Richardt G. Landgraf

A selective cyclooxygenase-2 inhibitor suppresses the growth of endometriosis with an antiangiogenic effect in a rat model

OBJECTIVE To analyze the antiangiogenic effects of the selective cyclooxygenase-2 (COX-2) inhibitor parecoxib on the growth of endometrial implants in a rat model of peritoneal endometriosis. DESIGN Pharmacologic interventions in an experimental model of peritoneal endometriosis. SETTING Research laboratory in the Federal University of Rio de Janeiro. ANIMAL(S) Twenty female Sprague-Dawley rats with experimentally induced endometriosis. INTERVENTION(S) After implantation and establishment of autologous endometrium onto the peritoneum abdominal wall, rats were randomized into groups and treated with parecoxib or the vehicle by IM injection for 30 days. MAIN OUTCOME MEASURE(S) Vascular density, the expression of vascular endothelial growth factor (VEGF) and its receptor Flk-1, the distribution of activated macrophages, the expression of COX-2, and the prostaglandin concentration in the endometriotic lesions treated with parecoxib were analyzed. RESULT(S) The treatment significantly decreased the implant size, and histologic examination indicated mostly atrophy and regression. A reduction in microvessel density and in the number of macrophages, associated with decreased expression of VEGF and Flk-1, also were observed. The treatment group showed a low concentration of prostaglandin E(2). CONCLUSION(S) These results suggest that the use of COX-2 selective inhibitors could be effective to suppress the establishment and growth of endometriosis, partially through their antiangiogenic activity.

Metformin attenuates the exacerbation of the allergic eosinophilic inflammation in high fat-diet-induced obesity in mice.

A positive relationship between obesity and asthma has been well documented. The AMP-activated protein kinase (AMPK) activator metformin reverses obesity-associated insulin resistance (IR) and inhibits different types of inflammatory responses. This study aimed to evaluate the effects of metformin on the exacerbation of allergic eosinophilic inflammation in obese mice. Male C57BL6/J mice were fed for 10 weeks with high-fat diet (HFD) to induce obesity. The cell infiltration and inflammatory markers in bronchoalveolar lavage (BAL) fluid and lung tissue were evaluated at 48 h after ovalbumin (OVA) challenge. HFD obese mice displayed peripheral IR that was fully reversed by metformin (300 mg/kg/day, two weeks). OVA-challenge resulted in higher influx of total cell and eosinophils in lung tissue of obese mice compared with lean group. As opposed, the cell number in BAL fluid of obese mice was reduced compared with lean group. Metformin significantly reduced the tissue eosinophil infiltration and prevented the reduction of cell counts in BAL fluid. In obese mice, greater levels of eotaxin, TNF-α and NOx, together with increased iNOS protein expression were observed, all of which were normalized by metformin. In addition, metformin nearly abrogated the binding of NF-κB subunit p65 to the iNOS promoter gene in lung tissue of obese mice. Lower levels of phosphorylated AMPK and its downstream target acetyl CoA carboxylase (ACC) were found in lung tissue of obese mice, which were restored by metformin. In separate experiments, the selective iNOS inhibitor aminoguanidine (20 mg/kg, 3 weeks) and the anti-TNF-α mAb (2 mg/kg) significantly attenuated the aggravation of eosinophilic inflammation in obese mice. In conclusion, metformin inhibits the TNF-α-induced inflammatory signaling and NF-κB-mediated iNOS expression in lung tissue of obese mice. Metformin may be a good pharmacological strategy to control the asthma exacerbation in obese individuals.

Insulin Suppresses LPS-induced iNOS and COX-2 Expression and NF-κB Activation in Alveolar Macrophages

The development of septic shock is a common and frequently lethal consequence of gram-negative infection. Mediators released by lung macrophages activated by bacterial products such as lipopolysaccharide (LPS) contribute to shock symptoms. We have shown that insulin down-regulates LPS-induced TNF production by alveolar macrophages (AMs). In the present study, we investigated the effect of insulin on the LPS-induced production of nitric oxide (NO) and prostaglandin (PG)-E2, on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, and on nuclear factor kappa B (NF-ĸB) activation in AMs. Resident AMs from male Wistar rats were stimulated with LPS (100 ng/mL) for 30 minutes. Insulin (1 mU/mL) was added 10 min before LPS. Enzymes expression, NF-ĸB p65 activation and inhibitor of kappa B (I-ĸB)α phosphorylation were assessed by immunobloting; NO by Griess reaction and PGE2 by enzyme immunoassay (EIA). LPS induced in AMs the expression of iNOS and COX-2 proteins and production of NO and PGE2, and, in parallel, NF-ĸB p65 activation and cytoplasmic I-ĸBα phosphorylation. Administration of insulin before LPS suppressed the expression of iNOS and COX-2, of NO and PGE2 production and Nuclear NF-ĸB p65 activation. Insulin also prevented cytoplasmic I-ĸBα phosphorylation. These results show that in AMs stimulated by LPS, insulin prevents nuclear translocation of NF-ĸB, possibly by blocking I-ĸBα degradation, and supresses the production of NO and PGE2, two molecules that contribute to septic shock.

Antineoplasic activity of Copaifera multijuga oil and fractions against ascitic and solid Ehrlich tumor.

The aim of this study was to investigate the effect of chronic treatment with C. multijuga oil on Ehrlich tumor evolution. C. multijuga was fractionated in a KOH impregnated silica gel column chromatography to give three distinct fractions, i.e., hexanic, chloroformic, and methanolic, mainly composed by hydrocarbon sesquiterpenes, oxygenated sesquiterpenes and acidic diterpenes, respectively. Results demonstrated that the C. multijuga oil, the hexanic, and chloroformic fractions did not develop toxic effects. The oil, hexanic and chloroformic fractions (doses varying between 100 and 200mg/kg) showed antineoplasic properties against Ehrlich ascitic tumor (EAT) and solid tumor during 10 consecutive days of treatment inhibiting ascitic tumor cell number, reverting medulla and blood cell counts to values similar to control group, and inhibiting the increase on several inflammatory mediators (total protein, PGE(2), nitric oxide, and TNF) on ascitic fluid. The treatment also inhibited the increase in paw volume on tumor-inoculated mice. In conclusion, C. multijuga as well as its fractions demonstrated antineoplasic effect even after oral administration confirming its use by traditional medicine.

Insulin Inhibits LPS-Induced Signaling Pathways in Alveolar Macrophages

The systemic inflammatory response syndrome (SIRS) is triggered by lipopolysaccharide (LPS) from Gram-negative bacteria. Insulin was shown to have a protective role in SIRS related to sepsis. Lungs are particularly affected in this condition and provide a second wave of mediators/cytokines which amplifies SIRS. The aim of the present study was to investigate the effect of insulin on the signaling pathways elicited by LPS in alveolar macrophages (AMs) and its consequence in cellular response to LPS measured as production of tumor necrosis factor (TNF). To this purpose, resident AMs from male Wistar rats were obtained by lung lavage and stimulated by LPS (100 ng/mL). Insulin (1 mU/mL) was added 10 min before LPS. Activation (phosphorylation) of signaling molecules by LPS was analyzed by western blot, 30 min after LPS stimulation. TNF was measured in the AMs culture supernatants by bioassay using L-929 tumor cells. Relative to controls, LPS induced a significant increase in the activation of ERK (3.6-fold), p38 (4.4-fold), Tyr-326 Akt (4.7-fold), Ser-473 Akt (6.9-fold), PKCα (4.7-fold) and PKCδ (2.3-fold). Treatment of AMs with insulin before LPS stimulation, significantly reduced the activation of ERK (54%), p38 (48%), Tyr-326 Akt (64%), Ser-473 Akt (41%), PKCα (62%) and PKCδ (39%). LPS induced TNF production in AMs which was also inhibited by insulin (60%). These results show that insulin down-regulates MAPK, PI3K and PKCs and inhibits a downstream effect of LPS, TNF production, in rat AMs stimulated with LPS and suggest that the protective effect of insulin in sepsis could be through modulation of signal transduction pathways elicited by LPS in lung macrophages.

Differential modulation of murine lung inflammation by bradykinin B1 and B2 selective receptor antagonists.

The effect of bradykinin receptor antagonists was studied in a mouse (C57Bl/6) model of allergic lung inflammation. Bradykinin B(2) receptor antagonist HOE-140 (D-Arg-[Hyp(3),Thi(5),Dtic(7)-Oic(8)]bradykinin) or bradykinin B(1) receptor antagonist R-954 (Ac-Orn[Oic(2),alphaMePhe(5),betaD-Nal(7)Ile(8)]des-Arg(9)-bradykinin) were given i.p. to ovalbumin sensitized mice 30 min before antigen challenge. After 24 h, bronchoalveolar lavage was performed for cell analysis and the lungs were removed for evaluation of airway hyperreactivity and histopathology. Treatment with HOE-140 caused a significant increase in bronchoalveolar lavage cell number: eosinophils (182%), neutrophils (98%), lymphocytes CD(4)(+) (192%), CD(8)(+) (236%), B220 (840%), Tgammadelta(+) (194%) and NK1.1(+) (246%). Hyperreactivity and mucus secretion were not significantly affected in this group. Treatment with R-954 significantly reduced eosinophil (79%) and neutrophil (83%) but has no effect on lymphocytes number in bronchoalveolar lavage fluid. Airway hyperreactivity and mucus secretion were reduced by this treatment (84% and 35%, respectively). These results show important modulatory effect of bradykinin B(1) and B(2) receptors on allergic lung inflammation.

Brabykinin B1 Receptor Antagonism Is Beneficial in Renal Ischemia-Reperfusion Injury

Previously we have demonstrated that bradykinin B1 receptor deficient mice (B1KO) were protected against renal ischemia and reperfusion injury (IRI). Here, we aimed to analyze the effect of B1 antagonism on renal IRI and to study whether B1R knockout or antagonism could modulate the renal expression of pro and anti-inflammatory molecules. To this end, mice were subjected to 45 minutes ischemia and reperfused at 4, 24, 48 and 120 hours. Wild-type mice were treated intra-peritoneally with antagonists of either B1 (R-954, 200 µg/kg) or B2 receptor (HOE140, 200 µg/kg) 30 minutes prior to ischemia. Blood samples were collected to ascertain serum creatinine level, and kidneys were harvested for gene transcript analyses by real-time PCR. Herein, B1R antagonism (R-954) was able to decrease serum creatinine levels, whereas B2R antagonism had no effect. The protection seen under B1R deletion or antagonism was associated with an increased expression of GATA-3, IL-4 and IL-10 and a decreased T-bet and IL-1β transcription. Moreover, treatment with R-954 resulted in lower MCP-1, and higher HO-1 expression. Our results demonstrated that bradykinin B1R antagonism is beneficial in renal IRI.

Emerging roles for eicosanoids in renal diseases.

Purpose of reviewEicosanoids are products of arachidonic acid metabolism which have important roles in renal homeostasis and disease. In recent years the development of genetically modified animals and new drugs targeting eicosanoids producing enzymes and receptors has unveiled new roles for eicosanoids in kidney function. This review provides an overview of eicosanoid biosynthesis and receptors and discusses recent findings on their role in acute and chronic renal diseases and in renal transplantation. Recent findingsProducts of the cyclooxygenases, 5-lipoxygenase, and cytochrome P450 pathways of arachidonic acid metabolism act through distinct receptors presented at different segment of the nephron. Apart from its role in renal physiology and hemodynamic, eicosanoids actively participate in the pathogenesis of acute and chronic renal diseases and have immunoregulatory role in kidney transplantation. SummaryThe new discoveries on the role of eicosanoids in kidney functions and the development of drugs targeting eicosanoids synthesis or action should help to envisage novel therapeutic approaches for patients suffering from renal diseases.

Lung Remodeling in a Mouse Model of Asthma Involves a Balance between TGF-β1 and BMP-7

A key event in chronic allergic asthma is the TGF-β-induced activation of fibroblasts into α-SMA-positive myofibroblasts which synthesize type-I collagen. In the present study we investigated the effect of the anti-fibrotic molecule BMP-7 in asthma. Balb/c mice were immunized i.p. with ovalbumin in alum and challenged every 2 days with ovalbumin aerosol (two or six challenges for acute and chronic protocols, respectively). The lung was evaluated for: α-SMA and type-I collagen by immunohistochemistry; BMP-7 and TGF- β1 gene expression by qRT-PCR; type-I collagen and Smads 2 and 3 by immunoblotting; mucus by PSA staining. Type-I collagen around bronchi, α-SMA, mucus secretion, TGF- β1 and BMP-7 gene expression were all increased in asthma. The TGF- β1/BMP-7 ratio was higher in the chronic group and correlated with higher levels of collagen. Fibroblasts isolated from asthmatic and healthy lungs produced type-I collagen upon stimulation with TGF- β1 via phosphorylation of Smad-2, Smad-3. Pre-treatment of the fibroblasts with BMP-7 reduced collagen production and Smads phosphorylation. Intranasal treatment of asthmatic mice with recombinant BMP-7 during the immunization protocol reduced lung inflammation and type I collagen deposition. These results suggest a protective role for BMP-7 in lung allergic inflammation, opposing the pro-fibrotic effects of TGF- β1.

Leptin Downregulates LPS-Induced Lung Injury: Role of Corticosterone and Insulin

Background/Aims: We investigated the effects of leptin in the development of lipopolysaccharide (LPS)-induced acute lung inflammation (ALI) in lean mice. Methods: Mice were administered leptin (1.0µg/g) or leptin (1.0µg/g) followed by LPS (1.5µg/g) intranasally. Additionally, some animals were given LPS (1.5µg/g) or saline intranasally alone, as a control. Tissue samples and fluids were collected six hours after instillation. Results: We demonstrated that leptin alone did not induce any injury. Local LPS exposure resulted in significant acute lung inflammation, characterized by a substantial increase in total cells, mainly neutrophils, in bronchoalveolar lavages (BAL). We also observed a significant lymphocyte influx into the lungs associated with enhanced lung expression of chemokines and cytokines (KC, RANTES, TNF-α, IFN-γ, GM-CSF and VEGF). LPS-induced ALI was characterized by the enhanced expression of ICAM-1 and iNOS in the lungs. Mice that received LPS showed an increase in insulin levels. Leptin, when administered prior to LPS instillation, abolished all of these effects. LPS induced an increase in corticosterone levels, and leptin potentiated this event. Conclusion: These data suggest that exogenous leptin may promote protection during sepsis, and downregulation of the insulin levels and upregulation of corticosterone may be important mechanisms in the amelioration of LPS-induced ALI.