Estrogen depletion modulates aortic prothrombotic signaling in normotensive and spontaneously hypertensive female rats.

AIM: In this study, we investigated how platelets and aorta contribute to the creation and maintenance of a prothrombotic state in an experimental model of postmenopausal hypertension in ovariectomized rats. METHODS: Bilateral ovariectomy was performed in both 14-week-old female spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. The animals were kept in phytoestrogen free diet. Vascular parameters, platelet, coagulation and aortic prothrombotic functions and mechanisms were assessed. RESULTS: Exacerbated platelet aggregation was observed in both SHR and WKY animals after ovariectomy. The mechanism was related to aortic COX2 downregulation and reduction in AMP, ADP, and ATP hydrolysis in serum and platelets. A procoagulant potential was observed in plasma from ovariectomized rats and this was confirmed by kallikrein and factor Xa generation in aortic rings. Aortic rings derived from ovariectomized SHR presented a greater thrombin generation capacity compared to equivalent rings from WKY animals. The mechanism involved tissue factor and PAR-1 upregulation as well as an increase in extrinsic coagulation and fibrinolysis markers in aorta and platelets. Aortic smooth muscle cells pre-treated with a plasma pool derived from estrogen-depleted animals developed a procoagulant profile with tissue factor upregulation. This procoagulant profile was dependent on inflammatory signalling, since NFκB inhibition attenuated the procoagulant activity and tissue factor expression. CONCLUSIONS: A prothrombotic phenotype was observed in both WKY and SHR ovariectomized rats being associated with platelet hyperreactivity and tissue factor upregulation in aorta and platelets. The mechanism involves proinflammatory signalling that supports greater thrombin generation in aorta and vascular smooth muscle cells.

Urine proteomic analysis reveals alterations in heme/hemoglobin and aminopeptidase metabolism during Lonomia obliqua venom-induced acute kidney injury.

AIMS: Accidental contact with the Lonomia obliqua caterpillar is a common event in southern Brazil. Envenomed victims present consumption coagulopathy, which can evolve to acute kidney injury (AKI). In the present study, we searched for AKI biomarkers and changes in molecular pathway signatures through urine proteomic analysis. METHODOLOGY: Male Wistar rats were injected with L. obliqua venom (1.5 mg/kg, via s.c.) or 0.9 % NaCl and distributed into metabolic cages. After 24 h, urine was obtained, and the set of differentially regulated proteins was analyzed by MudPIT technology in an OrbiTRAP mass spectrometer. RESULTS: L. obliqua venom leads to an increase in urine output and water and electrolyte excretion and to an increase in the albumin to creatine ratio in urine. The proteomic analysis revealed an up-regulation of tubular injury biomarkers, such as neutrophil-gelatinase associated lipocalin (NGAL) and cystatin C, in urine from envenomed rats. Several components related to the heme scavenging system were up-regulated or exclusively identified in urine from envenomed animals. There was an increase in urinary heme levels and hemoglobin subunits, hemopexin, haptoglobin, and biliverdin reductase. Similarly, kinin- and angiotensin-generating/degrading peptidases, such as kallikreins, neprilysin, plasmin, dipeptidyl peptidase IV, cathepsin D, kininogen, and neutral, basic, glutamyl, and acidic aminopeptidases, were also up-regulated in urine. CONCLUSIONS: L. obliqua envenomation induced tubular and glomerular injury, probably involving heme/hemoglobin toxicity and an imbalance in the kinin/angiotensin generating/degrading system.

Association of polypropylene scaffolds and mesenchymal stem cells for use in tissue engineering / Associação de arcabouço de polipropileno e células tronco mesenquimais para uso em engenharia de tecido

Tissue engineering replaces injured tissues by manipulating cells, making scaffolds, and using molecules that stimulate the tissue. Mesenchymal stem cells (MSCs) are good candidates for tissue engineering, as this is one of the cell types which are recruited to repair injured tissues. Scaffolds are structural devices that allow cell fixation and migration, with polypropylene meshes being an example. This study aims to evaluate the culture of adipose tissue-derived mesenchymal stem cells (ADSCs), isolated from C57Bl/6 GFP + mice, in two types of polypropylene meshes (macroporous and microporous) in conventional culture plates and plates coated with methacrylate, over a period of fifteen days. The objective was to obtain the best interaction protocol between the mesh and the cells. The choice of the best method was based on adherence, maintenance of adherence and viability during culture. The amount of ADSCs adhering was checked daily by counting in a Neubauer Chamber and by using a growth curve performed with the MTT assay. The ADSCs adhering to the meshes were visualized with DAPI, panotic, hematoxylin and eosin, immunohistochemistry (integrin), and immunofluorescence (actin). ADSCs adhere to all forms of culture and to the two types of polypropylene mesh. ADSCs adhered more to the microporous mesh, within the seven day period of culture and in the plates without methacrylate. Thus, polypropylene meshes offer a good scaffold for ADSCs to adhere to.

A engenharia de tecidos substitui tecidos danificados com a manipulação de células, confecção de arcabouços e a utilização de moléculas que estimulem o tecido. As células-tronco mesenquimais (MSCs) são boas candidatas para engenharia de tecido, pois são um dos tipos celulares recrutadas para a reparação de tecidos lesionados. O arcabouço deve ser um dispositivo estrutural que forneça uma estrutura para o crescimento e a diferenciação celular no sítio, sendo a tela de polipropileno um exemplo. O objetivo deste estudo foi avaliar o cultivo de células-tronco mesenquimais de tecido de adiposo (ADSCs), isoladas de camundongos C57Bl/6 GFP+, em dois tipos de telas de polipropileno (macroporosa e microporosa) em placas de cultura convencionais e revestidas com metacrilato, durante quinze dias, para obter o melhor protocolo de interação entre a tela e as células. A escolha do melhor método foi baseada na adesão, manutenção da adesão e viabilidade durante cultivo. A quantidade de ADSCs aderidas foi verificada diariamente em contagem em Câmara de Neubauer e através de uma curva de crescimento realizada através de ensaio de MTT. As ADSCs aderidas nas telas foram visualizadas com a marcação de DAPI, panótico, hematoxilina e eosina, imumo-histoquímica (integrina) e imunofluorescência (actina). Nas duas formas de cultivo e nos dois tipos de telas de polipropileno houve aderência das ADSCs. Houve maior aderência na tela microporosa, no período de sete dias de cultivo e em placas sem metacrilato. Conclui-se que a tela de polipropileno oferece um bom arcabouço para as ADSCs se aderirem.

Associação de arcabouço de polipropileno e células tronco mesenqimais para uso em engenharia de tecido / Association of polypropylene scaffolds and mesenchymal stem cells for use in tissue engineering

A engenharia de tecidos tem como objetivo substituir tecidos danificados, manipulando células, confecção de arcabouços e a utilização de moléculas que estimulem o tecido. A proposta deste estudo foi avaliar duas técnicas de cultivo de células-tronco mesenquimais (MSC) em diferentes placas de cultura, utilizando dois tipos de telas de polipropileno (macroporoso e microporoso), para obter as melhores condições de interação entre a tela e as células, e definir uma proposta de protético para engenharia de tecidos. As telas de polipropileno foram cultivadas com células-tronco mesenquimais de tecido adiposo (ADSCs) isoladas de camundongos C57B1/6 GFP+ durante quinze dias em placas revestidas com metacrilato ou não revestidas com metacrilato. A quantidade de ADSCs aderidas foram verificadas diariamente em Câmara de Neubauer e através de uma curva de crescimento realizada pelo ensaio MTT. As ADSCs aderidas às malhas foram visualizadas com marcação de DAPI, panóticas, hematoxilina e eosina imuno-histoquímica e imunofluorescência. O melhor protocolo foi na tela microporosa, no o período de sete dias de cultivo e em placas sem metacrilato. Conclui-se que a tela de polipropileno fornece um bom suporte para as ADSCs se aderirem podendo ser utilizada na engenharia de tecidos.

Tissue engineering replaces injured tissues by manipulating cells, making scaffolds, and using molecules that stimulate the tissue. Mesenchymal stem cells (MSCs) are good candidates for tissue engineering, as this is one of the cell types which are recruited to repair injured tissues. Scaffolds are structural devices that allow cell fixation and migration, with polypropylene meshes being an example. This study aims to evaluate the culture of adipose tissue-derived mesenchymal stem cells (ADSCs), isolated from C57Bl/6 GFP + mice, in two types of polypropylene meshes (macroporous and microporous) in conventional culture plates and plates coated with methacrylate, over a period of fifteen days. The objective was to obtain the best interaction protocol between the mesh and the cells. The choice of the best method was based on adherence, maintenance of adherence and viability during culture. The amount of ADSCs adhering was checked daily by counting in a Neubauer Chamber and by using a growth curve performed with the MTT assay. The ADSCs adhering to the meshes were visualized with DAPI, panotic, hematoxylin and eosin, immunohistochemistry (integrin), and immunofluorescence (actin). ADSCs adhere to all forms of culture and to the two types of polypropylene mesh. ADSCs adhered more to the microporous mesh, within the seven day period of culture and in the plates without methacrylate. Thus, polypropylene meshes offer a good scaffold for ADSCs to adhere to.

Adipose-derived stem cells improve full-thickness skin grafts in a rat model.

To investigate the effects of heterologous adipose-derived stem cells (ADSCs) on autologous full-thickness skin grafts, we designed a first-intention healing model using Wistar rats. We harvested and sutured two full-thickness skin grafts in the dorsal recipient beds of 15 rats, randomized into three groups. In the treatment group, 1 × 106 ADSCs resuspended in saline solution (200 µL) were administered subcutaneously to the skin graft. The control group received only saline solution subcutaneously, whereas the negative control group did not receive any treatment. Compressive dressings were maintained until postoperative day 5. The grafts were assessed by two observers, who checked for the presence of epidermolysis on day 14. Planimetry showed the relative areas of normal skin, redness, ulceration, and contraction. Graft samples were obtained on day 14 and stained with hematoxylin and eosin and Masson's trichrome. Epidermal analysis evaluated thickening, keratosis, acanthosis, hydropic degeneration, and inflammatory infiltrate. Dermal evaluation investigated the absence of hair follicles, granulation tissue formation, presence of inflammatory infiltrate, and collagen deposition. Immunohistochemistry was performed for dermal anti-VEGF and epidermal anti-Ki-67 staining. The ADSC group presented better macroscopic aspects, lower incidence of epidermolysis, and less loss of hair follicles. In addition, the ADSC group presented the lowest frequency of histopathological changes in the dermis and epidermis, as well as the largest subcutaneous and granulation tissue VEGF averages and the weakest Ki-67 staining of the epidermal basal layer. Subcutaneous administration of ADSCs may improve the integration of skin grafts, reducing the deleterious effects of ischemia and reperfusion injury.