o potencial de auxiliar na identificação de novos biomarcadores de gravidade e de novos alvos terapêuticos para estes pacientes. Estudos recentes demonstraram a participação do fator de crescimento do endotélio vascular (VEGF-A), do seu receptor solúvel (sFlt-1) e das angiopoietinas 1 e 2, proteínas envolvidas na angiogênese e na regulação da integridade da barreira endotelial na fisiopatogenia do choque séptico em pacientes não oncológicos internados em unidade de terapia intensiva. Neste trabalho, avaliamos prospectivamente a cinética do VEGF-A, do sFlt-1 e das angiopoietinas 1 e 2 durante as 48 horas inicias da neutropenia febril em 41 pacientes portadores de neoplasia hematológica submetidos a quimioterapia intensiva ou a regime de condicionamento para transplante de células progenitoras hematopoiéticas, através da dosagem dos mesmos por ensaio imuno-enzimático. Exploramos também a associação dos níveis séricos destes biomarcadores com a gravidade da sepse através da correlação com o MASCC, um índice desenvolvido para identificar pacientes com neutropenia febril de baixo risco, e com o SOFA, um escore de avaliação de disfunção orgânica em pacientes com sepse, ambos amplamente aceitos. A evolução para choque séptico foi associada a níveis significativamente maiores de VEGF-A, sFlt-1 e angiopoietina-2 48 horas após o início da neutropenia febril quando comparado aos valores em pacientes com sepse não complicada e a estimativa da acurácia diagnóstica sugere a capacidade de discriminar os pacientes que evoluíram com choque séptico. Estes biomarcadores também apresentaram correlação com os escores gravidade, sugerindo a relevância biológica da associação. Em conclusão, nossos achados sugerem que a avaliação destes biomarcadores em pacientes com neutropenia febril deve ser avaliada em estudos com maior número de pacientes, quanto ao seu potencial de incorporação na prática clínica. Além disso, os resultados reforçam o potencial terapêutico da intervenção nestas vias para o tratamento da sepse / Abstract: Patients with hematologic malignancy and neutropenia represent a group at high risk of sepsis and septic shock. In recent decades, target-specific therapeutic strategies for sepsis did not change significantly the survival of patients and treatment is still based on antibiotic therapy and supportive care, with high mortality rates. The breakdown of the endothelial barrier is a key event in the pathophysiology of septic shock and understanding of the mechanisms involved in this event has the potential to assist in the identification of new biomarkers and severity of new therapeutic targets for these patients. Recent studies have demonstrated the involvement of endothelial growth factor (VEGF-A), its soluble receptor (sFlt-1) and angiopoietins 1 and 2, proteins involved in angiogenesis and in regulation of endothelial barrier integrity in the pathogenesis of shock septic patients without cancer admitted to the intensive care unit. In this study, we prospectively evaluated the kinetics of VEGF-A, sFlt-1 and angiopoietins 1 and 2 during the initial 48 hours of febrile neutropenia in 41 patients with hematological malignancy undergoing intensive chemotherapy or conditioning regimen for stem cell transplantation hematopoietic cells by the same dosage by enzyme immunoassay. We also explored the association of serum levels of these biomarkers with the severity of sepsis through correlation with the MASCC, an index developed to identify patients with febrile neutropenia at low risk, and the SOFA score for assessment of organ dysfunction in patients with sepsis, both widely accepted. Progression to septic shock was associated with significantly higher levels of VEGF-A, sFlt-1 and angiopoietin-2 48 hours after the onset of febrile neutropenia when compared to values in patients with uncomplicated sepsis and the estimation of diagnostic accuracy suggests the ability to discriminate among patients who developed septic shock. These biomarkers also correlated with the severity scores, suggesting the biological relevance of the association / Doutorado / Clinica Medica / Doutor em Clínica Médica