RESUMO
Excess soluble fms-like tyrosine kinase 1 (sFlt-1), a soluble inhibitor of vascular endothelial growth factor pathway, has been demonstrated to promote endothelial dysfunction. Here, we demonstrate that sFlt-1 plasma levels correlate with respiratory symptom severity, expression of endothelial dysfunction biomarker, and incidence of organ failure in coronavirus disease 2019 patients. Clinical Trials Registration: NCT04394195.
Assuntos
COVID-19 , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Estado Terminal , Humanos , SARS-CoV-2 , Fator A de Crescimento do Endotélio VascularRESUMO
Peripheral arterial disease occurs more frequently and has a worse prognosis in patients with chronic kidney disease (CKD). The receptor for advanced glycation end products (RAGE) is involved in multiple aspects of uremia-associated vasculopathy. Previous data suggest that the RAGE pathway may promote soluble fms-like tyrosine kinase 1 (sFlt1) production, an anti-angiogenic molecule. Thus, we tested the hypothesis that the deletion of AgeR would decrease sFlt1 production and improve post-ischemic revascularization in uremic condition. We used a well-established CKD model (5/6 nephrectomy) in WT and AgeR-/- C57/Bl6 mice. Hindlimb ischemia was induced by femoral artery ligation. Revascularization was evaluated by complementary approaches: ischemic limb retraction, LASCA imagery, and capillary density. The production of sFlt1 was assessed at both RNA and protein levels. After hindlimb ischemia, uremic mice showed slower functional recovery (p < 0.01), decreased reperfusion (p < 0.01), lower capillary density (p = 0.02), and increased circulating sFlt1 levels (p = 0.03). AgeR deletion restored post-ischemic angiogenesis and was protective from sFlt1 increase in uremic mice. These findings show the main role of RAGE in post-ischemic angiogenesis impairment associated with CKD. RAGE may represent a key target for building new therapeutic approaches to improve the outcome of CKD patients with PAD.
Assuntos
Deleção de Genes , Isquemia/complicações , Neovascularização Fisiológica , Receptor para Produtos Finais de Glicação Avançada/deficiência , Uremia/complicações , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Animais , Biomarcadores/sangue , Linhagem Celular , Humanos , Ligantes , Masculino , Camundongos Endogâmicos C57BL , RNA/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Solubilidade , Regulação para CimaRESUMO
In paediatric acute lymphoblastic leukaemia (ALL), focus has shifted towards preventing treatment-related complications, including venous thromboembolism, the cause of significant mortality and morbidity. To better understand thrombogenic mechanisms during induction treatment, we studied the number, origin and procoagulant activity of extracellular vesicles (EVs) and P-selectin level throughout the induction course in 24 paediatric patients. EVs were mainly of platelet origin. We observed a significant increase in EV number, in platelet EV number and P-selectin level throughout the induction course. There was a correlation between higher EV and platelet EV number, P-selectin level, higher platelet count and leucocyte count. We also observed a correlation between higher EV procoagulant activity and higher platelet count and leucocyte count and higher P-selectin level. Older age and T phenotype were associated with a higher EV procoagulant activity. Platelet EV generation may play a role in thrombogenic complications in ALL patients and could serve as a biomarker to identify patients with a high risk of thrombosis. As a marker of platelet activation, P-selectin may be another relevant marker with the advantage of being easier to analyse in clinical practice.
Assuntos
Asparaginase/uso terapêutico , Vesículas Extracelulares , Quimioterapia de Indução , Selectina-P/sangue , Contagem de Plaquetas , Leucemia-Linfoma Linfoblástico de Células Precursoras , Tromboembolia Venosa , Antineoplásicos/uso terapêutico , Biomarcadores/sangue , Coagulação Sanguínea/efeitos dos fármacos , Coagulação Sanguínea/fisiologia , Plaquetas/metabolismo , Plaquetas/patologia , Criança , Correlação de Dados , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patologia , Feminino , Humanos , Quimioterapia de Indução/efeitos adversos , Quimioterapia de Indução/métodos , Contagem de Leucócitos/métodos , Contagem de Leucócitos/estatística & dados numéricos , Masculino , Projetos Piloto , Ativação Plaquetária/efeitos dos fármacos , Contagem de Plaquetas/métodos , Contagem de Plaquetas/estatística & dados numéricos , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Tromboembolia Venosa/sangue , Tromboembolia Venosa/diagnóstico , Tromboembolia Venosa/etiologiaRESUMO
BACKGROUND: Cell therapy has been proposed for patients with critical limb ischemia (CLI). Autologous bone marrow derived cells (BMCs) have been mostly used, mesenchymal stem cells (MSCs) being an alternative. The aim of this study was to characterize two types of MSCs and evaluate their efficacy. METHODS: MSCs were obtained from CLI-patients BMCs. Stimulated- (S-) MSCs were cultured in endothelial growth medium. Cells were characterized by the expression of cell surface markers, the relative expression of 6 genes, the secretion of 10 cytokines and the ability to form vessel-like structures. The cell proangiogenic properties was analysed in vivo, in a hindlimb ischemia model. Perfusion of lower limbs and functional tests were assessed for 28 days after cell infusion. Muscle histological analysis (neoangiogenesis, arteriogenesis and muscle repair) was performed. RESULTS: S-MSCs can be obtained from CLI-patients BMCs. They do not express endothelial specific markers but can be distinguished from MSCs by their secretome. S-MSCs have the ability to form tube-like structures and, in vivo, to induce blood flow recovery. No amputation was observed in S-MSCs treated mice. Functional tests showed improvement in treated groups with a superiority of MSCs and S-MSCs. In muscles, CD31+ and αSMA+ labelling were the highest in S-MSCs treated mice. S-MSCs induced the highest muscle repair. CONCLUSIONS: S-MSCs exert angiogenic potential probably mediated by a paracrine mechanism. Their administration is associated with flow recovery, limb salvage and muscle repair. The secretome from S-MSCs or secretome-derived products may have a strong potential in vessel regeneration and muscle repair. Trial registration NCT00533104.
Assuntos
Meios de Cultura/farmacologia , Células Endoteliais/citologia , Extremidades/irrigação sanguínea , Isquemia/terapia , Células-Tronco Mesenquimais/citologia , Adulto , Idoso , Animais , Artérias/crescimento & desenvolvimento , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Extremidades/patologia , Feminino , Membro Posterior/irrigação sanguínea , Humanos , Isquemia/patologia , Masculino , Transplante de Células-Tronco Mesenquimais , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Músculos/irrigação sanguínea , Músculos/patologia , Neovascularização Fisiológica , Organogênese , Fluxo Sanguíneo RegionalRESUMO
We have investigated the development of murine hindlimb ischemia from day 1 to day 55 after femoral artery ligation (FAL) using blood flow analysis, functional tests, histopathological staining, and in vivo Raman spectroscopy. FAL resulted in hindlimb blood deprivation and the loss of functionality as attested by the blood flow analysis and functional tests, respectively. The limbs recovered a normal circulation progressively without recovering complete functionality. Histological analysis showed changes in the morphology of muscle fibers with intense inflammation. From day 22 to day 55 post-ischemia, regeneration of the myofibers was observed. Raman spectroscopic results related to subcutaneous analysis made the identification of modification in the biochemical constituents of hindlimb muscles possible during disease progression. Ischemia was characterized by a quantitative increase in the lipid content and a decrease in the protein content. The lipid to protein ratio can be used as a spectroscopic marker to score the severity of ischemia. Multivariate statistical analysis PC-LDA (Principal Component-Linear Discriminant Analysis) was used to classify all the data measured for the normal and ischemic tissues. This classification illustrated an excellent separation between the control and ischemic tissues at any time during the course of ischemic development. In vivo Raman spectroscopy was then applied to assess the potential of this technique as a screening tool to explore an ischemic disease non-invasively (transcutaneously). For this purpose, the influence of skin on the diagnostic accuracy was evaluated; transcutaneous analysis revealed the accuracy of this technique, indicating its potential in the in situ monitoring of muscle structural changes during ischemia.
Assuntos
Membro Posterior/metabolismo , Isquemia/diagnóstico , Isquemia/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Pele/metabolismo , Animais , Circulação Sanguínea/fisiologia , Análise Discriminante , Membro Posterior/irrigação sanguínea , Masculino , Camundongos Endogâmicos BALB C , Análise Multivariada , Fibras Musculares Esqueléticas/patologia , Análise Espectral Raman/métodosRESUMO
Thromboembolic events are frequent and serious complications of acute lymphoblastic leukaemia treatment. The importance of chemotherapy in the pathogenesis of this increased risk is enhanced by the fact that thrombosis rarely occurs at diagnosis. Our study aims at investigating the effect of chemotherapy on pro-coagulant activity (PCA), phosphatidylserine (PS) exposure, tissue factor (TF) activity and derived extracellular vesicles (EV) of Jurkat cells. Jurkat cells were treated with two commonly used chemotherapeutics: Vincristine (VCR) or Daunorubicin (DNR), at relevant concentrations. PCA of cells and derived EV were evaluated using Thrombin generation Assay (TGA). Cells or EV were incubated with annexin V or anti TF antibodies to assess the respective contribution of TF and PS. PS exposure on cells was analysed by flow cytometry. Derived EV were evaluated in fluorescence microscopy and flow cytometry. Untreated Jurkat cells and EV support thrombin generation. Thrombin generation was abolished when PS activity was inhibited by annexin V. VCR treatment resulted in a time dependent increase of thrombin generation. After VCR exposure, TF activity increased as well as PS exposure increased on the cell surface. The increase in TF activity was abolished by annexin V indicating that PS was required. A spontaneous release of EV from Jurkat cells was observed and VCR treatment increased the number of generated EV. Our results indicate that VCR increased the PCA of Jurkat cells predominantly through PS exposure and increased EV generation. Lymphoid blasts derived EV could be biomarkers to determine high thrombotic risk ALL patients.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Vincristina/farmacologia , Anexina A5/farmacologia , Antineoplásicos Fitogênicos , Citometria de Fluxo , Humanos , Células Jurkat , Fosfatidilserinas/metabolismo , Tromboplastina/metabolismoRESUMO
BACKGROUND: Vascular endothelial injury during ischemia generates apoptotic cell death and precedes apoptosis of underlying tissues. We aimed at studying the role of extracellular adenosine triphosphate (ATP) on endothelial cells protection against hypoxia injury. METHODS: In a hypoxic model on endothelial cells, we quantified the extracellular concentration of ATP and adenosine. The expression of mRNA (ectonucleotidases, adenosine, and P2 receptors) was measured. Apoptosis was evaluated by the expression of cleaved caspase 3. The involvement of P2 and adenosine receptors and signaling pathways was investigated using selective inhibitors. RESULTS: Hypoxic stress induced a significant increase in extracellular ATP and adenosine. After a 2-h hypoxic injury, an increase of cleaved caspase 3 was observed. ATP anti-apoptotic effect was prevented by suramin, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), and CGS15943, as well as by selective A2A, A2B, and A3 receptor antagonists. P2 receptor-mediated anti-apoptotic effect of ATP involved phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinases (ERK1/2), mitoKATP, and nitric oxide synthase (NOS) pathways whereas adenosine receptor-mediated anti-apoptotic effect involved ERK1/2, protein kinase A (PKA), and NOS. CONCLUSIONS: These results suggest a complementary role of P2 and adenosine receptors in ATP-induced protective effects against hypoxia injury of endothelial. This could be considered therapeutic targets to limit the development of ischemic injury of organs such as heart, brain, and kidney.
Assuntos
Trifosfato de Adenosina/metabolismo , Apoptose , Células Endoteliais da Veia Umbilical Humana/metabolismo , Hipóxia/metabolismo , Receptores Purinérgicos P1/metabolismo , Receptores Purinérgicos P2/metabolismo , Adenosina/metabolismo , Apoptose/genética , Biomarcadores , Espaço Extracelular/metabolismo , Expressão Gênica , Humanos , Hipóxia/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Óxido Nítrico Sintase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , RNA Mensageiro/genética , Receptores Purinérgicos P1/genética , Receptores Purinérgicos P2/genética , Transdução de Sinais , Estresse Fisiológico/genéticaRESUMO
BACKGROUND: Cell therapy has been proposed as a salvage limb procedure in critical limb ischemia (CLI). In spite of the fact that clinical trials found some efficacy, the mechanism of action remains elusive. The objective of this study was to characterize two autologous cell therapy products (CTPs) obtained from patients with advanced peripheral arterial disease. METHODS: Bone marrow (BM-CTPs) (n = 20) and CTPs obtained by non-mobilized cytapheresis (peripheral blood [PB]-CTPs) (n = 20) were compared. CTPs were characterized by their cell composition, by the quantification of endothelial progenitor cells (EPCs) and mesenchymal stromal cells (MSCs) and by transcriptomic profiling. The angiogenic profile and the 6-month outcome of CLI patients are described. RESULTS: Patients presented inflammation syndrome and high levels of CXCL12, soluble stem cell factor and granulocyte colony-stimulating factor, whereas granulocyte macrophage colony-stimulating factor was low. Circulating CD34+ cells represented rare events. BM and PB-CTPs were heterogeneous. Mature cells and colony-forming unit-endothelial cells were in higher concentration in PB-CTPs, whereas CD34+ stem cells and EPCs were more abundant in BM-CTPs. MSCs were identified in both CTPs. Transcriptomic profiling revealed the strong angiogenic potential of BM-CTPs. Transcutaneous partial pressure of oxygen, C-reative protein and neutrophil content in CTPs are predictive of the clinical outcome at 6 months. DISCUSSION: Transcriptomic allows an accurate characterization of CTPs. BM-CTPs have the richest content in terms of stem cells and transcriptome. The high content of mature cells in PB-CTPs means that they work via a paracrine mechanism. The clinical outcome indicates the deleterious influence the patients' status and the limits of an autologous approach. In this respect, MSCs may allow an allogenic strategy.
Assuntos
Células da Medula Óssea/citologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Estado Terminal/terapia , Extremidades/irrigação sanguínea , Isquemia/terapia , Salvamento de Membro/métodos , Células-Tronco de Sangue Periférico/citologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Transplante de Medula Óssea , Citaferese/métodos , Feminino , Fator Estimulador de Colônias de Granulócitos/farmacologia , Humanos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Pessoa de Meia-Idade , Doença Arterial Periférica/terapia , Transplante de Células-Tronco de Sangue PeriféricoRESUMO
BACKGROUND: Cell therapy has been proposed as a salvage limb procedure in critical limb ischemia (CLI). Autologous cell therapy products (CTP) are obtained from patients with advanced peripheral arterial disease to be injected at the site of ischemia. Thrombogenicity of CTPs has not yet been assessed. The objectives were: 1) to assess thrombotic risk in candidates for cell therapy, 2) to evaluate two different CTPs in terms of thrombogenic potential, and 3) to evaluate clinical thrombotic events. STUDY DESIGN AND METHODS: In this ancillary study of a Phase I and II clinical trial, bone marrow (BM)-CTPs (n = 20) and CTPs obtained by cytapheresis (peripheral blood [PB]-CTPs; n = 20) were compared. Inflammatory and coagulation markers were measured at baseline and 24 hours after CTP implantation. CTP cell content and tissue factor (TF) expression (mRNA and protein) were analyzed. Thrombin generation assessed CTP-related thrombogenicity. RESULTS: All patients presented cardiovascular risk factors. At baseline, the patients' biologic profile was characterized by high levels of fibrinogen, C-reactive protein (CRP), D-dimer, interleukin (IL)-6, and plasmatic TF, whereas IL-10 was low. Although different in terms of cell composition, both BM- and PB-CTPs support low thrombin generation. Twenty-four hours after implantation, biologic markers remained stable in the PB-CTP group, except for IL-6. In the BM-CTP group, a significant increase of IL-6 but also of CRP and D-dimer was observed. Clinically, one single patient developed deep vein thrombosis 24 hours after the implantation of autologous PB-CTP. CONCLUSION: CTPs supported low thrombin generation and were well tolerated after calf implantation.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Isquemia/diagnóstico , Perna (Membro)/irrigação sanguínea , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Isquemia/terapia , Masculino , Pessoa de Meia-Idade , Fluxo Sanguíneo Regional/fisiologiaRESUMO
OBJECTIVE: Elastin is the major structural extracellular matrix component of the arterial wall that provides the elastic recoil properties and resilience essential for proper vascular function. Elastin-derived peptides (EDP) originating from elastin fragmentation during vascular remodeling have been shown to play an important role in cell physiology and development of cardiovascular diseases. However, their involvement in thrombosis has been unexplored to date. In this study, we investigated the effects of EDP on (1) platelet aggregation and related signaling and (2) thrombus formation. We also characterized the mechanism by which EDP regulate thrombosis. APPROACH AND RESULTS: We show that EDP, derived from organo-alkaline hydrolysate of bovine insoluble elastin (kappa-elastin), decrease human platelet aggregation in whole blood induced by weak and strong agonists, such as ADP, epinephrine, arachidonic acid, collagen, TRAP, and U46619. In a mouse whole blood perfusion assay over a collagen matrix, kappa-elastin and VGVAPG, the canonical peptide recognizing the elastin receptor complex, significantly decrease thrombus formation under arterial shear conditions. We confirmed these results in vivo by demonstrating that both kappa-elastin and VGVAPG significantly prolonged the time for complete arteriole occlusion in a mouse model of thrombosis and increased tail bleeding times. Finally, we demonstrate that the regulatory role of EDP on thrombosis relies on platelets that express a functional elastin receptor complex and on the ability of EDP to disrupt plasma von Willebrand factor interaction with collagen. CONCLUSIONS: These results highlight the complex nature of the mechanisms governing thrombus formation and reveal an unsuspected regulatory role for circulating EDP in thrombosis.
Assuntos
Elastina/fisiologia , Trombose/etiologia , Animais , Plaquetas/fisiologia , Catepsina A/sangue , Bovinos , Colágeno/sangue , Elastina/sangue , Elastina/química , Humanos , Camundongos , Neuraminidase/sangue , Oligopeptídeos/sangue , Oligopeptídeos/química , Oligopeptídeos/fisiologia , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/fisiologia , Agregação Plaquetária/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Proteólise , Receptores de Superfície Celular/sangue , Transdução de Sinais , Trombose/sangue , Remodelação Vascular/fisiologia , Fator de von Willebrand/metabolismoRESUMO
BACKGROUND: Several studies have reported the beneficial effects of anti-platelet drugs in cardioprotection against ischaemia-reperfusion injuries. To date, no studies have focused on the indirect cytoprotective effects of ticagrelor via adenosine receptor on the endothelium. METHOD: By evaluating cell viability and cleaved caspase 3 expression, we validated a model of endothelial cell apoptosis induced by hypoxia. In hypoxic endothelial cells treated with ticagrelor, we quantified the extracellular concentration of adenosine, and then we studied the involvement of adenosine pathways in the cytoprotective effect of ticagrelor. RESULTS: Our results showed that 10 µM ticagrelor induced an anti-apoptotic effect in our model associated with an increase of extracellular adenosine concentration. Similar experiments were conducted with cangrelor but did not demonstrate an anti-apoptotic effect. We also found that A2B and A3 adenosine receptors were involved in the anti-apoptotic effect of ticagrelor in endothelial cells exposed to 2 h of hypoxia stress. CONCLUSION: we described an endothelial cytoprotective mechanism of ticagrelor against hypoxia stress, independent of blood elements. We highlighted a mechanism triggered mainly by the increased extracellular bioavailability of adenosine, which activates A2B and A3 receptors on the endothelium.
Assuntos
Adenosina/metabolismo , Apoptose/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/patologia , Transdução de Sinais , Ticagrelor/farmacologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Biomarcadores/metabolismo , Hipóxia Celular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Óxido Nítrico Sintase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Purinérgicos P1/genética , Receptores Purinérgicos P1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacosRESUMO
Non-specific markers of inflammation such as C-reactive protein (CRP) are associated statistically with an increased risk of atherosclerosis through mechanisms that have not yet been fully elucidated. We investigated the effects of CRP on several aspects of human monocyte biology, a cell type involved in the initiation and progression of atherosclerosis. Blood monocytes isolated from healthy men and premenopausal women (n = 9/group) were exposed to purified CRP (25 microg/ml) for 12 hours. Changes in gene expression were analyzed using a custom-made array containing oligonucleotide sequences of 250 genes expressed by activated monocytes and confirmed by quantitative PCR. CRP increased significantly the expression of the cytokines interleukin (IL)-1alpha, IL-1beta and IL-6, and the chemokines GRO-alpha, GRO-beta and IL-8. CRP also displayed anti-inflammatory effects through upregulation of liver X receptor (LXR) alpha and activin receptor expression, and down-regulation of alpha 2-macroglobulin expression. Increased LXRalpha mRNA expression in both monocytes and the monocytic cell lineTHP-1 was associated with increased LXRalpha protein expression and nuclear translocation, as well as increased ABCA1 mRNA expression, a target gene of LXRalpha. Western blot analysis revealed CRP-induced nuclear translocation of NF-kappaB and activation of p42/44, MAP and Akt kinases. CRP-induced LXRalpha mRNA expression was inhibited by anti-CD64 (FcgammaRI) antibodies and by p42/44 and PI3 kinase inhibitors. This hypothesis-generating study demonstrates that CRP modulates the expression of genes that contribute to both pro- and anti-inflammatory responses in human monocytes. Among these novel anti-inflammatory effects, we show clearly that CRP activates the LXRalpha pathway.
Assuntos
Aterosclerose/metabolismo , Proteína C-Reativa/metabolismo , Citocinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Mediadores da Inflamação/metabolismo , Inflamação/metabolismo , Monócitos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Adulto , Aterosclerose/genética , Aterosclerose/prevenção & controle , Linhagem Celular Tumoral , Células Cultivadas , Citocinas/genética , Proteínas de Ligação a DNA/genética , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Inflamação/genética , Inflamação/prevenção & controle , Receptores de Lipopolissacarídeos/análise , Receptores X do Fígado , Masculino , Monócitos/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Receptores Nucleares Órfãos , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores Sexuais , Transdução de Sinais/genética , Fatores de Tempo , Regulação para CimaRESUMO
The lack of blood donations and the threat of infections from blood and blood products have led to extensive research into the development of blood substitutes. The latest generation of hemoglobin based oxygen carriers (HBOC) has been shown to induce side effects like hypertension, vasoconstriction, inflammation and oxidative stress. HBOC are able to restore volemia and transport oxygen after a hemorrhagic shock, the reperfusion leading to the restoration of the blood flow in vessels. We propose an innovative approach, more closely emulating clinical situations, to assess the impact of HBOC perfusion on endothelial cells (EC) in vitro. Through this approach we quantified levels of oxidative stress, vasoactive factors and inflammation. EC were cultivated under a laminar flow to reproduce the return of shear stress (SS) during the reperfusion. We showed that heme oxygenase I transcription correlated with changes in oxidatively modified heme and methemoglobin; all were lower under SS. SS induced increased nitric oxide production, which may have implications for the mechanism of in vivo vasoconstriction and hypertension. E-selectin changes under SS were greater than those of ICAM-1. Our results demonstrate how it is essential to include SS in assays attempting to understand the potential vascular side effects of HBOC perfusion.
Assuntos
Substitutos Sanguíneos/normas , Células Endoteliais/metabolismo , Hemoglobinas/metabolismo , Oxigênio/metabolismo , Estresse Mecânico , Células Cultivadas , Células Endoteliais/enzimologia , Regulação da Expressão Gênica , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Inflamação , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Metemoglobina/metabolismo , Oxirredução , Estresse Oxidativo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Selectinas/genética , Selectinas/metabolismo , Vasoconstritores/metabolismoRESUMO
OBJECTIVE: To examine differences in telomere (terminal restriction fragment [TRF]) length and pulse wave velocity (PWV)--an index of arterial stiffness--in patients with type 2 diabetes with and without microalbuminuria (MA). RESEARCH DESIGN AND METHODS: A total of 84 men with type 2 diabetes, 40 with MA and 44 without MA (aged 63.5 +/- 9.0 vs. 61.2 +/- 9.8 years), were studied. TRF length was determined in white blood cells. MA was defined as albumin excretion rate (AER) in the range of 30-300 mg/24 h in at least two of three 24-h urine collections. PWV was assessed using applanation tonometry. Markers of oxidative stress were also measured. RESULTS: TRF length was shorter in patients with MA than in those without MA (6.64 +/- 0.74 vs. 7.23 +/- 1.01 kb, respectively, P = 0.004). PWV was significantly higher in the patients with MA. Multivariate linear regression analysis in the total sample demonstrated an independent association between TRF length and age (P = 0.02), MA status (P = 0.04) or AER (P = 0.002), and plasma nitrotyrosine levels (P = 0.02). AER was associated significantly with PWV (P < 0.01). CONCLUSIONS: Subjects with type 2 diabetes and MA have shorter TRF length and increased arterial stiffness than those without MA. Additionally, TRF length is associated with age, AER, and nitrosative stress. As shorter TRF length indicates older biological age, the increased arterial stiffness in patients with type 2 diabetes who have MA may be due to the more pronounced "aging " of these subjects.