Cytoprotective role of heme oxygenase-1 and heme degradation derived end products in liver injury

The activation of heme oxygenase-1 (HO-1) appears to be an endogenous defensive mechanism used by cells to reduce inflammation and tissue damage in a number of injury models. HO-1, a stress-responsive enzyme that catabolizes heme into carbon monoxide (CO), biliverdin and iron, has previously been shown to protect grafts from ischemia/reperfusion and rejection. In addition, the products of the HO-catalyzed reaction, particularly CO and biliverdin/bilirubin, have been shown to exert protective effects in the liver against a number of stimuli, as in chronic hepatitis C and in transplanted liver grafts. Furthermore, the induction of HO-1 expression can protect the liver against damage caused by a number of chemical compounds. More specifically, the CO derived from HO-1-mediated heme catabolism has been shown to be involved in the regulation of inflammation; furthermore, administration of low concentrations of exogenous CO has a protective effect against inflammation. Both murine and human HO-1 deficiencies have systemic manifestations associated with iron metabolism, such as hepatic overload (with signs of a chronic hepatitis) and iron deficiency anemia (with paradoxical increased levels of ferritin). Hypoxia induces HO-1 expression in multiple rodent, bovine and monkey cell lines, but interestingly, hypoxia represses expression of the human HO-1 gene in a variety of human cell types (endothelial cells, epithelial cells, T cells). These data suggest that HO-1 and CO are promising novel therapeutic molecules for patients with inflammatory diseases. In this review, we present what is currently known regarding the role of HO-1 in liver injuries and in particular, we focus on the implications of targeted induction of HO-1 as a potential therapeutic strategy to protect the liver against chemically induced injury.FAPESP (Fundação de Apoio à Pesquisa do Estado de São Paulo), 07/07139-3FAPESP (Fundação de Apoio à Pesquisa do Estado de São Paulo), 10/02024-6CNP

In search of mechanisms associated with mesenchymal stem cell-based therapies for acute kidney injury

Acute kidney injury (AKI) is classically described as a rapid loss of kidney function. AKI affects more than 15% of all hospital admissions and is associated with elevated mortality rates. Although many advances have occurred, intermittent or continuous renal replacement therapies are still considered the best options for reversing mild and severe AKI syndrome. For this reason, it is essential that innovative and effective therapies, without side effects and complications, be developed to treat AKI and the end-stages of renal disease. Mesenchymal stem cell (MSC) based therapies have numerous advantages in helping to repair inflamed and damaged tissues and are being considered as a new alternative for treating kidney injuries. Numerous experimental models have shown that MSCs can act via differentiation-independent mechanisms to help renal recovery. Essentially, MSCs can secrete a pool of cytokines, growth factors and chemokines, express enzymes, interact via cell-to-cell contacts and release bioagents such as microvesicles to orchestrate renal protection. In this review, we propose seven distinct properties of MSCs which explain how renoprotection may be conferred: 1) anti-inflammatory; 2) pro-angiogenic; 3) stimulation of endogenous progenitor cells; 4) anti-apoptotic; 5) anti-fibrotic; 6) anti-oxidant; and 7) promotion of cellular reprogramming. In this context, these mechanisms, either individually or synergically, could induce renal protection and functional recovery. This review summarises the most important effects and benefits associated with MSC-based therapies in experimental renal disease models and attempts to clarify the mechanisms behind the MSC-related renoprotection. MSCs may prove to be an effective, innovative and affordable treatment for moderate and severe AKI. However, more studies need to be performed to provide a more comprehensive global understanding of MSC-related therapies and to ensure their safety for future clinical applications

Hydrogen peroxide (H2O2) induces leukemic but not normal hematopoietic cell death in a dose-dependent manner

Over the last few years, studies have suggested that oxidative stress plays a role in the regulation of hematopoietic cell homeostasis. in particular, the effects of hydrogen peroxide (H2O2) range from hematopoietic cell proliferation to cell death, depending on its concentration in the intracellular milieu. in this work, we evaluated the effects of an oxidative environment on normal and leukemic hematopoietic cells by stimulating normal human (umbilical cord blood) and murine (bone marrow) hematopoietic cells, as well as human myeloid leukemic cells (HL-60 lineage), upon H2O2 stimulus. Total cell populations and primitive subsets were evaluated for each cell type. H2O2 stimulus induces HL-60 cell death, whereas the viability of human and murine normal cells was not affected. the effects of H2O2 stimulus on hematopoietic stem/progenitor cell subsets were examined and the normal primitive cells were found to be unaffected; however, the percentage of leukemic stem cells (LSC) increased in response to H2O2, while clonogenic ability of these cells to generate myeloid clones was inhibited. in addition, H2O2 stimulus caused a decrease in the levels of p-AKT in HL-60 cells, which most likely mediates the observed decrease of viability. in summary, we found that at low concentrations, H2O2 preferentially affects both the LSC subset and total HL-60 cells without damage normal cells.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Dept Biophys, BR-04062023 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, BR-04062023 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Med, BR-04062023 São Paulo, BrazilIPCTRON Stem Cell Res Inst, BR-04037000 São Paulo, BrazilUniversidade Federal de São Paulo, BR-04039032 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, BR-04062023 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, BR-04062023 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Med, BR-04062023 São Paulo, BrazilUniversidade Federal de São Paulo, BR-04039032 São Paulo, BrazilFAPESP: 2009/52852-5Web of Scienc

Balance between the two kinin receptors in the progression of experimental focal and segmental glomerulosclerosis in mice

Focal and segmental glomerulosclerosis (FSGS) is one of the most important renal diseases related to end-stage renal failure. Bradykinin has been implicated in the pathogenesis of renal inflammation, whereas the role of its receptor 2 (B2RBK; also known as BDKRB2) in FSGS has not been studied. FSGS was induced in wild-type and B2RBK-knockout mice by a single intravenous injection of Adriamycin (ADM). in order to further modulate the kinin receptors, the animals were also treated with the B2RBK antagonist HOE-140 and the B1RBK antagonist DALBK. Here, we show that the blockage of B2RBK with HOE-140 protects mice from the development of FSGS, including podocyte foot process effacement and the re-establishment of slit-diaphragm-related proteins. However, B2RBK-knockout mice were not protected from FSGS. These opposite results were due to B1RBK expression. B1RBK was upregulated after the injection of ADM and this upregulation was exacerbated in B2RBK-knockout animals. Furthermore, treatment with HOE-140 downregulated the B1RBK receptor. the blockage of B1RBK in B2RBK-knockout animals promoted FSGS regression, with a less-inflammatory phenotype. These results indicate a deleterious role of both kinin receptors in an FSGS model and suggest a possible cross-talk between them in the progression of disease.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Clin & Expt Immunol Lab, Div Nephrol, BR-04023900 São Paulo, BrazilUniv São Paulo, Inst Biomed Sci 4, Dept Immunol, Lab Transplantat Immunobiol, BR-05508000 São Paulo, BrazilUniversidade Federal de São Paulo, Translat Med Div, Clin & Expt Immunol Lab, BR-04039002 São Paulo, BrazilInst Butantan, Lab Cellular Biol, BR-05503900 São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Biophys, BR-04023062 São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Microbiol Immunol & Parasitol, BR-04023062 São Paulo, BrazilINSERM, Unite Mixte Rech 699, F-75870 Paris, FranceAlbert Einstein Hosp, Inst Israelita Ensino & Pesquisa Albert Einst, Renal Transplantat Unit, BR-05521000 São Paulo, BrazilUniversidade Federal de São Paulo, Clin & Expt Immunol Lab, Div Nephrol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Translat Med Div, Clin & Expt Immunol Lab, BR-04039002 São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Biophys, BR-04023062 São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Microbiol Immunol & Parasitol, BR-04023062 São Paulo, BrazilFAPESP: 2012/05605-5FAPESP: 07/07139-3FAPESP: 12/02270-2CNPq: 140739/2008-4Web of Scienc

Identification and quantification of plasma heparanase isoforms in gastrointestinal carcinoma patients

Heparanase-1 (HPA1) is an endo-β-glucuronidase that degrades intradisaccharides glycosidic linkage between hexosamine and glucuronic acid into heparan sulfate chains, present on extracellular matrix and surface proteoglycans. HPA1 gene is located at 4q21.3 chromossome. Oligosaccharides generated by HPA1 are involved in cell proliferation, angiogenesis and cell diferentiation related with tumor development and metastasis. Heparanase-2 (HPA2) is encoded by 10q23-24 chromossome. There are three HPA2 isoforms containing 480, 534 and 592 aminoacids. All HPA2 isoforms have shown that they are intracellular, membrane associated proteins, containing C-terminal domain to the cytoplasm and do not present enzymatic activity. The objective of the present study is to identify and quantify HPA1 (isoforms 50 kDa and 65 kDa), and HPA2 isoforms expression in the plasma of gastrointestinal carcinoma patients, compared with individuals that do not present neoplasia (control group). Plasmatic proteins were identified by polyacrilamide gel electrophoresis (10% SDS-PAGE) and transferred to Hybond-CE membrane, incubated with primary anti-HPA1 H-80 or anti-HPA2 C-17 and developed using secondary antibody conjugated with HRP IgG peroxidase. Heparanases isoforms were quantified by densitometry (Scion Image) and confirmed by real time RT-PCR. Statistic analysis were performed using SPSS 13.0 program (SPSS, Chicago, IL). The results have shown that both HPA1 isoforms (HPA1 50 kDa and HPA1 65 kDa) and HPA2 isoforms were significantly increased in gastrointestinal carcinoma patients plasma, compared with control group. Real time analysis of HPA1 and HPA2 expression in the mononuclear fraction of blood demonstrated an increase HPA1 and HPA2 expression in gastrointestinal carcinoma patients that corroborates with plasma analysis. The obtained results demonstrated that both HPA1 and HPA2 have a fundamental role in gastrointestinal carcinogenesis and, therefore, to understand their physiological function it could help the development of possible new antitumor therapies.A heparanase-1 (HPA1) é uma endo-β-glucuronidase que degrada ligações glicosídicas intrassacarídicas entre a hexosamina e ácido glucurônico de cadeias de heparam sulfato, constituintes dos proteoglicanos de matriz extracelular e superfície celular. Seu gene encontra-se localizado no cromossomo 4 humano (4q21,3). A HPA1 gera oligossacarídeos que estão envolvidos na proliferação celular, angiogênese e diferenciação celular relacionados com o desenvolvimento de tumores e metástases. A heparanase-2 (HPA2) é codificada pelo cromossomo 10q23-24. Existem três isoformas da HPA2 de 480, 534 e 592 aminoácidos. As análises dessas proteínas evidenciam que todas essas isoformas são proteínas intracelulares, associadas à membrana, contendo a porção C-terminal voltada para o citoplasma e não apresentam atividade enzimática. O presente estudo tem por objetivo identificar e quantificar a expressão de HPA1 (isoformas 50kDa e 65 kDa), e isoformas da HPA2 no plasma de pacientes com carcinoma gastrointestinal, comparando-se com a expressão em indivíduos não acometidos por neoplasia (grupo controle). As proteínas plasmáticas foram identificadas por eletroforese em gel de poliacrilamida (SDS-PAGE 10%) e transferidas para membrana Hybond-CE, que foi incubada com anticorpo primário anti-HPA1 H-80 ou anti-HPA2 C-17 e revelada com anticorpo secundário conjugado com HRP IgG peroxidase. Isoformas das heparanases foram quantificadas por densitometria (Scion Image) e confirmada por RT-PCR em tempo real. Análises estatística foram realizadas utilizando o programa SPSS 13.0 Windows (SPSS, Chicago, IL). Os resultados demosntraram que ambas isoformas da HPA1 (HPA1 50 kDa e HPA1 65 kDa) e isoformas de HPA2 encontram-se significativamente aumentadas no plasma de pacientes com carcinoma gastrointestinal, comparando-se com o grupo controle. As análises em tempo real da expressão de HPA1 e HPA2 na fração mononuclear do sangue evidenciaram aumento de expressão de HPA1 e HPA2 em pacientes com carcinoma gastrointestinal corroborando com as análises do plasma. Os resultados obtidos demonstram que tanto HPA1 como HPA2 desempenham papel fundamental na carcinogênese gastrointestinal e, portanto, entender o papel fisiológico que desempenham possa fornecer informações para desenvolvimento de possivel terapia anti-tumoral.TEDEBV UNIFESP: Teses e dissertaçõe

Regression of albuminuria and hypertension and arrest of severe renal injury by a losartan-hydrochlorothiazide association in a model of very advanced nephropathy.

Treatments that effectively prevent chronic kidney disease (CKD) when initiated early often yield disappointing results when started at more advanced phases. We examined the long-term evolution of renal injury in the 5/6 nephrectomy model (Nx) and the effect of an association between an AT-1 receptor blocker, losartan (L), and hydrochlorothiazide (H), shown previously to be effective when started one month after Nx. Adult male Munich-Wistar rats underwent Nx, being divided into four groups: Nx+V, no treatment; Nx+L, receiving L monotherapy; Nx+LH, receiving the L+H association (LH), and Nx+AHHz, treated with the calcium channel blocker, amlodipine, the vascular relaxant, hydralazine, and H. This latter group served to assess the effect of lowering blood pressure (BP). Rats undergoing sham nephrectomy (S) were also studied. In a first protocol, treatments were initiated 60 days after Nx, when CKD is at a relatively early stage. In a second protocol, treatments were started 120 days after Nx, when glomerulosclerosis and interstitial fibrosis are already advanced. In both protocols, L treatment promoted only partial renoprotection, whereas LH brought BP, albuminuria, tubulointerstitial cell proliferation and plasma aldosterone below pretreatment levels, and completely detained progression of renal injury. Despite normalizing BP, the AHHz association failed to prevent renal damage, indicating that the renoprotective effect of LH was not due to a systemic hemodynamic action. These findings are inconsistent with the contention that thiazides are innocuous in advanced CKD. In Nx, LH promotes effective renoprotection even at advanced stages by mechanisms that may involve anti-inflammatory and intrarenal hemodynamic effects, but seem not to require BP normalization

Clinical characteristics and frequency of TLR4 polymorphisms in Brazilian patients with ankylosing spondylitis

ABSTRACT Objectives: Innate immunity is involved in the physiopathology of ankylosing spondylitis (AS), with the participation of Gram-negative bacteria, modulation of human leukocyte antigen (HLA) B27 and the involvement of pattern recognition receptors, such as Toll-like receptors (TLRs). The aim of this study was to investigate the clinical characteristics and frequency of TLR4 polymorphisms (Asp299Gly and Thr 399Ile) in a cohort of Brazilian patients with AS. Methods: A cross-sectional study was carried out involving 200 patients with a diagnosis of AS and a healthy control group of 200 individuals. Disease activity, severity and functional capacity were measured. The study of TLR4 polymorphisms was performed using the restriction fragment length polymorphism method. HLA-B27 was analyzed by conventional polymerase chain reaction. The IBM SPSS Statistics 20 program was used for the statistical analysis, with p-values less than 0.05 considered significant. Results: Mean age and disease duration were 43.1 ± 12.7 and 16.6 ± 9.2 years, respectively. The sample was predominantly male (71%) and non-Caucasian (52%). A total of 66% of the group of patients were positive for HLA-B27. The sample of patients was characterized by moderate functional impairment and a high degree of disease activity. No significant association was found between the two TLR4 polymorphisms and susceptibility to AS. Conclusions: TLR4 polymorphisms 399 and 299 were not more frequent in patients with AS in comparison to the health controls and none of the clinical variables were associated with these polymorphisms

Endothelial Cells Tissue-Specific Origins Affects Their Responsiveness to TGF-β2 during Endothelial-to-Mesenchymal Transition

The endothelial-to-mesenchymal transition (EndMT) is a biological process where endothelial cells (ECs) acquire a fibroblastic phenotype after concomitant loss of the apical-basal polarity and intercellular junction proteins. This process is critical to embryonic development and is involved in diseases such as fibrosis and tumor progression. The signaling pathway of the transforming growth factor β (TGF-β) is an important molecular route responsible for EndMT activation. However, it is unclear whether the anatomic location of endothelial cells influences the activation of molecular pathways responsible for EndMT induction. Our study investigated the molecular mechanisms and signaling pathways involved in EndMT induced by TGF-β2 in macrovascular ECs obtained from different sources. For this purpose, we used four types of endothelial cells (coronary artery endothelial cells, CAECs; primary aortic endothelial cells PAECs; human umbilical vein endothelia cells, HUVECs; and human pulmonary artery endothelial cells, HPAECs) and stimulated with 10 ng/mL of TGF-β2. We observed that among the ECs analyzed in this study, PAECs showed the best response to the TGF-β2 treatment, displaying phenotypic changes such as loss of endothelial marker and acquisition of mesenchymal markers, which are consistent with the EndMT activation. Moreover, the PAECs phenotypic transition was probably triggered by the extracellular signal⁻regulated kinases 1/2 (ERK1/2) signaling pathway activation. Therefore, the anatomical origin of ECs influences their ability to undergo EndMT and the selective inhibition of the ERK pathway may suppress or reverse the progression of diseases caused or aggravated by the involvement EndMT activation

Clinical characteristics and frequency of TLR4 polymorphisms in Brazilian patients with ankylosing spondylitis

ABSTRACT Objectives: Innate immunity is involved in the physiopathology of ankylosing spondylitis (AS), with the participation of Gram-negative bacteria, modulation of human leukocyte antigen (HLA) B27 and the involvement of pattern recognition receptors, such as Toll-like receptors (TLRs). The aim of this study was to investigate the clinical characteristics and frequency of TLR4 polymorphisms (Asp299Gly and Thr 399Ile) in a cohort of Brazilian patients with AS. Methods: A cross-sectional study was carried out involving 200 patients with a diagnosis of AS and a healthy control group of 200 individuals. Disease activity, severity and functional capacity were measured. The study of TLR4 polymorphisms was performed using the restriction fragment length polymorphism method. HLA-B27 was analyzed by conventional polymerase chain reaction. The IBM SPSS Statistics 20 program was used for the statistical analysis, with p-values less than 0.05 considered significant. Results: Mean age and disease duration were 43.1 ± 12.7 and 16.6 ± 9.2 years, respectively. The sample was predominantly male (71%) and non-Caucasian (52%). A total of 66% of the group of patients were positive for HLA-B27. The sample of patients was characterized by moderate functional impairment and a high degree of disease activity. No significant association was found between the two TLR4 polymorphisms and susceptibility to AS. Conclusions: TLR4 polymorphisms 399 and 299 were not more frequent in patients with AS in comparison to the health controls and none of the clinical variables were associated with these polymorphisms