Atherosclerosis morphology and pathogenesis

Atherosclerosis is a very common and important disease being the most important cause of mortality in Brazil. Indeed, in 1995, 23.3% of deaths, all ages, in our country, were the consequence of atherosclerosis. This percentage grows to 26.3% for S. Paulo and 32.7% for Rio Grande do Sul. Morphologically, there are 3 main types of lesions: fatty streaks, fibrous plaques, and complicated lesions. Fatty streaks are inocuous and occur early in life. In some persons, with age, they change into fibrous plaques that may lead to stenosis. They also may become complicated by erosion, calcification, hemorrhage and thrombosis. Atherosclerosis is initiated by endothelial functional alterations responsible for increase in permeability to macromolecules, adhesion, and migration of monocytes-macrophages and lymphocytes plus recruitment of platelets and smooth-muscle medial cells. Adhesion molecules, cytokines, growth factors, and free radicals are locally synthesized, favoring proliferation of extracellular matrix and progression of the lesion. Experimental, clinical, and epidemiological evidence point to the importance of lipids, mainly cholesterol-rich low-density lipoprotein (LDL), as one of the most important molecules involved in the genesis and progression of atherosclerosis. Patients with a genetic disorder of cholesterol metabolism (familial hyperlipidemia), caused by a decrease in the availability of receptors for LDL, develop severe atherosclerosis early in life. A series of other factors, such as age, diabetes melitus, diet, hypertension, lack of exercise, elevated hemocysteinemia, immunological disorders, and coagulation instability, are related to the progression of atherosclerosis. All of them are capable of altering the endothelium or increasing the offer of LDL. All the above-mentioned factors are systemic; but atherosclerosic lesions are focal, located at preferential sites such as the emergence of colaterals, bifurcations, and curvatures of arteries, all areas in which the laminar flow is disturbed. In these areas shear stress is diminished favoring the prolongation of permanence time of lipid particles, cells, cytokines, growth factors, etc., in the vicinity of the endothelium. Moreover, the endothelium has sensors that act as transducers of mechanical forces in biological responses. Experimental data demonstrate that the number and quality of adhesion molecules, cytokines, and growth factors synthetized, as well as the local production of radicals, and pro and anticoagulation factors may change with shear stress favoring or not the local establishment and progression of atherosclerotic lesions.

Endocytic activity in the thrombocytes of the turtle Phrynopys hilarii (freshwater South American species)

The phagocytic process in cells depends on lysosomal enzymes, high-energy metabolism and cellular recognition. In this paper, we investigated the presence of energy and recognition factors in thrombocytes of turtle Phrynopys hilarii (a freshwater South American species). Turtle thrombocytes (P. hilarii) present glycogen - possibly β particles - dispersed in their cytoplasm and glycoproteins in the cell surface, as well as a large number of enzymes involved in the endocytic process (Pellizzon, 1996). The activity of these enzymes depends on high-energy metabolism and on cellular recognition provided by specific glycoconjugates (Alberts et al., 1994). This metabolic characterization is demonstrated by the large amount of glycogen particles observed in the cytoplasm by Thiéry's method. Glycogen labeling was also observed when concanavalin A-peroxidase was used as a marker for thrombocytes and for endocyted charcoal particles. Our results show that these cells have phagocytic ability, suggesting that their function in blood circulation is not limited to aggregation but may also involve a great potential for phagocytosis.

Sand-water slurry erosion of API 5L X65 pipe steel as quenched from intercritical temperature

The objective of this study was to analyze the erosion of API 5L X65 pipe steel whose microstructure consisted of ferrite and martensite obtained by quenching from intercritical temperature (770 °C). Jet impingement tests with sand-water slurry were used. The changes in mechanical properties, caused by heat treatment carried out, did not induce changes in either the mechanism or erosion resistance. The erosion rate increased with angle of attack until 30° and later decreased until 90°. The microtexture of the eroded surfaces, at angles of attack of 30° and 90°, were similar for both conditions and were composed of craters and platelets at several stages of evolution. The erosion mechanism was by extrusion with the forming and forging of platelets.

The thrombocyte aggregation process in the turtle Phrynopys hilarii (Chelonia). An ultrastructural study

This study investigates the thrombocyte aggregation process in the South American fresh water turtle (Phrynopys hilarii) using electron microscopy. Blood was taken from surgically exposed lateral neck vessels often turtles Phrynopys hilarii during the spring and summer seasons, when the mean temperature is 37°C. Blood samples were fixed with Karnovsky solution for processing by transmission electron microscopy. The turtle thrombocytes were spindle-shaped with lobulated nuclei. Prominent vesicles and canaliculi were found throughout the cytoplasm. The cytoplasm organelles showed an agranular endoplasmatic reticulum, Golgi complex near the centrioles and scattered free ribosomes. These cells are similar to bird thrombocytes but distinct from fish and frog thrombocytes. Blood clotting time was 5 min ± 30 sec measured by the Lee and White method. Structural alterations resulting from the aggregation process occurred after activation. Thrombocytes developed numerous filopodial projections, an increased number of vacuoles and changed from spindle to spherical shape. P. hilarii thrombocytes have different morphologic characteristics compared to other non-mammalian vertebrate cells. These cells can participate in the aggregation process, as observed in birds.

A complexa fisiopatologia dos episódios vasooclusivos na anemia falciforme

Hemolytic anemia and vasoocclusion are the cardinal clinical features of sickle cell anemia. Vasoocclusion is a complex process involving not only the polymerization of deoxygenated sickle hemoglobin tetramers, but also interactions between sickle erythrocytes, vascular endothelium, platelets, leukocytes, and plasma proteins. The increased adherence of sickle erythrocytes to endothelium has been implicated as an early step in vasoocclusion. Other researchers have focused on leukocytes and platelets which might also contribute to disturbed blood flow. Microvascular occlusion results in acute painful crises, whereas macrovascular occlusion seems to be the cause of organ failure. The anemia results from the markedly shortened circulatory survival of sickle erythrocytes, together with a limited erythropoietic response. The erythropoiesis increases intensively, but it is not enough to balance the increased rate of erythrocytes destruction to maintain normal levels of total erythrocytes and hemoglobin concentrations; mainly by the low oxygen affinity of hemoglobin S and increased 2,3-Diphosphoglycerate. It is very difficult to separate processes leading to anemia or to vasoocclusion. Understanding the involvement of multiple blood componentes in vasoocclusion may elucidate the clinical manifestations and complications of sickle cell anemia, and may give new insights into the preventive and curative therapy.

Standardization of platelet parameters determination in blood samples collected with EDTA

The Abbott Cell-Dyn 3000 automated hematological analyzer prepares a histogram of platelet volume, which is measured by a technique using electrical impedance, inferring from its platelet count (PLT), mean platelet volume (MPV) and platelet distribution width (PDW). This equipment also calculates the plateletcrit (PCT). These platelet parameters may be important to evaluate platelet function, but they require standardization, because platelets swell when in contact with ethylenediaminetetra-acetic acid salts, hence an increase in blood sample storage time produces artificially increased results. To assess the effect of storage time on MPV, PLT, PDW and PCT, blood samples from 23 sickle cell anemia patients during steady state (Group I) and 50 from healthy controls (Group II) were placed in Vacutainer® tubes with dipotassium ethylenediaminetetra-acetic acid and measured over a period of 1440 minutes (24 h) at the following times: immediately after the venipuncture (time 0), 15, 30, 60, 120, 240, 360, 480 and 1440 minutes. The mean values of MPV and PCT were significantly increased (p<0.00001) along the storage time in both groups. The mean values of PLT and PDW were practically stable (p>0.05) throughout the storage time in both groups.

Fator de crescimento derivado de plaquetas humanas obtido por uma metodologia rápida e de baixo custo

Human platelet-derived growth factor (PDGF) was purified from lysates of clinically outdated human platelets by ionic exchange chromatography in CM-Sepharose. The eluated fraction was submitted to the Immunoblot/Slot Blot assay using anti-PDGF-AA and anti-PDGF-BB polyclonal antibodies and was evaluated as to its biological activity through the test of [H 3]-thymidine incorporation in NIH/3T3 cell line fibroblasts in culture. The Immunoblot/Slot Blot assay using anti-PDGF-AA and anti-PDGF-BB antibodies proved the presence of the PDGF in chromatographic cationic fraction. The comparison of biological activities between fiblobrast stimulation assay using recombinant PDGF-AB and partially purified PDGF was demonstrated in 165.796 and 157.567 cpm, respectively. This result, proved the potent mitogenic effect of partially purified PDGF and consequently their evidence about the wound healing activity.

Morphological, cytochemical, and ultrastructural study of thrombocytes and leukocytes in neotropical fish, Brycon orbignyanus Valenciennes, 1850 (Characidae, Bryconinae)

Morphological, cytochemical and ultrastructural studies are important to demonstrate the function of the blood cells, which is very little understood in teleosts. In peripheral blood of piracanjuba' Brycon orbignyanus, thrombocytes, lymphocytes, monocytes, neutrophils and heterophils were studied and characterized. Thrombocytes had a fusiform or oval shape with PAS-positive granules. Lymphocytes presented small size with sparse basophilic cytoplasm. Monocytes were large in size, presented basophilic cytoplasm that may be foamy or vacuolated, with non-specific esterase staining. The neutrophils presented lightly neutrophilic granule cytoplasm, with positivity for PAS and peroxidase. The heterophils were large in size, with eosinophilic and basophilic granules cytoplasm and PAS-positive. Transmission electron microscopy study demonstrated that the thrombocytes, lymphocytes and monocytes features were similar to other teleosts. In ultrastructural study only one type of neutrophils was observed. Cytochemical findings indicated that neutrophils and monocytes of B. orbignyanus may be involved in phagocytosis, and neutrophils play an important microbicidal role.

Comportamento do implante contendo bone morphogenetic protein (bmp) associado ao plasma rico em plaquetas na reparação de fraturas orbitárias

Purpose: This study intends to evaluate BMP (Bone Morphogenetic Protein) implant and BMP implant plus PRP (Platelet Rich Plasma) in rabbit orbital fractures, searching for tissue reaction, by radiological and morfometrical analysis. Methods:Third six white rabbits were submitted to orbital floor fracture and distributed in three groups: G1, with rabbits receiving a plate containing decalcified bone matrix and BMP; G2, with rabbits receiving the implant with BMP wrapped by PRP; G3, the control group where it was made the fracture only. The animals were evaluated radiologically after surgery and at sacrifice time in 7, 30, 90 and 180th day after surgery. After sacrifice, a block containing the right orbital tissue was extracted and prepared to morphological and morphometrical analysis. Results: An intensive linfomononuclear inflammatory reaction was observed at 7th day in G1 e G2, witch decreased after the 30th day; mesenchimal cells, osteoblasts, new bone and progressive cavitation of the implant were also observed, besides signs of calcium deposition by radiological study. In the control group fibrosis at the site of fracture was identified only. Conclusion: BMP seemed a good orbital implant producing new bone at the implant site and correcting bone defect.There was not observed acceleration of osteoinduction when the implant was associated with PRP.

Biochemical and hematological study of goats envenomed with natural and 60Co-irradiated bothropic venom

Venoms from snakes of the Bothrops genus are proteolytic, coagulant, hemorrhagic and nephrotoxic, causing edema, necrosis, hemorrhage and intense pain at the bite site, besides systemic alterations. Many adjuvants have been added to the venom used in the sensitization of antiserum-producer animals to increase antigenic induction and reduce the envenomation pathological effects. Gamma radiation from 60Co has been used as an attenuating agent of the venoms toxic properties. The main objective was to study, comparatively, clinical and laboratory aspects of goats inoculated with bothropic (Bothrops jararaca) venom, natural and irradiated from a 60Co source. Twelve goats were divided into two groups of six animals: GINV, inoculated with 0.5mg/kg of natural venom; and GIIV, inoculated with 0.5mg/kg of irradiated venom. Blood samples were collected immediately before and one, two, seven, and thirty days after venom injection. Local lesions were daily evaluated. The following exams were carried out: blood tests; biochemical tests of urea, creatinine, creatine kinase (CK), aspartate amino-transferase (AST) and alanine amino-transferase (ALT); clotting time; platelets count; and total serum immunoglobulin measurement. In the conditions of the present experiment, irradiated venom was less aggressive and more immunogenic than natural venom.

Avoiding leukocyte contamination and early platelet activation in platelet-rich plasma.

The objective of this study was to describe a new platelet-rich plasma (PRP) protocol with a reduced concentration of leukocytes and intact platelets. We collected 8 mL of venous blood (VB) from marginal ear veins of 10 male New Zealand white rabbits in acid dextrose citrate Vacutainer tubes. Tubes were centrifuged at 302g for 10 minutes. All plasma was collected in plastic tubes to avoid buffy-coat contamination and centrifuged at 2862g for 5 minutes. A 10% calcium chloride activator (10 PRP:2 CaCl2) was added to the lower third of this plasma (PRP), and the PRP gel was obtained. Mean platelet count was 317.7 x 10(3) +/- 39.9/microL in VB and 1344.9 x 10(3) +/- 347.5/microL in PRP. Leukocyte counts were 3.96 x 10(3) +/- 2.01/microL and 0.46 x 10(3) +/- 0.45/microL in VB and PRP, respectively. Mean platelet enrichment was 327.4 +/- 97.8%. All differences were statistically significant (P > .05). This protocol is practical and reproducible, resulting in a high concentration of intact platelets to help tissue repair and low levels of leukocytes.

Modulation of the pharmacological effects of enzymatically-active PLA 2 by BTL-2, an isolectin isolated from the Bryothamnion triquetrum red alga

Background. An interaction between lectins from marine algae and PLA 2 from rattlesnake was suggested some years ago. We, herein, studied the effects elicited by a small isolectin (BTL-2), isolated from Bryothamnion triquetrum, on the pharmacological and biological activities of a PLA 2 isolated from rattlesnake venom (Crotalus durissus cascavella), to better understand the enzymatic and pharmacological mechanisms of the PLA 2 and its complex. Results. This PLA2 consisted of 122 amino acids (approximate molecular mass of 14 kDa), its pI was estimated to be 8.3, and its amino acid sequence shared a high degree of similarity with that of other neurotoxic and enzymatically-active PLA2s. BTL-2 had a molecular mass estimated in approximately 9 kDa and was characterized as a basic protein. In addition, BTL-2 did not exhibit any enzymatic activity. The PLA2 and BTL-2 formed a stable heterodimer with a molecular mass of approximately 24-26 kDa, estimated by molecular exclusion HPLC. In the presence of BTL-2, we observed a significant increase in PLA2 activity, 23% higher than that of PLA2 alone. BTL-2 demonstrated an inhibition of 98% in the growth of the Gram-positive bacterial strain, Clavibacter michiganensis michiganensis (Cmm), but only 9.8% inhibition of the Gram-negative bacterial strain, Xanthomonas axonopodis pv passiflorae (Xap). PLA2 decreased bacterial growth by 27.3% and 98.5% for Xap and Cmm, respectively, while incubating these two proteins with PLA2-BTL-2 inhibited their growths by 36.2% for Xap and 98.5% for Cmm. PLA2 significantly induced platelet aggregation in washed platelets, whereas BTL-2 did not induce significant platelet aggregation in any assay. However, BTL-2 significantly inhibited platelet aggregation induced by PLA2. In addition, PLA 2 exhibited strong oedematogenic activity, which was decreased in the presence of BTL-2. BTL-2 alone did not induce oedema and did not decrease or abolish the oedema induced by the 48/80 compound. Conclusion. The unexpected results observed for the PLA2-BTL-2 complex strongly suggest that the pharmacological activity of this PLA2 is not solely dependent on the presence of enzymatic activity, and that other pharmacological regions may also be involved. In addition, we describe for the first time an interaction between two different molecules, which form a stable complex with significant changes in their original biological action. This opens new possibilities for understanding the function and action of crude venom, an extremely complex mixture of different molecules. © 2008 Oliveira et al; licensee BioMed Central Ltd.

Evaluation of blood compatibility of plasma deposited heparin-like films and SF6 plasma treated surfaces

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Evaluations hematological and biochemical by the use of sodium diclofenac, meloxicam and firocoxib in rats

This work has evaluated the hematological and biochemical profile by the use of sodium diclofenac, meloxicam and firocoxib in Wistar rats. The rats were distributed in groups: G1 (control), G2 (diclofenac sodium: 15 mg/kg), G3 (meloxicam: 2.0 mg/ kg), G4 (meloxicam: 10.0 mg/ kg), G5 (firocoxib: 5.0 mg/ kg) e G6 (firocoxib: 25.0 mg/ kg). The drugs were administered intragastrically (gavage) once a day, during five days and evaluated in three moments: M1 (48 hours after the beginning of the treatment), M2 (96 hours after the beginning of the treatment) and M3 (72 hours after the ending of the treatment). In each moment of each group, five to seven animals were evaluated and laboratory exams were performed. There were no significant changes observed in the biochemical and hematological parameters by the use of meloxicam and firocoxib. One of the effects of the sodium diclofenac was eritrogram variation as hematocrit, erythrocytes, hemoglobin decrease during the treatment. In addition, the platelets and total white blood cells counts did not change except for basophil. There was no changes in AST, ALP, GGT, urea, creatinine, sodium, potassium values. However, the values of protein, globulin and albumin decreased. It was concluded that diclofenac sodium does not provide large variations in the hemogram and biochemical profile than the meloxicam and firocoxib do not provide delletery effects in laboratories tests.

Efeitos terapêuticos da suplementação de L-arginina nas doenças cardiovasculares e endócrino-metabólicas

Nitric oxide is synthesized from L-arginine and plays an important role in blood pressure regulation, platelets aggregation and atherosclerosis development. Most of the studies have shown that the beneficial effects of the L-arginine supplementation are related to an increasing of nitric oxide bioavailability to the cells, improving the endothelial dysfunction, decreasing oxidative stress, ameliorating lipid profile and insulin resistance. However some studies show conflicting results. Considering the role of the endothelium on the pathogenesis of the cardiovascular diseases as well as on the endocrine-metabolic diseases, this review will update studies involving the role of nitric oxide and its signaling pathways in the regulation of vascular function. Furthermore, this review will focus on the main results of the clinical trials using oral L-arginine supplementation, with or without physical exercise, in an attempt to obtain beneficial effects on the cardiovascular and endocrine-metabolic systems in patients and healthy subjects.