Mechanisms of Vascular Damage by Hemorrhagic Snake Venom Metalloproteinases: Tissue Distribution and In Situ Hydrolysis

Background: Envenoming by viper snakes constitutes an important public health problem in Brazil and other developing countries. Local hemorrhage is an important symptom of these accidents and is correlated with the action of snake venom metalloproteinases (SVMPs). The degradation of vascular basement membrane has been proposed as a key event for the capillary vessel disruption. However, SVMPs that present similar catalytic activity towards extracellular matrix proteins differ in their hemorrhagic activity, suggesting that other mechanisms might be contributing to the accumulation of SVMPs at the snakebite area allowing capillary disruption. Methodology/Principal Findings: In this work, we compared the tissue distribution and degradation of extracellular matrix proteins induced by jararhagin (highly hemorrhagic SVMP) and BnP1 (weakly hemorrhagic SVMP) using the mouse skin as experimental model. Jararhagin induced strong hemorrhage accompanied by hydrolysis of collagen fibers in the hypodermis and a marked degradation of type IV collagen at the vascular basement membrane. In contrast, BnP1 induced only a mild hemorrhage and did not disrupt collagen fibers or type IV collagen. Injection of Alexa488-labeled jararhagin revealed fluorescent staining around capillary vessels and co-localization with basement membrane type IV collagen. The same distribution pattern was detected with jararhagin-C (disintegrin-like/cysteine-rich domains of jararhagin). In opposition, BnP1 did not accumulate in the tissues. Conclusions/Significance: These results show a particular tissue distribution of hemorrhagic toxins accumulating at the basement membrane. This probably occurs through binding to collagens, which are drastically hydrolyzed at the sites of hemorrhagic lesions. Toxin accumulation near blood vessels explains enhanced catalysis of basement membrane components, resulting in the strong hemorrhagic activity of SVMPs. This is a novel mechanism that underlies the difference between hemorrhagic and non-hemorrhagic SVMPs, improving the understanding of snakebite pathology.

Induction of Heme Oxygenase-1 Can Halt and Even Reverse Renal Tubule-Interstitial Fibrosis

Background: The tubule-interstitial fibrosis is the hallmark of progressive renal disease and is strongly associated with inflammation of this compartment. Heme-oxygenase-1 (HO-1) is a cytoprotective molecule that has been shown to be beneficial in various models of renal injury. However, the role of HO-1 in reversing an established renal scar has not yet been addressed. Aim: We explored the ability of HO-1 to halt and reverse the establishment of fibrosis in an experimental model of chronic renal disease. Methods: Sprague-Dawley male rats were subjected to unilateral ureteral obstruction (UUO) and divided into two groups: non-treated and Hemin-treated. To study the prevention of fibrosis, animals were pre-treated with Hemin at days -2 and -1 prior to UUO. To investigate whether HO-1 could reverse established fibrosis, Hemin therapy was given at days 6 and 7 post-surgery. After 7 and/or 14 days, animals were sacrificed and blood, urine and kidney tissue samples were collected for analyses. Renal function was determined by assessing the serum creatinine, inulin clearance, proteinuria/creatininuria ratio and extent of albuminuria. Arterial blood pressure was measured and fibrosis was quantified by Picrosirius staining. Gene and protein expression of pro-inflammatory and pro-fibrotic molecules, as well as HO-1 were performed. Results: Pre-treatment with Hemin upregulated HO-1 expression and significantly reduced proteinuria, albuminuria, inflammation and pro-fibrotic protein and gene expressions in animals subjected to UUO. Interestingly, the delayed treatment with Hemin was also able to reduce renal dysfunction and to decrease the expression of pro-inflammatory molecules, all in association with significantly reduced levels of fibrosis-related molecules and collagen deposition. Finally, TGF-beta protein production was significantly lower in Hemin-treated animals. Conclusion: Treatment with Hemin was able both to prevent the progression of fibrosis and to reverse an established renal scar. Modulation of inflammation appears to be the major mechanism behind HO-1 cytoprotection.

The Function and Three-Dimensional Structure of a Thromboxane A(2)/Cysteinyl Leukotriene-Binding Protein from the Saliva of a Mosquito Vector of the Malaria Parasite

The highly expressed D7 protein family of mosquito saliva has previously been shown to act as an anti-inflammatory mediator by binding host biogenic amines and cysteinyl leukotrienes (CysLTs). In this study we demonstrate that AnSt-D7L1, a two-domain member of this group from Anopheles stephensi, retains the CysLT binding function seen in the homolog AeD7 from Aedes aegypti but has lost the ability to bind biogenic amines. Unlike any previously characterized members of the D7 family, AnSt-D7L1 has acquired the important function of binding thromboxane A(2) (TXA(2)) and its analogs with high affinity. When administered to tissue preparations, AnSt-D7L1 abrogated Leukotriene C(4) (LTC(4))-induced contraction of guinea pig ileum and contraction of rat aorta by the TXA(2) analog U46619. The protein also inhibited platelet aggregation induced by both collagen and U46619 when administered to stirred platelets. The crystal structure of AnSt-D7L1 contains two OBP-like domains and has a structure similar to AeD(7). In AnSt-D7L1, the binding pocket of the C-terminal domain has been rearranged relative to AeD7, making the protein unable to bind biogenic amines. Structures of the ligand complexes show that CysLTs and TXA(2) analogs both bind in the same hydrophobic pocket of the N-terminal domain. The TXA(2) analog U46619 is stabilized by hydrogen bonding interactions of the omega-5 hydroxyl group with the phenolic hydroxyl group of Tyr 52. LTC(4) and occupies a very similar position to LTE(4) in the previously determined structure of its complex with AeD7. As yet, it is not known what, if any, new function has been acquired by the rearranged C-terminal domain. This article presents, to our knowledge, the first structural characterization of a protein from mosquito saliva that inhibits collagen mediated platelet activation.

Novel pathogenic mutations and skin biopsy analysis in Knobloch syndrome

Purpose: To facilitate future diagnosis of Knobloch syndrome (KS) and better understand its etiology, we sought to identify not yet described COL18A1 mutations in KS patients. In addition, we tested whether mutations in this gene lead to absence of the COL18A1 gene product and attempted to better characterize the functional effect of a previously reported missense mutation. Methods: Direct sequencing of COL18A1 exons was performed in KS patients from four unrelated pedigrees. We used immunofluorescent histochemistry in skin biopsies to evaluate the presence of type XVIII collagen in four KS patients carrying two already described mutations: c. 3277C>T, a nonsense mutation, and c. 3601G>A, a missense mutation. Furthermore, we determined the binding properties of the mutated endostatin domain p.A1381T (c.3601G>A) to extracellular matrix proteins using ELISA and surface plasmon resonance assays. Results: We identified four novel mutations in COL18A1, including a large deletion involving exon 41. Skin biopsies from KS patients revealed lack of type XVIII collagen in epithelial basement membranes and blood vessels. We also found a reduced affinity of p.A1381T endostatin to some extracellular matrix components. Conclusions: COL18A1 mutations involved in Knobloch syndrome have a distribution bias toward the coding exons of the C-terminal end. Large deletions must also be considered when point mutations are not identified in patients with characteristic KS phenotype. We report, for the first time, lack of type XVIII collagen in KS patients by immunofluorescent histochemistry in skin biopsy samples. As a final point, we suggest the employment of this technique as a preliminary and complementary test for diagnosis of KS in cases when mutation screening either does not detect mutations or reveals mutations of uncertain effect, such as the p.A1381T change.

Exercise prevents the effects of experimental arthritis on the metabolism and function of immune cells

Active lymphocytes (LY) and macrophages (M Phi) are involved in the pathophysiology of rheumatoid arthritis (RA) Due to its anti-inflammatory effect. physical exercise may be beneficial in RA by acting on the immune system (IS) Thus, female Wistar rats with type II collagen-induced arthritis (CIA) were submitted to swimming training (6 weeks. 5 days/week. 60 min/day) and some biochemical and immune parameters, such as the metabolism of glucose and glutamine and function of LY and M. were evaluated In addition, plasma levels of some hormones and of interleukin-2 (IL-2) were also determined Results demonstrate that CIA increased lymphocyte proliferation (1.9- and 1 7-fold, respectively, in response to concanavalin A (ConA) and lipopolysaccharide (LPS)), as well as macrophage H(2)O(2) production (1 6-fold), in comparison to control Exercise training prevented the activation of immune cells, induced by CIA. and established a pattern of substrate utilization similar to that described as normal for these cells. Exercise also promoted an elevation of plasma levels of corticosterone (22 2%), progesterone (1 7-fold) and IL-2 (2 6-fold) Our data suggest that chronic exercise is able to counterbalance the effects of CIA on cells of the IS. reinforcing the proposal that the benefits of exercise may not be restricted to aerobic capacity and/or strength improvement Copyright (C) 2010 John Wiley & Sons, Ltd

Anabolic steroid associated to physical training induces deleterious cardiac effects

DO CARMO, E. C., T. FERNANDES, D. KOIKE, N. D. DA SILVA JR., K. C. MATTOS, K. T. ROSA, D. BARRETTI, S. F. S. MELO, R. B. WICHI, M. C. C. IRIGOYEN, and E. M. DE OLIVEIRA. Anabolic Steroid Associated to Physical Training Induces Deleterious Cardiac Effects. Med. Sci. Sports Exerc., Vol. 43, No. 10, pp. 1836-1848, 2011. Purpose: Cardiac aldosterone might be involved in the deleterious effects of nandrolone decanoate (ND) on the heart. Therefore, we investigated the involvement of cardiac aldosterone, by the pharmacological block of AT1 or mineralocorticoid receptors, on cardiac hypertrophy and fibrosis. Methods: Male Wistar rats were randomized into eight groups (n = 14 per group): Control (C), nandrolone decanoate (ND), trained (T), trained ND (TND), ND + losartan (ND + L), trained ND + losartan (TND + L), ND + spironolactone (ND + S), and trained ND + spironolactone (TND + S). ND (10 mg.kg(-1).wk(-1)) was administered during 10 wk of swimming training (five times per week). Losartan (20 mg.kg(-1).d(-1)) and spironolactone (10 mg.kg(-1).d(-1)) were administered in drinking water. Results: Cardiac hypertrophy was increased 10% by using ND and 17% by ND plus training (P < 0.05). In both groups, there was an increase in the collagen volumetric fraction (CVF) and cardiac collagen type III expression (P < 0.05). The ND treatment increased left ventricle-angiotensin-converting enzyme I activity, AT1 receptor expression, aldosterone synthase (CYP11B2), and 11-beta hydroxysteroid dehydrogenase 2 (11 beta-HSD2) gene expression and inflammatory markers, TGF beta and osteopontin. Both losartan and spironolactone inhibited the increase of CVF and collagen type III. In addition, both treatments inhibited the increase in left ventricle-angiotensin-converting enzyme I activity, CYP11B2, 11 beta-HSD2, TGF beta, and osteopontin induced by the ND treatment. Conclusions: We believe this is the first study to show the effects of ND on cardiac aldosterone. Our results suggest that these effects may be associated to TGF beta and osteopontin. Thus, we conclude that the cardiac aldosterone has an important role on the deleterious effects on the heart induced by ND.

PKC beta II inhibition attenuates myocardial infarction induced heart failure and is associated with a reduction of fibrosis and pro-inflammatory responses

Protein kinase C beta II (PKC beta II) levels increase in the myocardium of patients with end-stage heart failure (HF). Also targeted overexpression of PKC beta II in the myocardium of mice leads to dilated cardiomyopathy associated with inflammation, fibrosis and myocardial dysfunction. These reports suggest a deleterious role of PKC beta II in HF development. Using a post-myocardial infarction (MI) model of HF in rats, we determined the benefit of chronic inhibition of PKC beta II on the progression of HF over a period of 6 weeks after the onset of symptoms and the cellular basis for these effects. Four weeks after MI, rats with HF signs that were treated for 6 weeks with the PKC beta II selective inhibitor (beta IIV5-3 conjugated to TAT(47-57) carrier peptide) (3 mg/kg/day) showed improved fractional shortening (from 21% to 35%) compared to control (TAT(47-57) carrier peptide alone). Formalin-fixed mid-ventricle tissue sections stained with picrosirius red, haematoxylin and eosin and toluidine blue dyes exhibited a 150% decrease in collagen deposition, a two-fold decrease in inflammation and a 30% reduction in mast cell degranulation, respectively, in rat hearts treated with the selective PKC beta II inhibitor. Further, a 90% decrease in active TGF beta 1 and a significant reduction in SMAD2/3 phosphorylation indicated that the selective inhibition of PKC beta II attenuates cardiac remodelling mediated by the TGF-SMAD signalling pathway. Therefore, sustained selective inhibition of PKC beta II in a post-MI HF rat model improves cardiac function and is associated with inhibition of pathological myocardial remodelling.

The role of local and systemic renin angiotensin system activation in a genetic model of sympathetic hyperactivity-induced heart failure in mice

Sympathetic hyperactivity (SH) and renin angiotensin system (RAS) activation are commonly associated with heart failure (HF), even though the relative contribution of these factors to the cardiac derangement is less understood. The role of SH on RAS components and its consequences for the HF were investigated in mice lacking alpha(2A) and alpha(2C) adrenoceptor knockout (alpha(2A)/alpha(2C) ARKO) that present SH with evidence of HF by 7 mo of age. Cardiac and systemic RAS components and plasma norepinephrine (PN) levels were evaluated in male adult mice at 3 and 7 mo of age. In addition, cardiac morphometric analysis, collagen content, exercise tolerance, and hemodynamic assessments were made. At 3 mo, alpha(2A)/alpha(2C)ARKO mice showed no signs of HF, while displaying elevated PN, activation of local and systemic RAS components, and increased cardiomyocyte width (16%) compared with wild-type mice (WT). In contrast, at 7 mo, alpha(2A)/alpha(2C)ARKO mice presented clear signs of HF accompanied only by cardiac activation of angiotensinogen and ANG II levels and increased collagen content (twofold). Consistent with this local activation of RAS, 8 wk of ANG II AT(1) receptor blocker treatment restored cardiac structure and function comparable to the WT. Collectively, these data provide direct evidence that cardiac RAS activation plays a major role underlying the structural and functional abnormalities associated with a genetic SH-induced HF in mice.

Baroreflex Sensitivity Impairment Is Associated With Cardiac Diastolic Dysfunction in Rats

Background: Studies have shown that the autonomic dysfunction accompanied by impaired baroreflex sensitivity was associated with higher mortality. However, the influence of decreased baroreflex sensitivity on cardiac function, especially in diastolic function, is not well understood. This study evaluated the morpho-functional changes associated with baroreflex impairment induced by chronic sinoaortic denervation (SAD). Methods and Results: Animals were divided into sinoaortic denervation (SAD) and control (C) groups. Baroreflex sensitivity was evaluated by tachycardic and bradycardic responses, induced by vasoactive drugs. Cardiac function was studied by echocardiography and by left ventricle (LV) catheterization. LV collagen content and the expression of regulatory proteins involved in intracellular Ca(2+) homeostasis were quantified. Results showed higher LV mass in SAD versus C animals. Furthermore, an increase in deceleration time of E-wave in the SAD versus the C group (2.14 +/- 0.07 ms vs 1.78 +/- 0.03 ms) was observed. LV end-diastolic pressure was increased and the minimum dP/dt was decreased in the SAD versus the C group (12 +/- 1.5 mm Hg vs 5.3 +/- 0.2 mm Hg and 7,422 +/- 201 vs 4,999 +/- 345 mm Hg/s, respectively). SERCA/NCX ratio was lower in SAD than in control rats. The same was verified in SERCA/PLB ratio. Conclusions: The results suggest that baroreflex dysfunction is associated with cardiac diastolic dysfunction independently of the presence of other risk factors. (J Cardiac Fail 2011;17:519-525)

MicroRNAs 29 are involved in the improvement of ventricular compliance promoted by aerobic exercise training in rats

Soci UPR, Fernandes T, Hashimoto NY, Mota GF, Amadeu MA, Rosa KT, Irigoyen MC, Phillips MI, Oliveira EM. MicroRNAs 29 are involved in the improvement of ventricular compliance promoted by aerobic exercise training in rats. Physiol Genomics 43: 665-673, 2011. First published March 29, 2011; doi:10.1152/physiolgenomics.00145.2010.-MiRNAs regulate cardiac development, hypertrophy, and angiogenesis, but their role in cardiac hypertrophy (CH) induced by aerobic training has not previously been studied. Aerobic training promotes physiological CH preserving cardiac function. This study assessed involvement of miRNAs-29 in CH of trained rats. Female Wistar rats (n = 7/group) were randomized into three groups: sedentary (S), training 1 (T1), training 2 (T2). T1: swimming sessions of 60 min/5 days/wk/10 wk. T2: similar to T1 until 8th wk. On the 9th wk rats swam 2x/day, and on the 10th wk 3x/day. MiRNAs analysis was performed by miRNA microarray and confirmed by real-time PCR. We assessed: markers of training, CH by ratio of left ventricle (LV) weight/body wt and cardiomyocytes diameter, pathological markers of CH (ANF, skeletal alpha-actin, alpha/beta-MHC), collagen I and III (COLIAI and COLIIIAI) by real-time PCR, protein collagen by hydroxyproline (OH-proline) concentration, CF and CH by echocardiography. Training improved aerobic capacity and induced CH. MiRNAs-1, 133a, and 133b were downregulated as observed in pathological CH, however, without pathological markers. MiRNA-29c expression increased in T1 (52%) and T2 (123%), correlated with a decrease in COLIAI and COLIIIAI expression in T1 (27%, 38%) and T2 (33%, 48%), respectively. MiRNA-29c was inversely correlated to OH-proline concentration (r = 0.61, P = 0.05). The E/A ratio increased in T2, indicating improved LV compliance. Thus, these results show that aerobic training increase miR-29 expression and decreased collagen gene expression and concentration in the heart, which is relevant to the improved LV compliance and beneficial cardiac effects, associated with aerobic high performance training.

Effect of fibre morphology on flocculation of fibre-cement suspensions

The objective of the present research was to evaluate the effect of fibre morphology (e.g., length, width, fibrillation, broken ends, content of fines and number of fibres per gram) on flocculation and drainage properties of fibre-cement suspensions and on physical properties of the fibre-cement composites. Mechanical refining was used to change the morphological properties of Eucalyptus and Pinus pulps. Results show that the mechanical refining increased the size of the formed flocs and decreased the concentration of free small particles (with dimensions between 1 and 20 pm) as a consequence of the increased fibrillation and content of fines, which increased the capacity of the fibres to capture the mineral particles. High levels of refining were necessary for Pinus pulp to obtain cement retention values similar to those obtained by unrefined Eucalyptus pulp. This is due to the higher number of fibres per gram in Eucalyptus pulp than in Pinus pulp. Pulp refining improved the packing of the particles and, although decreased the drainage rate. it contributed to a less porous structure, which improved the microstructure of the composite. (C) 2009 Elsevier Ltd. All rights reserved.

Experimentation and numerical simulation of steel fibre reinforced concrete pipes

The results concerning on an experimental and a numerical study related to SFRCP are presented. Eighteen pipes with an internal diameter of 600 mm and fibre dosages of 10, 20 and 40 kg/m(3) were manufactured and tested. Some technological aspects were concluded. Likewise, a numerical parameterized model was implemented. With this model, the simulation of the resistant behaviour of SFRCP can be performed. In this sense, the results experimentally obtained were contrasted with those suggested by means MAP reaching very satisfactory correlations. Taking it into account, it could be said that the numerical model is a useful tool for the optimal design of the SFRCP fibre dosages, avoiding the need of the systematic employment of the test as an indirect design method. Consequently, the use of this model would reduce the overall cost of the pipes and would give fibres a boost as a solution for this structural typology.

Texture and mechanical properties evolution of a deep drawing medium carbon steel during cold rolling and subsequent recrystallization

Medium carbon steels are mostly used for simple applications; however, new applications have been developed for which good sheet metal formability is required. These types of steels have an inherent low formability. A medium-carbon hot-rolled SAE 1050 steel was selected for this study. It has been cold rolled with thickness reductions varying between 7 and 80%. The samples obtained were used to evaluate the strain hardening curve. For samples with a 50 and 80% thickness reduction, an annealing heat treatment was performed to achieve recrystallization. The material was characterized in the ""as-received"", cold rolled and annealed conditions using several methods: optical metallography, X-ray diffraction (texture), Vickers hardness, and tensile testing. For large thickness reductions, the SAE 1050 steel presented low elongation, less than 2%, and yield strength (YS) and tensile strength (TS) around 1400 MPa. Texture in the ""as-received"" condition showed strong components on the {001} plane, in the < 100 >, < 210 > and (110) directions. After cold rolling, the texture did not present any significant changes for small thickness reductions, however. It changed completely for large ones, where gamma, < 111 >//ND, alpha, < 110 > HRD, and gamma prime, < 223 >//ND, fibres were strengthened. After annealing, the microstructure of the SAE 1050 steel was characterized by recrystallized ferrite and globular cementite. There was little change in the alpha fibre for the 50% reduction, whereas for the 80% reduction, its intensity increased. Both gamma and gamma prime fibres vanished upon annealing for 50 and 80% reductions alike. (c) 2008 Elsevier B.V. All rights reserved.

Age-related association of rDNA and telomeres with the nuclear matrix in mouse hepatocytes

Transcribed sequences have been suggested to be associated with the nuclear matrix, differing from non-transcribing sequences, which have been reported to be contained in DNA loops. However, although a dozen of genes have their expression level affected by aging, data on chromatin-nuclear matrix interactions under this physiological condition are still scarce. In the present study, liver imprints from young, adult and old mice were subjected to FISH (fluorescence in situ hybridization) for 45S rDNA and telomeric sequences, with or without a lysis treatment to produce extended chromatin fibres. There was an increased amount of 45S rDNA sequences located in DNA loops as the animals grow older, while telomeric sequences were always observed in DNA loops irrespective of the animal age. We assume that active rRNA genes associate with the nuclear matrix, while DNA loops contain silent sequences. Transcription of each 45S rDNA repeat unit is suggested to be dependent on its interaction with the nuclear matrix.

Sensory evaluation and physicochemical optimisation of soy-based desserts using response surface methodology

The objective of this study was to develop a dessert that contains soy protein (SP) (1%, 2%, 3%) and guava juice (GJ) (22%, 27%, 32%) using Response Surface Methodology (RSM) as the optimisation technique. Water activity, physical stability, colour, acidity, pH, iron, and carotenoid contents were analysed. Affective tests were performed to determine the degree of liking of colour, creaminess, and acceptability. The results showed that GJ increased the values of redness, hue angle, chromaticity, acidity, and carotenoid content, while SP reduced water activity. Optimisation suggested a dessert containing 32% GJ and 1.17% SP as the best proportion of these components. This sample was considered a source of fibres, ascorbic acid, copper, and iron and garnered scores above the level of `slightly liked` for sensory attributes. Moreover, RSM was shown to be an adequate approach for modelling the physicochemical parameters and the degree of liking of creaminess of desserts. (C) 2010 Elsevier Ltd. All rights reserved.