Hematopoietic-to-mesenchymal transition of adipose tissue macrophages is regulated by integrin ß1 and fabricated fibrin matrices.

Some bona fide adult adipocytes arise de novo from a bone marrow-derived myeloid lineage. These studies further demonstrate that adipose tissue stroma contains a resident population of myeloid cells capable of adipocyte and multilineage mesenchymal differentiation. These resident myeloid cells lack hematopoietic markers and express mesenchymal and progenitor cell markers. Because bone marrow mesenchymal progenitor cells have not been shown to enter the circulation, we hypothesized that myeloid cells acquire mesenchymal differentiation capacity in adipose tissue. We fabricated a 3-dimensional fibrin matrix culture system to define the adipose differentiation potential of adipose tissue-resident myeloid subpopulations, including macrophages, granulocytes and dendritic cells. Our data show that multilineage mesenchymal potential was limited to adipose tissue macrophages, characterized by the acquisition of adipocyte, osteoblast, chondrocyte and skeletal muscle myocyte phenotypes. Fibrin hydrogel matrices stimulated macrophage loss of hematopoietic cell lineage determinants and the expression of mesenchymal and progenitor cell markers, including integrin ß1. Ablation of integrin ß1 in macrophages inhibited adipocyte specification. Therefore, some bona fide adipocytes are specifically derived from adipose tissue-resident macrophages via an integrin ß1-dependent hematopoietic-to-mesenchymal transition, whereby they become capable of multipotent mesenchymal differentiation. The requirement for integrin ß1 highlights this molecule as a potential target for controlling the production of marrow-derived adipocytes and their contribution to adipose tissue development and function.

Interface between breast cancer cells and the tumor microenvironment using platelet-rich plasma to promote tumor angiogenesis - influence of platelets and fibrin bundles on the behavior of breast tumor cells.

Cancer progression is associated with an evolving tissue interface of direct epithelial-tumor microenvironment interactions. In biopsies of human breast tumors, extensive alterations in molecular pathways are correlated with cancer staging on both sides of the tumor-stroma interface. These interactions provide a pivotal paracrine signaling to induce malignant phenotype transition, the epithelial-mesenchymal transition (EMT). We explored how the direct contact between platelets-fibrin bundles primes metastasis using platelet-rich plasma (PRP) as a source of growth factors and mimics the provisional fibrin matrix between actively growing breast cancer cells and the tumor stroma. We have demonstrated PRP functions, modulating cell proliferation that is tumor-subtype and cancer cell-type-specific. Epithelial and stromal primary cells were prepared from breast cancer biopsies from 21 women with different cancer subtypes. Cells supplemented with PRP were immunoblotted with anti-phospho and total Src-Tyr-416, FAK-Try-925, E-cadherin, N-cadherin, TGF-ß, Smad2, and Snail monoclonal antibodies. Breast tumor cells from luminal B and HER2 subtypes showed the most malignant profiles and the expression of thrombin and other classes of proteases at levels that were detectable through FRET peptide libraries. The angiogenesis process was investigated in the interface obtained between platelet-fibrin-breast tumor cells co-cultured with HUVEC cells. Luminal B and HER2 cells showed robust endothelial cell capillary-like tubes ex vivo. The studied interface contributes to the attachment of endothelial cells, provides a source of growth factors, and is a solid substrate. Thus, replacement of FBS supplementation with PRP supplementation represents an efficient and simple approach for mimicking the real multifactorial tumor microenvironment.

Laparotomia paramediana direita com cecostomia em equino com eventração: relato de caso / Right paramedian laparotomy with cecostomy in equine eventration: report of case / Laparotomía paramediana derecha con el caballo colostomía con eventración: reporte de un caso

A eventração é a ruptura da parede abdominal com saída de vísceras, ficando contida apenas pela pele.O trauma é a etiologia mais frequente na maioria dos casos e pode ocasionar estrangulação das porções acometidas.Após a lesão ocorre deposição de fibrina e construção de aglutinações fibrinosas, que serão degradadas em poucos dias ou transformadas em aderências fibrosas permanentes. O tratamento é cirúrgico e consiste na redução do conteúdo eventrado e reconstituição da parede abdominal. O propósito deste trabalho é reportar um caso de. eventração equina com aderência de ceco, atendido no setor de Clínica Médica e Terapêutica de Equídeos da Faculdade de Medicina Veterinária e Zootecnia (FAMEZ), Universidade Federal de Mato Grosso do Sul (UFMS). O animal foi submetido à laparotomia paramediana direita com cecostomia e redução da eventração. O equino recebeu alta após 30 dias da intervenção com a ferida cirúrgica cicatrizada e sem evidências de complicações pós-operatórias.

The eventration is the rupture of the abdominal wall with output viscera, being restrained only by theskin. Trauma is the most frequent cause in most cases and may lead to strangulation of affected portions. Alter theinjury, the fibrin deposition and clumps fibrinous construction may be degraded in few days or transformed intopermanent fibrous adhesion. The treatment is surgical and consists to reduce the gut content and reconstitute abdominal wall. The purpose of this study is to report an equine eventration with cecal adhesion, atlended at Large Animal Medical and Surgical Clinic, Department of Veterinary Medicine and Zootechny College (FAMEZ), Federal University of Mato Grosso do Sul (UFMS). The animal under went laparotomy with right paramedian cecostomy andreducing herniation. The horse was discharged after 30 days of intervention with the healed wound and no evidenceof post-operative complications.

A eventración es Ia ruptura de Ia pared abdominal con salida de Ias viseras, quedandó sostenida apenaspor lapiel. EI trauma es Ia etologia mas frecuente en Ia mayoria de los casos que puede ocasionar estrangulacion deIas porciones acometidas despues de Ia lesion ocurre deposição de fibrina y construccion de glutinaco es fibrinosasque seran degradads en pocos dias o transformadas en aderência fibrosas permanente. EI tratamento es cirúrgico que consiste en Ia reduccion dei contenido eventrada y Ia regeneracion de Ia pared abdominal. EI proposito de estetrabajo es reportar un caso de eventración equina com aderência de ceco atendido en el sector de clinica medica y terapêutica de equideos de Ia Facultad de Medicina Veterinária y Zootecnia (FAMEZ), Universidade Federal de MatoGrosso do Sul (UFMS). EI animal fue sometido a laparotomia paramediana derecha con cecostomia y reduccion deIa eventración. EI equino tuvo de alta aios 30 dias de intervencion con Ia herida cirúrgica cicatrisada y sin evidenciade complicacion pos-operatória.

Laparotomia paramediana direita com cecostomia em equino com eventração: relato de caso / Right paramedian laparotomy with cecostomy in equine eventration: report of case / Laparotomía paramediana derecha con el caballo colostomía con eventración: reporte de un caso

A eventração é a ruptura da parede abdominal com saída de vísceras, ficando contida apenas pela pele.O trauma é a etiologia mais frequente na maioria dos casos e pode ocasionar estrangulação das porções acometidas.Após a lesão ocorre deposição de fibrina e construção de aglutinações fibrinosas, que serão degradadas em poucos dias ou transformadas em aderências fibrosas permanentes. O tratamento é cirúrgico e consiste na redução do conteúdo eventrado e reconstituição da parede abdominal. O propósito deste trabalho é reportar um caso de. eventração equina com aderência de ceco, atendido no setor de Clínica Médica e Terapêutica de Equídeos da Faculdade de Medicina Veterinária e Zootecnia (FAMEZ), Universidade Federal de Mato Grosso do Sul (UFMS). O animal foi submetido à laparotomia paramediana direita com cecostomia e redução da eventração. O equino recebeu alta após 30 dias da intervenção com a ferida cirúrgica cicatrizada e sem evidências de complicações pós-operatórias.(AU)

The eventration is the rupture of the abdominal wall with output viscera, being restrained only by theskin. Trauma is the most frequent cause in most cases and may lead to strangulation of affected portions. Alter theinjury, the fibrin deposition and clumps fibrinous construction may be degraded in few days or transformed intopermanent fibrous adhesion. The treatment is surgical and consists to reduce the gut content and reconstitute abdominal wall. The purpose of this study is to report an equine eventration with cecal adhesion, atlended at Large Animal Medical and Surgical Clinic, Department of Veterinary Medicine and Zootechny College (FAMEZ), Federal University of Mato Grosso do Sul (UFMS). The animal under went laparotomy with right paramedian cecostomy andreducing herniation. The horse was discharged after 30 days of intervention with the healed wound and no evidenceof post-operative complications.(AU)

A eventración es Ia ruptura de Ia pared abdominal con salida de Ias viseras, quedandó sostenida apenaspor lapiel. EI trauma es Ia etologia mas frecuente en Ia mayoria de los casos que puede ocasionar estrangulacion deIas porciones acometidas despues de Ia lesion ocurre deposição de fibrina y construccion de glutinaco es fibrinosasque seran degradads en pocos dias o transformadas en aderência fibrosas permanente. EI tratamento es cirúrgico que consiste en Ia reduccion dei contenido eventrada y Ia regeneracion de Ia pared abdominal. EI proposito de estetrabajo es reportar un caso de eventración equina com aderência de ceco atendido en el sector de clinica medica y terapêutica de equideos de Ia Facultad de Medicina Veterinária y Zootecnia (FAMEZ), Universidade Federal de MatoGrosso do Sul (UFMS). EI animal fue sometido a laparotomia paramediana derecha con cecostomia y reduccion deIa eventración. EI equino tuvo de alta aios 30 dias de intervencion con Ia herida cirúrgica cicatrisada y sin evidenciade complicacion pos-operatória.(AU)

Generalidades del sistema de la coagulación y pruebas para su estudio / Overwiew of the Coagulation System and laboratory tests for its study

En la década de los años sesenta, se describió la cascada de la coagulación como una secuencia de eventos enzimáticos iniciada por dos vías, la intrínseca y la extrínseca, las cuales convergían en una vía común para generar una enzima multifuncional, denominada trombina. La principal función de esta enzima consistía en transformar el fibrinógeno, en fibrina, una proteína que se polimeriza espontáneamente para formar la base estructural del coágulo. Posteriormente, se propuso el Modelo Celular según el cual la coagulación no es la consecuencia de vías de activación enzimáticas secuenciales, sino de una red de interacciones entre proteínas plasmáticas y transmembranas, así como, varios tipos celulares, que permiten la formación de complejos enzimáticos altamente eficientes con la finalidad de generar trombina. Esta revisión explica en detalle ambos enfoques, además, aborda las diferentes funciones que cumple la trombina dentro de la hemostasia y los mecanismos de inhibición que regulan la coagulación. Finalmente, se describen diferentes pruebas empleadas en la actualidad para evaluar la funcionalidad del sistema de coagulación, como: el tiempo de tromboplastina parcial activado, el tiempo de protrombina, el tiempo de trombina, el tiempo de reptilasa, el tiempo de coagulación por ecarina y el uso de sustratos cromogénicos para evaluar cada factor de la coagulación. Finalmente, dado a que la generación de trombina es clave dentro de la coagulación y a que el potencial de generar trombina puede indicar propensión a desarrollar eventos trombóticos o hemorrágicos, en este trabajo se presentan los métodos existentes para determinar la generación de trombina.

In the sixties, the clotting cascade was proposed, which describes the coagulation process as a sequence of enzymatic events initiated by two different pathways, the intrinsic and the extrinsic pathways, converging on a common pathway, to generate a multifunctional enzyme, thrombin, whose main function is to convert fibrinogen into fibrin, a protein that polymerizes spontaneously to form the building block of a hemostatic clot. Later, it was proposed a cell-based model of the hemostasis according to that coagulation does not occur as a consequence of linear sequential enzyme activation pathways, but rather via a network of simultaneous interactions between plasmatic and transmembrane proteins, as well as several cellular types, that allow the formation of highly efficient enzymatic complexes that lead to thrombin generation. In this review, we summarize these two approaches highlighting the functions of thrombin within the hemostasis and the inhibition mechanisms that regulate the blood coagulation. Moreover, we described different tests that are used to assess the function of the coagulation system, such as: activated partial thromboplastin time, prothrombin time, thrombin time, reptilase time, ecarin clotting time, and the use of chromogenic substrates to evaluate individual coagulation factors. Finally, because of thrombin generation is a fundamental part of the blood coagulation and, an estimation of how well a particular individual can generate thrombin may correlate with either a risk of bleeding or thrombosis, we also include the existing methods to evaluate the potential of thrombin generation in an individual.

Overwiew of the Coagulation System and laboratory tests for its study. / Generalidades del sistema de la coagulación y pruebas para su estudio.

In the sixties, the clotting cascade was proposed, which describes the coagulation process as a sequence of enzymatic events initiated by two different pathways, the intrinsic and the extrinsic pathways, converging on a common pathway, to generate a multifunctional enzyme, thrombin, whose main function is to convert fibrinogen into fibrin, a protein that polymerizes spontaneously to form the building block of a hemostatic clot. Later, it was proposed a cell-based model of the hemostasis according to that coagulation does not occur as a consequence of linear sequential enzyme activation pathways, but rather via a network of simultaneous interactions between plasmatic and transmembrane proteins, as well as several cellular types, that allow the formation of highly efficient enzymatic complexes that lead to thrombin generation. In this review, we summarize these two approaches highlighting the functions of thrombin within the hemostasis and the inhibition mechanisms that regulate the blood coagulation. Moreover, we described different tests that are used to assess the function of the coagulation system, such as: activated partial thromboplastin time, prothrombin time, thrombin time, reptilase time, ecarin clotting time, and the use of chromogenic substrates to evaluate individual coagulation factors. Finally, because of thrombin generation is a fundamental part of the blood coagulation and, an estimation of how well a particular individual can generate thrombin may correlate with either a risk of bleeding or thrombosis, we also include the existing methods to evaluate the potential of thrombin generation in an individual.

The effects of fibrin and fibrin-agarose on the extracellular matrix profile of bioengineered oral mucosa.

Several studies have developed efficient oral mucosa constructs using different types of scaffold. However, the changes in the morphology and gene and protein expression profile that could occur in these artificial constructs remain unknown. This study compared the histology and expression of several extracellular matrix molecules in human artificial oral mucosa developed using two different types of scaffolds: fibrin and fibrin-agarose. To that end, bioengineered oral mucosa stromas were constructed from biopsy samples of human oral mucosa and the substitute generated was analyzed at different periods of time in culture. Histological analysis was carried out by light and transmission electron microscopy and the expression of collagen types I, III, and VI, the proteoglycans decorin and biglycan, and the different chains of laminin, were assessed by immunoperoxidase technique. This study found that fibrin scaffolds accelerated fibroblast growth and remodeling of the scaffold, thus enhancing collagen fibrillogenesis. In the fibrin-agarose scaffold, the morphology and organization of the fibroblasts did not change during the culture period. All extracellular matrix proteins analyzed were expressed in both scaffolds. However, in fibrin scaffolds, these proteins were widely distributed and replaced the scaffold during the follow-up period. These results show that the substitutes generated showed histological and molecular similarities with native human oral mucosa stroma. In addition, it was observed that the nature of the biomaterial influenced the behaviour of the oral stromal fibroblasts, thereby modulating their growth, protein synthesis, and collagen fibrillogenesis.

Alterations of fibrin network structure mediated by dermatan sulfate.

Dermatan sulfate (DS) is well-known for its anticoagulant activity through binding to heparin cofactor II (HCII) to enhance thrombin inhibition. It has also been reported that DS has a profibrinolytic effect. We have evaluated the effects of DS solutions (4-20 µg/mL) on the formation (by kinetic studies), structure (by electron microscopy and compaction assays) and lysis (with urokinase-type plasminogen activator) of plasma fibrin networks. The results showed that DS significantly prolonged the lag phase and decreased the fibrin formation rate and the optical density of the final networks versus control, in a concentration dependent way. DS-associated networks presented a minor network percentage compared with control, composed of lower number of fibers per field, which resulted significantly thinner and longer. Moreover, DS rendered gels more sensible to rupture by centrifugal force and more susceptible to lysis. When fibrin formation kinetic assays were performed with purified fibrinogen instead of plasma, in the absence of HCII, the optical density of final DS-associated networks was statistically lower than control. Therefore, a direct effect of DS on the thickness of fibers was observed. Since in all in vitro assays low DS concentrations were used, it could be postulated that the fibrin features described above are plausible to be found in in vivo thrombi and therefore, DS would contribute to the formation of less thrombogenic clots.

Blood cells attachment after root conditioning and PRP application: an in vitro study.

AIM: Root conditioning is aimed at smear layer removal and at dental matrix collagen exposure, which may promote periodontal regeneration. This in vitro study assessed smear layer removal, collagen fiber exposure and the influence of PRP (platelet-rich plasma) application on adhesion of blood cells to the root surface using scanning electron microscopy (SEM). MATERIALS AND METHODS: Scaled root samples (n = 160) were set in five groups and conditioned with: group I - control group (saline solution); group II (EDTA 24%); group III (citric acid 25%); group IV (tetracycline hydrochloride 50 mg/ml); group V (sodium citrate 30%). Eighty samples were assessed using the root surface modification index (RSMI). The other eighty samples were set in two groups. The first group (n = 40) received PRP gel application with a soft brush and the second group (n = 40) received PRP application and then a blood drop. The fibrin clot formation was assessed in the first group and the blood cells adhesion was assessed in the second group using the BEAI (blood elements adhesion index). A previously trained, calibrated, and blind examiner evaluated photomicrographs. Statistical analysis was performed using the Kruskal-Wallis's and Dunn's tests. RESULTS: Group III attained the best results for RSMI and BEAI. Moreover, it was the only group showing fibrin clot formation. CONCLUSION: Citric acid was the most efficient conditioner for smear layer removal, collagen fiber exposure and blood cell adhesion. Moreover, it was the only group showing fibrin clot formation after PRP application. CLINICAL SIGNIFICANCE: This study demonstrated that root conditioning followed by PRP application may favor blood cell adhesion on root surface which may optimize periodontal healing.

Influence of titanium surface treatments on formation of the blood clot extension.

The aim of this in vitro study was to evaluate the influence of 3 different implant surface treatments on the extension of human blood clot formation. For this purpose, the 3 types of surfaces (as-machined; test group 1, titanium discs blasted with aluminum oxide particles and washed with nitric acid; test group 2, titanium discs blasted with titanium oxide particles and washed with maleic acid) obtained were evaluated regarding topography and blood clot extension formation. Data suggest that different treatments applied on implant surfaces confer different mechanical and chemical properties, and that titanium discs blasted with aluminum oxide particles and washed with nitric acid exhibited the widest blood clot extension (P < .001).

Effects of Er,Cr:YSGG laser irradiation on root surfaces for adhesion of blood components and morphology.

OBJECTIVE: This in vitro study evaluated the effects of Er,Cr:YSGG laser irradiation on root surfaces for adhesion of blood components and morphology. BACKGROUND DATA: No previous research has evaluated the biocompatibility of root surfaces irradiated by Er,Cr:YSGG laser. MATERIALS AND METHODS: Fifteen teeth were studied from nonsmoking patients with severe periodontal disease. Sixty root surface specimens were obtained by selecting four from each tooth. Specimens were divided into three groups of 20 each, according to treatments. Group 1 (G1) was treated by scaling and root planing (SRP), group 2 (G2) was irradiated by Er,Cr:YSGG laser, and group 3 (G3) was treated by SRP and Er,Cr:YSGG laser irradiation. Blood was placed on each of 10 specimens from each of the three groups, to evaluate adhesion of blood components to the root surfaces. A morphological analysis was made of the root surfaces of the other 10 specimens from each group. All were photomicrographed in a scanning electron microscope (SEM) and classified according to the index of blood component adhesion and modified index for analysis of morphology. Statistical processing was done with the Kruskal-Wallis and Mann-Whitney tests (p < 0.05). RESULTS: No statistical differences for adhesion of blood components to root surfaces were found between the groups (p = 0.359). However, morphological analysis disclosed that all root surfaces irradiated by Er,Cr:YSGG laser (100%) were rougher than surfaces that were not irradiated (G1-G2: p = 0.0003 and G1-G3: p = 0.0003). CONCLUSION: Er,Cr:YSGG laser irradiation produced rougher root surfaces than treatment by SRP. However, it did not interfere with the adhesion of blood components to the root surfaces.

Effect of Er:YAG and Diode lasers on the adhesion of blood components and on the morphology of irradiated root surfaces.

OBJECTIVE: The aim of this study was to evaluate in vitro, by scanning electron microscopy (SEM), the adhesion of blood components on root surfaces irradiated with Er:YAG (2.94 microm) and GaAlAs Diode (808 nm) lasers and the effects on the morphology of irradiated root surfaces. METHODS: One hundred samples of human teeth were obtained. They were previously planed and scaled with manual instruments and divided into five groups of 20 samples each: G1 (control group) - absence of treatment; G2--Er:YAG laser (7.6 J/cm2); G3--Er:YAG laser (12.9 J/cm2); G4--Diode laser (90 J/cm2) and G5--Diode laser (108 J/cm2). After these treatments, 10 samples of each group received a blood tissue but the remaining 10 did not. After laboratory treatments, the samples were obtained by SEM, the photomicrographs were analysed by the score of adhesion of blood components and the results were statistically analysed (Kruskall-Wallis and Mann-Whitney test). RESULTS: In relation to the adhesion of blood components, the study showed no significant differences between the control group and the groups treated with Er:YAG laser (p = 0.9633 and 0.6229). Diode laser radiation was less effective than control group and Er:YAG laser radiation (p < 0.01). CONCLUSIONS: None of the proposed treatments increased the adhesion of blood components in a significant way when compared to the control group. Although the Er:YAG laser did not interfere in the adhesion of blood components, it caused more changes on the root surface, whereas the Diode laser inhibited the adhesion.

Different clotting mechanisms of Bothrops jararaca snake venom on human and rabbit plasmas.

Bothrops jararaca venom is approximately 3.5 times more effective at coagulating rabbit plasma than human plasma. To investigate this difference B. jararaca venom was treated with several enzymatic inhibitors and the minimum coagulant dose was determined both on plasma anticoagulated with sodium citrate or a mixture of sodium citrate and heparin, and on fibrinogen (both human and rabbit). On human plasma, the thrombin-like component of the venom accounted for c. 60% of the coagulant activity, such activity was negligible on rabbit plasma. The venom had little clotting activity on rabbit fibrinogen. The factor II- and X-activator components could be inhibited by EDTA, EGTA and 2-mercaptoethanol, whereas the thrombin-like activity was inhibited by PMSF. These differences show that (using human plasma) B. jararaca clotting activity is mainly due to the thrombin-like component, whereas the factor II- and X-activator components are more important on rabbit plasma. The delayed action of the thrombin-like enzyme on rabbit fibrinogen may be attributed to the difference between rabbit and human fibrinopeptide A. Thus, the increased coagulant activity on rabbit plasma may be due to a faster rate of activation of factor X, V or II by snake venom enzymes.

Estudo comparativo da atividade fibrinolítica do plasma de rato em fibrina humana, bovina e murina / A comparative study of fibrinolytic activity of rat plasm in human, bovine e murine fibrin

Vários estudos mostram que o sangue humano lisa espontaneamente enquanto o sangue de outras espécies animais necessitam de altas concentraçöes de ativadores. Nossos resultados mostram que o plasma de rato näo tratado é capaz de lisar a fibrina murina, mas näo a fibrina humana ou bovina. Porém, quando tratamos estes animais com adrenalina, um conhecido ativador do sistema fibrinolítico, o plasma destes animais é capaz de digerir a fibrina humana e bovina. Estes resultados mostram que o uso da fibrina adequada para cada espécie de animal estudado pode evitar resultados discrepantes nos diferentes laboratórios que estudam atividade fibrinolítica

Fibrin-dependent fibrinolytic activity during extracorporeal circulation.

Cardiopulmonary By-Pass (CPB) Surgery may at times induce a haemostatic defect, at present not too well understood, causing severe bleeding from the operative site and chest tube drain. We present here some data on antigen increase in tissue Plasminogen Activator (tPA) and D 2 Dimer (D2D) detected during CPB and apparently not compensated by enhanced Plasminogen Activator Inhibitor type 1 (PAI 1) activity. tPA concentration (antigenic) ranged around 6.15 ng/ml (SD 5.6) before thoracotomy and 5.8 g/ml (SD 4.74) 5-10 minutes after a heparin 250 IU/Kg bolus injection. During CPB, tPA increased to 20.34 ng/ml (SD 9.17) before protamine infusion, and 16.93 ng/ml (SD 8.13) after heparin neutralization. As the D2D went up to 2000-4000 ng/ml (before/after protamine) and it was not correlated by fibrinogen consumption or FDP production, we find these observations suggestive of fibrin-dependent fibrinolytic activity, as an acquired haemostatic defect developed during CPB.