Claudio M. Costa-Neto

Participation of kallikrein–kinin system in different pathologies

The general description of kinins refers to these peptides as molecules involved in vascular tone regulation and inflammation. Nevertheless, in the last years a series of evidences has shown that local hormonal systems, such as the kallikrein-kinin system, may be differently regulated and are of pivotal importance to pathophysiological control. The combined interpretations of many recent studies allow us to conclude that the kallikrein-kinin system plays broader and richer roles than those classically described until recently. In this review, we report findings concerning the participation of the kallikrein-kinin system in inflammation, cancer, and in pathologies related to cardiovascular, renal and central nervous systems.

Angiotensin II Facilitates Breast Cancer Cell Migration and Metastasis

Breast cancer metastasis is a leading cause of death by malignancy in women worldwide. Efforts are being made to further characterize the rate-limiting steps of cancer metastasis, i.e. extravasation of circulating tumor cells and colonization of secondary organs. In this study, we investigated whether angiotensin II, a major vasoactive peptide both produced locally and released in the bloodstream, may trigger activating signals that contribute to cancer cell extravasation and metastasis. We used an experimental in vivo model of cancer metastasis in which bioluminescent breast tumor cells (D3H2LN) were injected intra-cardiacally into nude mice in order to recapitulate the late and essential steps of metastatic dissemination. Real-time intravital imaging studies revealed that angiotensin II accelerates the formation of metastatic foci at secondary sites. Pre-treatment of cancer cells with the peptide increases the number of mice with metastases, as well as the number and size of metastases per mouse. In vitro, angiotensin II contributes to each sequential step of cancer metastasis by promoting cancer cell adhesion to endothelial cells, trans-endothelial migration and tumor cell migration across extracellular matrix. At the molecular level, a total of 102 genes differentially expressed following angiotensin II pre-treatment were identified by comparative DNA microarray. Angiotensin II regulates two groups of connected genes related to its precursor angiotensinogen. Among those, up-regulated MMP2/MMP9 and ICAM1 stand at the crossroad of a network of genes involved in cell adhesion, migration and invasion. Our data suggest that targeting angiotensin II production or action may represent a valuable therapeutic option to prevent metastatic progression of invasive breast tumors.

Bradykinin-related peptides from Phyllomedusa hypochondrialis

Bradykinin related peptides (BRPs) present in the water-soluble secretion and freshly dissected skin fragments of Phyllomedusa hypochondrialis were investigated by mass spectrometry techniques. Eighteen BRPs, along with their post-translational modifications, were characterized in the secretion by de novo MS/MS sequencing and direct MALDI imaging experiments of the frog skin. These molecules revealed strong sequence similarities to the main plasma kinin of some mammals and reptiles. Such a diversity of molecules, within the same peptide family, belonging to a single amphibian species may be related to functional specializations of these peptides and a variety of corresponding receptors that might be present in a number of different predators. Also, a novel analog, [Val]1,[Thr]6-bradykinyl-Gln,Ser had its biological activity positively detected in cell culture expressing the human bradykinin B2 receptor and in guinea pig ileum preparations.

Effect of angiotensin converting enzyme inhibitor enalapril on body weight and composition in young rats

Obesity is considered a worldwide public health problem showing an increased prevalence in developing countries, with urgent need for new and more efficient drugs and therapies. Enalapril, an angiotensin-I converting enzyme inhibitor (ACEi), is classically used in anti-hypertensive therapies, however, earlier publications have shown that this drug could also have significant impact on body weight in rats as well as in humans, besides reducing blood pressure. The effect of this drug in the white adipose tissue has been neglected for long time, even considering that most components of the renin-angiotensin and kallikrein-kinin system are expressed in this tissue. Furthermore, the adipose tissue is considered today as one of the most important sites for endocrine/inflammatory regulation of appetite and energy output and AngII has been linked to the metabolism in this tissue. Therefore, we analyzed the influence of chronic enalapril treatment in normotensive rats at earlier ages, evaluating body weight, energy homeostasis, lipid profile and serum levels of the hormones leptin and insulin, in the presence of a standard or a palatable hyperlipidic diet regimen for one month. Our results show that enalapril treatment is able to reduce body fat on both diets, without alteration in serum lipid profile. Furthermore, animals receiving enalapril showed reduction in food intake, leptin level and energy intake. In summary, these findings show for the first time that the ACEi enalapril reduces body fat in young normotensive rats and highlights a novel target to treat obesity and associated diseases.

New bioactive metabolites produced by Phomopsis cassiae, an endophytic fungus in Cassia spectabilis

Two new metabolites, ethyl 2,4-dihydroxy-5,6-dimethylbenzoate (1) and phomopsilactone (2) were isolated from Phomopsis cassiae, an endophytic fungus in Cassia spectabilis. Their structures were elucidated by 1D and 2D NMR, MS and IR spectral data. Compounds 1 and 2 displayed strong antifungal activity against the phytopatogenic fungi Cladosporium cladosporioides and C. sphaerospermum, as well as cytotoxicity against human cervical tumor cell line (HeLa), in in vitro assays.

Angiotensin‐(1–7) decreases LPS‐induced inflammatory response in macrophages

It has been previously shown that besides its classical role in blood pressure control the renin–angiotensin system, mainly by action of angiotensin II on the AT1 receptor, exerts pro‐inflammatory effects such as by inducing the production of cytokines. More recently, alternative pathways to this system were described, such as binding of angiotensin‐(1–7) to receptor Mas, which was shown to counteract some of the effects evoked by activation of the angiotensin II–AT1 receptor axis. Here, by means of different molecular approaches we investigated the role of angiotensin‐(1–7) in modulating inflammatory responses triggered in mouse peritoneal macrophages. Our results show that receptor Mas transcripts were up‐regulated by eightfold in LPS‐induced macrophages. Interestingly, macrophage stimulation with angiotensin‐(1–7), following to LPS exposure, evoked an attenuation in expression of TNF‐α and IL‐6 pro‐inflammatory cytokines; where this event was abolished when the receptor Mas selective antagonist A779 was also included. We then used heterologous expression of the receptor Mas in HEK293T cells to search for the molecular mechanisms underlying the angiotensin‐(1–7)‐mediated anti‐inflammatory responses by a kinase array; what suggested the involvement of the Src kinase family. In LPS‐induced macrophages, this finding was corroborated using the PP2 compound, a specific Src kinase inhibitor; and also by Western blotting when we observed that Ang‐(1–7) attenuated the phosphorylation levels of Lyn, a member of the Src kinase family. Our findings bring evidence for an anti‐inflammatory role for angiotensin‐(1–7) at the cellular level, as well as show that its probable mechanism of action includes the modulation of Src kinases activities. J. Cell. Physiol. 227: 2117–2122, 2012.

Pt(II) and Ag(I) complexes with acesulfame: Crystal structure and a study of their antitumoral, antimicrobial and antiviral activities

Two new complexes of platinum(II) and silver(I) with acesulfame were synthesized. Acesulfame is in the anionic form acesulfamate (ace). The structures of both complexes were determined by X-ray crystallography. For K(2)[PtCl(2)(ace)(2)] the platinum atom is coordinated to two Cl(-) and two N-acesulfamate atoms forming a trans-square planar geometry. Each K(+) ion interacts with two oxygen atoms of the S(O)(2) group of each acesulfamate. For the polymeric complex [Ag(ace)](n) the water molecule bridges between two crystallographic equivalent Ag1 atoms which are related each other by a twofold symmetry axis. Two Ag1 atoms, related to each other by a symmetry centre, make bond contact with two equivalent oxygen atoms. These bonds give rise to infinite chains along the unit cell diagonal in the ac plane. The in vitro cytotoxic analyses for the platinum complex using HeLa (human cervix cancer) cells show its low activity when compared to the vehicle-treated cells. The Ag(I) complex submitted to in vitro antimycobacterial tests, using the Microplate Alamar Blue (MABA) method, showed a good activity against Mycobacterium tuberculosis, responsible for tuberculosis, with a minimal inhibitory concentration (MIC) value of 11.6microM. The Ag(I) complex also presented a promising activity against Gram negative (Escherichia coli and Pseudomonas aeruginosa) and Gram positive (Enterococcus faecalis) microorganisms. The complex K(2)[PtCl(2)(ace)(2)] was also evaluated for antiviral properties against dengue virus type 2 (New Guinea C strain) in Vero cells and showed a good inhibition of dengue virus type 2 (New Guinea C strain) replication at 200microM, when compared to vehicle-treated cells.

Inhibition of the renin-angiotensin system prevents seizures in a rat model of epilepsy

The RAS (renin-angiotensin system) is classically involved in BP (blood pressure) regulation and water-electrolyte balance, and in the central nervous system it has been mostly associated with homoeostatic processes, such as thirst, hormone secretion and thermoregulation. Epilepsies are chronic neurological disorders characterized by recurrent epileptic seizures that affect 1-3% of the worlds population, and the most commonly used anticonvulsants are described to be effective in approx. 70% of the population with this neurological alteration. Using a rat model of epilepsy, we found that components of the RAS, namely ACE (angiotensin-converting enzyme) and the AT1 receptor (angiotensin II type 1 receptor) are up-regulated in the brain (2.6- and 8.2-fold respectively) following repetitive seizures. Subsequently, epileptic animals were treated with clinically used doses of enalapril, an ACE inhibitor, and losartan, an AT1 receptor blocker, leading to a significant decrease in seizure severities. These results suggest that centrally acting drugs that target the RAS deserve further investigation as possible anticonvulsant agents and may represent an additional strategy in the management of epileptic patients.

Functional rescue of a defective angiotensin II AT1 receptor mutant by the Mas protooncogene

Earlier studies with Mas protooncogene, a member of the G-protein-coupled receptor family, have proposed this gene to code for a functional AngII receptor, however further results did not confirm this assumption. In this work we investigated the hypothesis that a heterodimeration AT(1)/Mas could result in a functional interaction between both receptors. For this purpose, CHO or COS-7 cells were transfected with the wild-type AT(1) receptor, a non-functional AT(1) receptor double mutant (C18F-K20A) and Mas or with WT/Mas and C18F-K20A/Mas. Cells single-expressing Mas or C18F/K20A did not show any binding for AngII. The co-expression of the wild-type AT(1) receptor and Mas showed a binding profile similar to that observed for the wild-type AT(1) expressed alone. Surprisingly, the co-expression of the double mutant C18F/K20A and Mas evoked a total recovery of the binding affinity for AngII to a level similar to that obtained for the wild-type AT(1). Functional measurements using inositol phosphate and extracellular acidification rate assays also showed a clear recovery of activity for AngII on cells co-expressing the mutant C18F/K20A and Mas. In addition, immunofluorescence analysis localized the AT(1) receptor mainly at the plasma membrane and the mutant C18F-K20A exclusively inside the cells. However, the co-expression of C18F-K20A mutant with the Mas changed the distribution pattern of the mutant, with intense signals at the plasma membrane, comparable to those observed in cells expressing the wild-type AT(1) receptor. These results support the hypothesis that Mas is able to rescue binding and functionality of the defective C18F-K20A mutant by dimerization.

Identification of a bioactive compound isolated from Brazilian propolis type 6.

A prenylated benzophenone, hyperibone A, was isolated from the hexane fraction of Brazilian propolis type 6. Its structure was determined by spectral analysis including 2D NMR. This compound exhibited cytotoxic activity against HeLa tumor cells (IC(50)=0.1756microM), strong antimicrobial activity (MIC range-0.73-6.6microg/mL; MBC range-2.92-106microg/mL) against Streptococcus mutans, Streptococcus sobrinus, Streptococcus oralis, Staphylococcus aureus, and Actinomyces naeslundii, and the results of its cytotoxic and antimicrobial activities were considered good.