Paula Peixoto Campos

Mechanisms of wound healing responses in lupus-prone New Zealand White mouse strain

Host responses to synthetic implants are analogous to healing, the process of repair that follows injury. Normally, the processes of wound healing follow well‐established patterns but conditions such as autoimmune diseases profoundly affect tissue repair. We have analyzed sponge‐induced wound healing responses in lupus‐prone New Zealand White and control (Balb/c) mouse strains by measuring inflammation, extracellular matrix deposition, angiogenesis, and cytokine production in polyether–polyurethane sponge implanted subcutaneously in male mice of these two strains. Although there was no difference in the gross appearance of the implants, further analysis of the wound healing responses, induced from 7 to 21 days post implantation, disclosed important differences between the New Zealand White and Balb/c strains. The intensity of inflammation (circulating tumor necrosis factor‐α and inflammatory leukocytes levels) was lower but implant fibrosis (collagen and transforming growth factor‐β1) was higher in New Zealand White, compared with Balb/c mice. Angiogenesis (hemoglobin, vascular endothelial growth factor, and vascularity) in New Zealand White implants peaked earlier than in Balb/c mice. In conclusion, we have shown that wound healing responses are clearly different in this strain of lupus‐prone mice and suggest that this pattern of repair was critically influenced by impaired inflammation and accelerated angiogenesis in the New Zealand White strain.

Sponge‐induced angiogenesis and inflammation in PAF receptor‐deficient mice (PAFR‐KO)

To determine biological functions of platelet‐activating factor (PAF) in chronic inflammation, we have investigated the kinetics of angiogenesis, inflammatory cells recruitment and cytokine production in sponge‐induced granuloma in wild type and PAF receptor‐deficient mice (PAFR‐KO). Angiogenesis as determined by morphometric analysis and hemoglobin content was significantly higher in the implants of PAFR‐KO mice at all time points. Treatment with PAF receptor antagonist UK74505 (30 mg kg−1) also increased angiogenesis in sponge implants. Neutrophils and macrophages accumulation, as determined by myeloperoxidase and N‐acetylglucosaminidase activities in the supernatant of implanted sponges were markedly decreased in PAFR‐KO mice. Surprisingly, the levels of the proinflammatory chemokines, keratinocyte‐derived chemokine and chemokine monocyte chemoattractant protein 1 were higher in the implants of the transgenic animals. We have shown that angiogenesis was stimulated in PAFR‐KO mice whereas inflammation was decreased, indicating that PAF is an endogenous regulator of new blood vessels formation in the inflammatory microenvironment induced by the sponge implant.

Genetic background determines mouse strain differences in inflammatory angiogenesis.

Inflammation and angiogenesis are key components of fibrovascular tissue growth, a biological event underlying both physiological (wound healing) and pathological conditions (tumor development, chronic inflammation). We investigated these components in three frequently used mouse strains (Swiss, Balb/c and C57BL/6J) to verify the influence of genetic background on the kinetics of inflammatory cell recruitment/activation, neovascularization, extracellular matrix deposition, and cytokine production in polyether-polyurethane sponge implanted subcutaneously in male mice of these strains. The kinetics of neutrophil recruitment/activation as assessed by myeloperoxidase (MPO) activity was 2- and 3-fold higher in Balb/c implants at day 1 compared with Swiss and C57BL/6J implants, respectively. Macrophage accumulation/activation as NAG (n-acetyl β-glucosaminidase) activity was higher in Swiss implants. The levels the monocyte chemoattractant protein 1 (CCL2(MCP-1)) peaked at day 10 in the three types of implants but was produced more by C57BL/6J mice. Angiogenesis (hemoglobin, vascular endothelial growth factor-VEGF, and number of vessels) differed among the strains. Swiss implants had the highest hemoglobin content but the lowest VEGF levels. In contrast, Balb/c implants had higher VEGF levels but lower hemoglobin. Collagen deposition and transforming growth factor β-1; TGFβ-1 levels also varied among the groups. Swiss and Balb/c implants had progressive increase in TGFβ-1 from 4 to 14 days, while C57BL/6J implants achieved the peak at day 10 and fell at day 14. These findings emphasize the major contribution of genetic background in the temporal pattern and intensity of inflammatory angiogenesis components that may have functional consequences in physiological and pathological conditions where these processes co-exist.

Cardiac oxidative stress is involved in heart failure induced by thiamine deprivation in rats

Thiamine is an important cofactor of metabolic enzymes, and its deficiency leads to cardiovascular dysfunction. First, we characterized the metabolic status measuring resting oxygen consumption rate and lactate blood concentration after 35 days of thiamine deficiency (TD). The results pointed to a decrease in resting oxygen consumption and a twofold increase in blood lactate. Confocal microscopy showed that intracellular superoxide (approximately 40%) and H(2)O(2) (2.5 times) contents had been increased. In addition, biochemical activities and protein expression of SOD, glutathione peroxidase, and catalase were evaluated in hearts isolated from rats submitted to thiamine deprivation. No difference in SOD activity was detected, but protein levels were found to be increased. Catalase activity increased 2.1 times in TD hearts. The observed gain in activity was attended by an increased catalase protein level. However, a marked decrease in glutathione peroxidase activity (control 435.3 + or - 28.6 vs. TD 199.4 + or - 30.2 nmol NADPH x min(-1) x ml(-1)) was paralleled by a diminution in the protein levels. Compared with control hearts, we did observe a greater proportion of apoptotic myocytes by TdT-mediated dUTP nick end labeling (TUNEL) and caspase-3 reactivity techniques. These results indicate that during TD, reactive oxygen species (ROS) production may be enhanced as a consequence of the installed acidosis. The perturbation in the cardiac myocytes redox balance was responsible for the increase in apoptosis.

Cilostazol and pentoxifylline decrease angiogenesis, inflammation, and fibrosis in sponge-induced intraperitoneal adhesion in mice.

AIMS Adhesion formation following abdominal intervention is an abnormal peritoneal healing process. Our aim was to investigate the effects of controlling adhesion development by inhibiting its key components (angiogenesis, inflammation and fibrosis) using phosphodiesterase (PDE) inhibitors. MAIN METHODS Two PDE inhibitors including cilostazol a PDE3 inhibitor (40 and 400 mg/kg), and pentoxifylline (PTX), a PDE 1-5 inhibitor (50 and 500 mg/kg) were used for a period of 7 days to inhibit angiogenesis, inflammation, and fibrosis in a murine model of sponge-induced peritoneal adhesion. Angiogenesis was assessed by hemoglobin content, vascular endothelial growth factor (VEGF) levels, and morphometric analysis. Accumulation of neutrophils and macrophages was determined by measuring myeloperoxidase (MPO) and N-acetylglucosaminidase (NAG) activities, respectively. Levels of TNF-alpha were also determined. Fibrosis was assessed by determining the amount of collagen in the implant; TGF-beta1 levels in the implant were also measured. KEY FINDINGS Our results show that the treatments attenuated the main components of the adhesion tissue by reducing the amount of fibrovascular tissue that infiltrated the sponge matrix (wet weight). Hemoglobin content and VEGF levels were also decreased by approximately 40%. Neutrophil accumulation was unaffected by the compounds. However, NAG activity was reduced by pentoxifylline, but not by cilostazol. These compounds also decreased the levels of the pro-inflammatory and pro-fibrogenic cytokines TNF-alpha and TGF-beta1, respectively, and collagen synthesis. SIGNIFICANCE Our results suggest that cilostazol and PTX decreased the development of peritoneal adhesions in the model, which might be associated with cyclic nucleotide modulation. Therapies to intervene in these pathways may be beneficial for the prevention of these lesions.

Phosphatidylinositol 3-kinase-δ up-regulates L-type Ca2+ currents and increases vascular contractility in a mouse model of type 1 diabetes

BACKGROUND AND PURPOSE Vasculopathies represent the main cause of morbidity and mortality in diabetes. Vascular malfunctioning in diabetes is associated with abnormal vasoconstriction and Ca2+ handling by smooth muscle cells (SMC). Phosphatidylinositol 3‐kinases (PI3K) are key mediators of insulin action and have been shown to modulate the function of voltage‐dependent L‐type Ca2+ channels (CaV1.2). In the present work, we investigated the involvement of PI3K signalling in regulating Ca2+ current through CaV1.2 (ICa,L) and vascular dysfunction in a mouse model of type I diabetes.

Cardiac structural changes and electrical remodeling in a thiamine-deficiency model in rats

AIMS Thiamine is an important cofactor present in many biochemical reactions, and its deprivation can lead to heart dysfunction. Little is known about the influence of thiamine deprivation on the electrophysiological behavior of the isolated heart cells and information about thiamine deficiency in heart morphology is controversial. Thus, we decided to investigate the major repolarizing conductances and their influence in the action potential (AP) waveform as well as the changes in the heart structure in a set of thiamine deficiency in rats. MAIN METHODS Using the patch-clamp technique, we investigated inward (I(K1)) and outward K(+) currents (I(to)), T-type and L-type Ca(2+) currents and APs. To evaluate heart morphology we used hematoxylin and eosin in transversal heart sections. KEY FINDINGS Thiamine deficiency caused a marked decrease in left ventricle thickness, cardiomyocyte number, cell length and width, and membrane capacitance. When evaluating I(to) we did not find difference in current amplitude; however an acceleration of I(to) inactivation was observed. I(K1) showed a reduction in the amplitude and slope conductance, which implicated a less negative resting membrane potential in cardiac myocytes isolated from thiamine-deficient rats. We did not find any difference in L-type Ca(2+) current density. T-type Ca(2+) current was not observed. In addition, we did not observe significant changes in AP repolarization. SIGNIFICANCE Based on our study we can conclude that thiamine deficiency causes heart hypotrophy and not heart hypertrophy. Moreover, we provided evidence that there is no major electrical remodeling during thiamine deficiency, a feature of heart failure models.

Hepatotoxicity of Pentavalent Antimonial Drug: Possible Role of Residual Sb(III) and Protective Effect of Ascorbic Acid

ABSTRACT Pentavalent antimonial drugs such as meglumine antimoniate (Glucantime [Glu; Sanofi-Aventis, São Paulo, Brazil]) produce severe side effects, including cardiotoxicity and hepatotoxicity, during the treatment of leishmaniasis. We evaluated the role of residual Sb(III) in the hepatotoxicity of meglumine antimoniate, as well as the protective effect of the antioxidant ascorbic acid (AA) during antimonial chemotherapy in a murine model of visceral leishmaniasis. BALB/c mice infected with Leishmania infantum were treated intraperitoneally at 80 mg of Sb/kg/day with commercial meglumine antimoniate (Glu) or a synthetic meglumine antimoniate with lower Sb(III) level (MA), in association or not with AA (15 mg/kg/day), for a 20-day period. Control groups received saline or saline plus AA. Livers were evaluated for hepatocytes histological alterations, peroxidase activity, and apoptosis. Increased proportions of swollen and apoptotic hepatocytes were observed in animals treated with Glu compared to animals treated with saline or MA. The peroxidase activity was also enhanced in the liver of animals that received Glu. Cotreatment with AA reduced the extent of histological changes, the apoptotic index, and the peroxidase activity to levels corresponding to the control group. Moreover, the association with AA did not affect the hepatic uptake of Sb and the ability of Glu to reduce the liver and spleen parasite loads in infected mice. In conclusion, our data supports the use of pentavalent antimonials with low residue of Sb(III) and the association of pentavalent antimonials with AA, as effective strategies to reduce side effects in antimonial therapy.

Kinetics of implant-induced inflammatory angiogenesis in abdominal muscle wall in mice.

Injury of skeletal abdominal muscle wall is a common medical condition and implantation of synthetic or biological material is a procedure to repair musculofascial defects. We proposed to characterize the dynamics of inflammatory cell recruitment, newly formed blood vessels, cytokine production and fibrogenesis in the abdominal skeletal muscle in response to polyether-polyurethane sponge implants in mice. At 2, 4, 7 and 10days after implantation the muscle tissue underneath the sponge matrix was removed for the assessment of the angiogenic response (hemoglobin content, vascular endothelial growth factor and morphometric analysis of the number of vessels) and inflammation (myeloperoxidase and n-acethyl-B-d-glucosaminidase activities, cytokines). In addition, muscle fibrogenesis was determined by the levels of TGF-β1 and collagen deposition. Hemoglobin content, wash out rate of sodium fluorescein (indicative of blood flow) and the number of vessels increased in the abdominal muscle bearing the synthetic matrix in comparison with the intact muscle. Neutrophil recruitment peaked in the muscle at day 2, followed by macrophage accumulation at day 4 post-injury. The levels of the cytokines, VEGF, TNF-α, CCL-2/MCP-1 were higher in the injured muscle compared with the intact muscle and peaked soon after muscle injury (days 2 to 4). Collagen levels were higher in sponge-bearing muscle compared with the non-bearing tissue soon after injury (day 2). The implantation technique together with the inflammatory and vascular parameters used in this study revealed inflammatory, angiogenic and fibrogenic events and mechanisms associated with skeletal muscle responses to synthetic implanted materials.

Amperometric Detection of Lactose Using β-Galactosidase Immobilized in Layer-by-Layer Films

A direct, low-cost method to determine the concentration of lactose is an important goal with possible impact in various types of industry. In this study, a biosensor is reported that exploits the specific interaction between lactose and the enzyme β-galactosidase (β-Gal) normally employed to process lactose into glucose and galactose for lactose-intolerant people. The biosensor was made with β-Gal immobilized in layer-by-layer (LbL) films with the polyelectrolyte poly(ethylene imine) (PEI) and poly(vinyl sufonate) (PVS) on an indium tin oxide (ITO) electrode modified with a layer of Prussian Blue (PB). With an ITO/PB/(PEI/PVS)1(PEI/β-Gal)30 architecture, lactose could be determined with an amperometric method with sensitivity of 0.31 μA mmol(-1) cm(-2) and detection limit of 1.13 mmol L(-1), which is sufficient for detecting lactose in milk and for clinical exams. Detection occurred via a cascade reaction involving glucose oxidase titrated as electrolytic solution in the electrochemical cell, while PB allowed for operation at 0.0 V versus saturated calomel electrode, thus avoiding effects from interfering species. Sum-frequency generation spectroscopy data for the interface between the LbL film and a buffer containing lactose indicated that β-Gal lost order, which is the first demonstration of structural effects induced by the molecular recognition interaction with lactose.