Thyroid hormones affect decidualization and angiogenesis in the decidua and metrial gland of rats / Hormônios tireoidianos afetam a decidualização e a angiogênese na decídua e glândula metrial de ratas

This study aimed to evaluate the effects of thyroid hormone on the decidua and metrial gland of rats and to examine the expression of angiogenic factors. 72 adult, female rats were divided into hypothyroid, T4-treated2, and control groups. At 10, 14 and 19 days of gestation (DG), the decidua and metrial gland were collected for histomorphometric and immunohistochemical evaluation of the expression of VEGF, Flk-1 and Tie-2. Hypothyroidism reduced the area of the decidua at 10 and 19 DG. Furthermore, VEGF was increased at 10 and 14 DG, and Flk-1 only at 14 DG, but both was reduced at 19 DG in the metrial gland without significantly changing the area occupied by blood vessels. Rats treated with T4 showed an increase in the decidua blood vessels at 10 and 19 DG. However, at 10 DG, excess T4 resulted in increased of Flk-1 in the decidua and metrial gland. Hypothyroidism increased the Tie-2 at 10 and 19 DG in the decidua and metrial gland. In conclusion, hypothyroidism reduces the area of the decidua and increases the expression of VEGF, Tie-2 and Flk-1. The excess of T4 promotes tissue angiogenesis by increasing the number of vessels in the decidua because of the increased expression of Flk-1.(AU)

Este estudo teve como objetivo avaliar os efeitos dos hormônios tireoidianos sobre a decídua e a glândula metrial pela análise da expressão de fatores angiogênicos em ratas. 72 ratas adultas, fêmeas foram distribuídas nos grupos hipotiroideo, tratado com T4 e controle. Aos 10, 14 e 19 dias de gestação (DG), a decídua e a glândula metrial foram coletadas para avaliação histomorfométrica e imunoistoquímica da expressão de VEGF, Flk-1 e Tie-2. O hipotireoidismo reduziu a área da decídua aos 10 e 19 DG. Além disso, o VEGF aumentou aos 10 e 14 DG e o Flk-1 apenas aos 14 DG, mas ambos foram reduzidos aos 19 DG na glândula metrial sem alterar significativamente a área ocupada pelos vasos sanguíneos. As ratas tratadas com T4 apresentaram aumento do número de vasos sanguíneos na decídua aos 10 e 19 DG. Além disso, aos 10 DG, o excesso de T4 resultou no aumento de Flk-1 na decídua e na glândula metrial. O hipotireoidismo aumentou o Tie-2 em 10 e 19 DG na decídua e na glândula metrial. Desta forma, pode-se concluir que o hipotireoidismo reduz a área da decídua e aumenta a expressão de VEGF, Tie-2 e Flk-1. O excesso de T4 promove a angiogênese tecidual ao aumentar o número de vasos na decídua devido ao aumento da expressão de Flk-1.(AU)

Involvement of mast cells, CD68+ and VEGF+ expressions in response to Himatanthus drasticus commercial latex in mice wound healing model / Envolvimento de mastócitos, expressão de CD68+ e VEGF+ em resposta ao látex comercial de Himatanthus drasticus em modelo de cicatrização em camundongos

This study aimed to evaluate Himatanthus drasticus latex in a mice wound healing experimental model. Animals were divided into four groups (n=7) according to the treatments: GI - saline 0.9% (control), GII - mineral oil (vehicle), GIII - H. drasticus commercial latex (HdCL) and GIV - H. drasticus mixed isolated fraction (MIF, 1 mg/mL). The treatments were applied topically once daily, 50 µL for 14 consecutive days. Macroscopic lesions were evaluated, considering parameters such as swelling, redness, granulation tissue and reepithelialization. VEGF+, CD68+ expressions and mast cells (Toluidin blue stain) were evaluated. HdCL induced higher contraction and exuberant granulation tissue (P > 0.05). HdCL showed a mild inflammatory process while MIF induced intense infiltrate inflammatory predominantly by lymphocytes, vascular congestion, bleeding and did not presented full reepithelialization. Reorganization of collagen fibers (red picrosirius stain) was observed. CD68+ expression and mast cells were presented as moderate, intense and mild in GI, GIII and GIV, respectively. Neovascularization occurred in all groups, while VEGF+ expression was intense in MIF in relation to HdCL. We concluded that HdCL presents wound healing potential, through modulation of mast cells, CD68+ and VEGF+ expressions that can be associated to triterpenes presence according MIF isolated from HdCL.(AU)

Objetivou-se avaliar o látex de Himatanthus drasticus em feridas induzidas experimentalmente em camundongos. Os animais foram divididos em quatro grupos (n=7): GI - salina 0,9% (controle), GII - óleo mineral (veículo), GIII - látex comercial de H. drasticus (HdCL) e GIV - fração isolada mista de H. drasticus (MIF, 1mg/mL). Os tratamentos foram aplicados topicamente uma vez ao dia (50µL), durante 14 dias consecutivos. Lesões macroscópicas, as expressões de VEGF+, CD68+ e a participação dos mastócitos (coloração azul de toluidina) foram avaliadas. HdCL induziu maior contração e tecido de granulação exuberante (P >0,05). HdCL induziu leve processo inflamatório enquanto MIF promoveu intenso infiltrado inflamatório predominantemente linfocítico, congestão vascular, hemorragia e reepitelização parcial. Observou-se reorganização das fibras colágenas (coloração picrosírius). A expressão de CD68+ e os mastócitos apresentaram-se moderados, intensos e leves em GI, GIII e GIV, respectivamente. A neovascularização foi observada em todos os grupos, enquanto a expressão de VEGF+ foi mais intensa em MIF em relação a HdCL. Conclui-se que HdCL apresenta potencial de cicatrização por meio da modulação dos mastócitos e das expressões de CD68+ e VEGF+, o que pode estar associado à presença de triterpenos de acordo com MIF isolada de HdCL.(AU)

Serum and plasma vascular endothelial growth factor in healthy dogs / Fator de crescimento do endotélio vascular no soro e plasma de cães saudáveis

Vascular endothelial growth factor (VEGF) is an angiogenic factor with a key role in physiological and pathological process. It can be measured in several organic fluids, including serum and plasma samples. The aim of this work was to investigate the concentration of serum and plasma VEGF of healthy dogs in order to recommend optimal handling of biological samples for accurate measurement of VEGF. Blood samples of thirty dogs were collected into sterile EDTA tube for plasma analysis and into clot activator tubes for serum analysis. The tubes were centrifuged within 90 minutes of collection at 1400 xg for 10 minutes. VEGF concentration was determined using the quantitative method (ELISA). Serum VEGF level was 26.5 + 13.3pg/mL and plasma VEGF was 11.7 + 16.4 pg/mL (p = 0.0003). There was a positive correlation between serum VEGF and platelets (r = 0.37, p = 0.03) and a negative correlation between serum VEGF and hemoglobin (r = -0.38, p = 0.03) and between plasma VEGF and  hemoglobin (r = -0.34, p = 0.06). When compared with serum samples it was concluded that plasma samples could be used as an optimal fluid for measuring VEGF in dogs.

O fator de crescimento do endotélio vascular (VEGF) é um fator angiogênico com papel importante em processos patológicos e fisiológicos. O VEGF pode ser quantificado em diversos fluidos orgânicos, incluindo amostras de soro e plasma. O presente trabalho investigou a concentração do VEGF no soro e no plasma de cães saudáveis a fim de recomendar o manejo ótimo de amostras biológicas para a determinação dos níveis do VEGF. Amostras de sangue de 30 cães saudáveis foram coletadas em tubos estéreis contendo EDTA para análise do plasma e em tubos com ativador de coagulação para análise do soro. Os tubos foram centrifugados após 90 minutos da coleta a 1.400 x g por 10 minutos. A concentração do VEGF foi determinada com o método quantitativo de ELISA. O nível sérico médio de VEGF foi de 26,5+13,3pg/mL e o plasmático foi 11,7 + 16.4 pg/mL (p = 0,0003). Houve correlação positiva entre o VEGF do soro com as plaquetas (r = 0,37, p = 0,03) e correlação negativa entre o VEGF do soro com hemoglobina (r = -0,38, p = 0,03) e entre VEGF do plasma com hemoglobina (r = -0,34, p = 0,06). A comparação dos resultado obtidos nos exames de plasma e soro indicou que amostras de plasma podem ser utilizadas como ótimo fluido para quantificação do VEGF de cães.

Isolation & characterization of angiogenic factors from cultured cells & conditioned media.

Angiogenic factors were isolated by ion exchange chromatography from three established cell lines viz., HEp 2, HeLa and CHO, a primary culture of mouse mammary adenocarcinoma and from conditioned media of HEp2 and cultured mammary adenocarcinoma cells. The angiogenic activity was assayed by chicken chorioallantoic membrane assay. The angiogenic factor eluted at 0.5 M, exhibited lambda max at 258 +/- 1 nm, contained protein and nucleic acid. The cellular angiogenic factor showed a ratio of 1:1 for protein and nucleic acid whereas the secreted angiogenic factor had 3-5 parts of protein to 1 part of nucleic acid. The angiogenic factor from HEp2 and CHO cells did not bind to heparin-agarose. Microheterogeneity of the angiogenic factors was established by SDS-PAGE. Antiserum raised against the cellular angiogenic factor from HEp2 cells, showed a titre of 1:1600 by ELISA. The angiogenic factor was directly localized on whole cells by ELISA. Cellular as well as secreted angiogenic factors crossreacted with the antibody. Neutralizing effect of the antiserum on induction of angiogenesis was detected on chicken chorioallantoic membrane.

Isolation & characterization of tumour angiogenesis factor from solid tumours & body fluids from cancer patients.

Tumour angiogenesis factor (TAF) was isolated from malignant solid tumours (10) and from pleural and peritoneal fluids (10) collected from cancer patients. Normal tissues and body fluids from individuals with no clinical history of cancer did not show any detectable levels of TAF. Also, no angiogenic activity was detectable in the benign tumour samples studied (2). The TAFs isolated were all ribonucleoproteins. Molecular weight determination by SDS-PAGE (9%) of the TAFs isolated by DEAE cellulose chromatography of tumour extracts showed them to be 18,000 dalton (D) ribonucleoproteins, while the TAFs isolated by immunoaffinity chromatography (using immobilized anti-TAF IgG) of solid tumour extracts and body fluids had a molecular weight of 38,000 D. The TAFs isolated by both the methods were found to be angiogenic by the chick chorioallantoic membrane and the mouse intradermal assays. Immunoaffinity chromatography could be used for the one-step purification of TAF from solid tumour extracts as well as from body fluids.