Actions of the Kunitz-type serine protease inhibitor Amblyomin-X on VEGF-A-induced angiogenesis

Amblyomin-X is a Kunitz-type serine protease inhibitor (Kunitz-type SPI) designed from the cDNA library of the Amblyomma cajennense tick, which displays in vivo anti-tumor activities. Here, the mechanisms of actions of Amblyomin-X in vascular endothelial growth factor A (VEGF-A)-induced angiogenesis were characterized. Topical application of Amblyomin-X (10 or 100 ng/10 mu l; each 48 h) inhibited VEGF-A-induced (10 ng/10 mu l; each 48 h) angiogenesis in the dorsal subcutaneous tissue in male Swiss mice. Moreover, similar effect was observed in the VEGF-A-induced angiogenesis in the chicken chorioallantoic membrane (CAM). Additional in vitro assays in t-End cells showed that Amblyomin-X treatment delayed the cell cycle, by maintaining them in G0/G1 phase, and inhibited cell proliferation and adhesion, tube formation and membrane expression of the adhesion molecule platelet-endothelial cell adhesion molecule-1 (PECAM-I), regardless of mRNA synthesis. Together, results herein reveal the role of Kunitz-type SPI on in vivo VEGF-A-induced angiogenesis, by exerting modulatory actions on endothelial cell proliferation and adhesion, especially on membrane expression of PECAM-1. These data provide further mechanisms of actions of Kunitz-type SPI, corroborating their relevance as scientific tools in the design of therapeutic molecules. (C) 2012 Elsevier Ltd. All rights reserved.

In vivo hydroquinone exposure causes tracheal hyperresponsiveness due to TNF secretion by epithelial cells

Hydroquinone (HQ) is the main oxidative substance in cigarette smoke and a toxic product of benzene biotransformation. Although the respiratory tract is an inlet pathway of HQ exposure, its effect on airway muscle responsiveness has not been assessed. We thus investigated the effects of low dose in vivo HQ-exposure on tracheal responsiveness to a muscarinic receptor agonist. Male Swiss mice were exposed to aerosolised 5% ethanol/saline solution (HQ vehicle; control) or 0.04 ppm HQ (1 h/day for 5 days) and tracheal rings were collected 1 h after the last exposure. HQ exposure caused tracheal hyper-responsiveness to methacholine (MCh), which was abolished by mechanical removal of the epithelium. This hyperresponsiveness was not dependent on neutrophil infiltration, but on tumour necrosis factor (TNF) secretion by epithelial cells. This conclusion was based on the following data: (1) trachea from HQ-exposed mice presented a higher amount of TNF, which was abrogated following removal of the epithelium; (2) the trachea hyperresponsiveness and TNF levels were attenuated by in vivo chlorpromazine (CPZ) treatment, an inhibitor of TNF synthesis. The involvement of HQ-induced TNF secretion in trachea mast cell degranulation was also demonstrated by the partial reversion of tracheal hyperresponsiveness in sodium cromoglicate-treated animals, and the in vivo HQ-exposure-induced degranulation of trachea connective tissue and mucosal mast cells, which was reversed by CPZ treatment. Our data show that in vivo HQ exposure indirectly exacerbates the parasympathetic-induced contraction of airway smooth muscle cells, mediated by TNF secreted by tracheal epithelial cells, clearly showing the link between environmental HQ exposure and the reactivity of airways. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Interleukin-4 Modulates the Inflammatory Response in Ifosfamide-Induced Hemorrhagic Cystitis

We investigated whether interleukin-4 (IL-4) is present and capable of reducing inflammatory changes seen in ifosfamide-induced hemorrhagic cystitis. Male Swiss mice were treated with saline or ifosfamide alone or ifosfamide with the classical protocol with mesna and analyzed by changes in bladder wet weight (BWW), macroscopic and microscopic parameters, exudate, and hemoglobin quantification. In other groups, IL-4 was administered intraperitoneally 1 h before ifosfamide. In other experimental groups, C57BL/6 WT (wild type) and C57BL/6 WT IL-4 (-/-) knockout animals were treated with ifosfamide and analyzed for changes in BWW. Quantification of bladder IL-4 protein by ELISA in control, ifosfamide-, and mesna-treated groups was performed. Immunohistochemistry to tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) as well as protein identification by Western blot assay for inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was carried out on ifosfamide- and IL-4-treated animals. In other experimental groups, antiserum against IL-4 was given 30 min before ifosfamide. In IL-4-treated animals, the severity of hemorrhagic cystitis was significantly milder than in animals treated with ifosfamide only, an effect that was reverted with serum anti-IL-4. Moreover, knockout animals for IL-4 (-/-) exhibit a worse degree of inflammation when compared to C57BL/6 wild type. Exogenous IL-4 also attenuated TNF-alpha, IL-1 beta, iNOS, and COX-2 expressions in ifosfamide-treated bladders. IL-4, an anti-inflammatory cytokine, attenuates the inflammation seen in ifosfamide-induced hemorrhagic cystitis.

Mikania glomerata Sprengel (Asteraceae) influences the mutagenicity induced by doxorubicin without altering liver lipid peroxidation or antioxidant levels

As shown in numerous studies, natural compounds may exert adverse effects, mainly when associated with some drugs. The hydroalcoholic extract of Mikania glomerata is the pharmaceutical form present in commercially available syrup used for the treatment of respiratory diseases in popular Brazilian medicine. The objective of the present investigation was (1) to evaluate the preventive effects of standardized hydroalcoholic extract of M. glomerata (MEx) against antitumoral drug doxorubicin (DXR)-induced micronucleated polychromatic erythrocytes (MNPCE) in a subchronic assay in mice, and (2) to determine the liver content of malondialdehyde (MDA) and the antioxidants glutathione (GSH) and vitamin E (VE). Male Swiss mice were treated for 30 d with MEx added to drinking water, combined or not with DXR (90 mg/kg body weight) injected intraperitoneally (ip) 24 h before analysis. The results demonstrated that MEx produced no genotoxic damage, but significantly increased the frequency of MNPCE induced by DXR, indicating a drug-drug interaction. This rise was not accompanied by lipid peroxidation or antioxidants level reduction, as measured by MDA, GSH, and VE. Despite the presence of coumarin (a known antioxidant), MEx may exert adverse effects probably in association with mutagenic compounds, although this effect on DNA damage did not involve oxidative stress.

A bradykinin-potentiating peptide (BPP-10c) from Bothrops jararaca induces changes in seminiferous tubules

Abstract: Background: The testis-specific isoform of angiotensin-converting enzyme (tACE) is exclusively expressed in germ cells during spermatogenesis. Although the exact role of tACE in male fertility is unknown, it clearly plays a critical function in spermatogenesis. The dipeptidase domain of tACE is identical to the C-terminal catalytic domain of somatic ACE (sACE). Bradykinin potentiating peptides (BPPs) from snake venoms are the first natural sACE inhibitors described and their structure–activity relationship studies were the basis for the development of antihypertensive drugs such as captopril. In recent years, it has been showed that a number of BPPs – including BPP-10c – are able to distinguish between the N- and C-active sites of sACE, what is not applicable to captopril. Considering the similarity between tACE and sACE (and since BPPs are able to distinguish between the two active sites of sACE), the effects of the BPP-10c and captopril on the structure and function of the seminiferous epithelium were characterized in the present study. BPP-10c and captopril were administered in male Swiss mice by intraperitoneal injection (4.7 μmol/kg for 15 days) and histological sections of testes were analyzed. Classification of seminiferous tubules and stage analysis were carried out for quantitative evaluation of germ cells of the seminiferous epithelium. The blood-testis barrier (BTB) permeability and distribution of claudin-1 in the seminiferous epithelium were analyzed by hypertonic fixative method and immunohistochemical analyses of testes, respectively. Results: The morphology of seminiferous tubules from animals treated with BPP-10c showed an intense disruption of the epithelium, presence of atypical multinucleated cells in the lumen and degenerated germ cells in the adluminal compartment. BPP-10c led to an increase in the number of round spermatids and total support capacity of Sertoli cell in stages I, V, VII/VIII of the seminiferous epithelium cycle, without affecting BTB permeability and the distribution of claudin-1 in the seminiferous epithelium. Interestingly, no morphological or morphometric alterations were observed in animals treated with captopril. Conclusions: The major finding of the present study was that BPP-10c, and not captopril, modifies spermatogenesis by causing hyperplasia of round spermatids in stages I, V, and VII/VIII of the spermatogenic cycle.

Long-term type 1 diabetes impairs decidualization and extracellular matrix remodeling during early embryonic development in mice

Introduction: Endometrial decidualization and associated extracellular matrix (ECM) remodeling are critical events to the establishment of the maternal-fetal interface and successful pregnancy. Here, we investigated the impact of type 1 diabetes on these processes during early embryonic development, in order to contribute to the understanding of the maternal factors associated to diabetic embryopathies. Methods: Alloxan-induced diabetic Swiss female mice were bred after different periods of time to determine the effects of diabetes progression on the development of gestational complications. Furthermore, the analyses focused on decidual development as well as mRNA expression, protein deposition and ultrastructural organization of decidual ECM. Results: Decreased number of implantation sites and decidual dimensions were observed in the group mated 90-110 days after diabetes induction (D), but not in the 50-70D group. Picrosirius staining showed augmentation in the fibrillar collagen network in the 90e110D group and, following immunohistochemical examination, that this was associated with increase in types I and V collagens and decrease in type III collagen and collagen-associated proteoglycans biglycan and lumican. qPCR, however, demonstrated that only type I collagen mRNA levels were increased in the diabetic group. Alterations in the molecular ratio among distinct collagen types and proteoglycans were associated with abnormal collagen fibrillogenesis, analyzed by transmission electron microscopy. Conclusions: Our results support the concept that the development of pregnancy complications is directly related with duration of diabetes (progression of the disease), and that this is a consequence of both systemic factors (i.e. disturbed maternal endocrine-metabolic profile) and uterine factors, including impaired decidualization and ECM remodeling