Cellular prion protein (PrP(C)) and superoxide dismutase (SOD) in vascular cells under oxidative stress

The PrP(C) is expressed in several cell types but its physiological function is unknown. Some studies associate the PrP(C) with copper metabolism and the antioxidant activity of SOD. Our hypothesis was that changes in PrP(C) expression lead to abnormal copper regulation and induce SOD downregulation in the vascular wall. Objectives: to study whether the PrP(C) expression undergoes induction by agents that trigger endoplasmic reticulum stress (ERS) and, in this context, to evaluate the SOD activity. Methods: To trigger ERS, in vitro, rabbit aortic smooth muscle cells were challenged for 4, 8 and 18 hours, with angiotensin-II, tunicamycin and 7-ketocholesterol. For in vivo studies rabbit aortic arteries were subjected to injury by balloon catheter. Results: In vitro baseline SOD activity, determined through inhibition of cytochrome-c reduction, was 13.9 +/- 1.2 U/mg protein, angiotensin-II exposed for 8 hours produced an increase in SOD activity, and cellular copper concentration was about 9 times greater only under these conditions. Western blotting analysis for SOD isoenzymes showed an expression profile that was not correlated with the enzymatic activity. PrP(C) expression decreased after exposure to all agents after different incubation periods. RT-PCR assay showed increased mRNA expression for PrP(C) only in cells stimulated for 8 hours with the different stressors. The PrP(C) mRNA expression in rabbit aortic artery fragments, subjected to balloon catheter injury, showed a pronounced increase immediately after overdistension. The results obtained indicated a PrP(C) protection factor during the early part of the ERS exposure period, but did not demonstrate a SOD-like profile for the PrP(C). (C) 2009 Elsevier GmbH. All rights reserved.

Increased Vascular Permeability, Angiogenesis and Wound Healing Induced by the Serum of Natural Latex of the Rubber Tree Hevea brasiliensis

Increases in vascular permeability and angiogenesis are crucial events to wound repair, tumoral growth and revascularization of tissues submitted to ischemia. An increased vascular permeability allows a variety of cytokines and growth factors to reach the damaged tissue. Nevertheless, the angiogenesis supply tissues with a wide variety of nutrients and is also important to metabolites clearance. It has been suggested that the natural latex from Hevea brasiliensis showed wound healing properties and angiogenic activity. Thus, the purpose of this work was to characterize its angiogenic activity and its effects on vascular permeability and wound healing. The serum fraction of the latex was separated from the rubber with reduction of the pH. The activity of the dialyzed serum fraction on the vascular permeability injected in subcutaneous tissue was assayed according Mile`s method. The angiogenic activity was determined using a chick embryo chorioallantoic membrane assay and its effects on the wound-healing process was determined by the rabbit ear dermal ulcer model. The serum fraction showed evident angiogenic effect and it was effective in enhancing vascular permeability. In dermal ulcers, this material significantly accelerated wound healing. Moreover, the serum fraction boiled and treated with proteases lost these activities. These results are in accordance with the enhancement of wound healing observed in clinical trials carried out with a biomembrane prepared with the same natural latex. Copyright (C) 2009 John Wiley & Sons, Ltd.

Immunization with pVAXhsp65 Decreases Inflammation and Modulates Immune Response in Experimental Encephalomyelitis

Background: A DNA vaccine (pVAXhsp65) containing the gene of a heat-shock protein (hsp65) from Mycobacterium leprae showed high immunogenicity and protective efficacy against tuberculosis in BALB/c mice. A possible deleterious effect related to autoimmunity needed to be tested because hsp65 is highly homologous to the correspondent mammalian protein. In this investigation we tested the effect of a previous immunization with DNAhsp65 in the development of experimental autoimmune encephalomyelitis (EAE), a rat model of multiple sclerosis. Methods: Female Lewis rats were immunized with 3 pVAXhsp65 doses by intramuscular route. Fifteen days after the last DNA dose the animals were evaluated for specific immunity or submitted to induction of EAE. Animals were evaluated daily for weight loss and clinical score, and euthanized during the recovery phase to assess the immune response and inflammatory infiltration at the central nervous system. Results: Immunization with pVAXhsp65 induced a specific immune response characterized by production of IgG(2b) anti-hsp65 antibodies and IFN-gamma secretion. Previous immunization with pVAXhsp65 did not change EAE clinical manifestations (weight and clinical score). However, the vaccine clearly decreased brain and lumbar spinal cord inflammation. In addition, it downmodulated IFN-gamma and IL-10 production by peripheral lymphoid organs. Conclusion: Our data demonstrated that this vaccine does not trigger a deleterious effect on EAE development and also points to a potential protective effect. Copyright (C) 2010 S. Karger AG, Basel

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. (c) 2007 Elsevier B.V. All rights reserved.

Determination of Kininogens Levels and Kallikrein/Kininase II Activities in Patients with Thromboangiitis Obliterans

Some components of the kinin system such as plasma kallikrein levels, the activities of tissue kallikrein (including saliva) and kininase II and the concentrations of kininogen fractions (low-molecular weight/LKg and high-molecular weight/HKg) were evaluated in the plasma of patients with thromboangiitis obliterans (TAO) presenting clinical symptoms of the condition. Twenty TAO were diagnosed by means of the traditional Shionoya and Olin criteria and later classified into non-smokers (n = 11) and active smokers (n = 9). Fifty-three normal, non-smoking/smoking individuals (control) were also studied. Kininogen levels were determined by ELISA; the activities of kallikreins and kininase II were determined using selective substrates. The levels of enzymes (kallikreins and kininase II) and protein (kininogens) were significantly higher in patients with TAO who were active smokers compared to the control groups (no matter whether control individuals were active smokers or non-smokers, P < 0.001 for all comparisons). Interestingly, regardless of the time of disease onset, a significant increase in the levels of these components of the kinin system was also observed in patients when TAO active smokers were compared with TAO ex-smokers (P < 0.01 for all analysed parameters). Activation of the kinin system in patients with TAO may indicate the involvement of vasodilatation in an attempt to control vascular changes, thereby favouring the deposition of immune complexes at the vascular level because of nicotine stimulation. Moreover, our results corroborate the idea that TAO can be an autoimmune disorder with specific mechanisms.

Antenatal steroid and tracheal occlusion restore vascular endothelial growth factor receptors in congenital diaphragmatic hernia rat model

OBJECTIVE: Investigate the effects of antenatal steroids and tracheal occlusion on pulmonary expression of vascular endothelial growth factor receptors in rats with nitrofen-induced congenital diaphragmatic hernia. STUDY DESIGN: Fetuses were exposed to nitrofen at embryonic day 9.5. Subgroups received dexamethasone or were operated on for tracheal occlusion, or received combined treatment. Morphologic variables were recorded. To analyze vascular endothelial growth factor receptor 1 and vascular endothelial growth factor receptor 2 expression, we performed Western blotting and immunohistochemistry. Morphologic variables were analyzed by analysis of variance and immunohistochemistry by Kruskal-Wallis test. RESULTS: Congenital diaphragmatic hernia decreased body weight, total lung weight, and lung-to-body weight ratio. Tracheal occlusion increased total lung weight and lung-to-body weight ratio (P < .05). Fetuses with congenital diaphragmatic hernia had reduced vascular endothelial growth factor receptor 1 and vascular endothelial growth factor receptor 2 expression, whereas steroids and tracheal occlusion increased their expression. Combined treatment increased expression of receptors, but had no additive effect. CONCLUSION: Vascular endothelial growth factor signaling disruption may be associated with pulmonary hypertension in congenital diaphragmatic hernia. Tracheal occlusion and steroids provide a pathway for restoring expression of vascular endothelial growth factor receptors.

Bovine Peritoneum Protection Role on Intestinal Adhesions to a Vascular Graft: A Morphological Analysis

The present study aimed to experimentally evaluate the protection role of glycerin preserved bovine peritoneum (BP) against intestinal adhesions to a vascular graft. Experiments were performed on 24 adult rabbits, randomly dived into two groups. All animals were submitted to a vascular graft over the infra-renal aorta and vena cava. Group I (12 animals) was submitted to a BP patch on the retroperitoneal opening, between the vascular prosthetic graft and the intestinal loops. Group II (12 animals) had the retroperitoneal opening sutured. After 7, 14, 28 and 60 days, 3 animals of each group were randomly killed and the retro peritoneum, with or without the BP patch, was removed for histological analysis. The histological analysis showed that the BP stimulated a moderate to intense inflammatory reaction at the beginning of the experiments and on the 60-day evaluation, the inflammatory reaction was mild, limited to the BP border with its histological structure preserved. In conclusion, the BP is a safe and cheap interposition material to be used between vascular grafts and intestinal loops, presenting a protection role against adhesions between them.

Role of mixed Th1 and Th2 serum cytokines on pathogenesis and prognosis of hantavirus pulmonary syndrome

The hantavirus pulmonary syndrome (HPS) is an emerging syndrome in the Americas. The disease results from intense immune activation and changes in vascular permeability. The aim of this study was to determine the profile of serum cytokines in HPS patients looking for correlation with the clinical parameters, severity and outcome of illness. Studying 21 HPS patients, we found that IL-6 may have an important role in the pathogenesis of HPS, being associated with fatal outcome. Our results also support a mixed Th1/Th2 immune response during the course of HPS and that the magnitude of Th1 response effector cytokines is correlated to HPS severity. The decreased levels of TGF-beta observed in HPS patients suggest that immunoregulatory activity could be damaged in these patients. (c) 2008 Elsevier Masson SAS. All rights reserved.

Combining two potential causes of metalloproteinase secretion causes abdominal aortic aneurysms in rats: a new experimental model

P>Progress in understanding the pathophysiology of abdominal aortic aneurysms (AAA) is dependent in part on the development and application of effective animal models that recapitulate key aspects of the disease. The objective was to produce an experimental model of AAA in rats by combining two potential causes of metalloproteinase (MMP) secretion: inflammation and turbulent blood flow. Male Wistar rats were randomly divided in four groups: Injury, Stenosis, Aneurysm and Control (40/group). The Injury group received a traumatic injury to the external aortic wall. The Stenosis group received an extrinsic stenosis at a corresponding location. The Aneurysm group received both the injury and stenosis simultaneously, and the Control group received a sham operation. Animals were euthanized at days 1, 3, 7 and 15. Aorta and/or aneurysms were collected and the fragments were fixed for morphologic, immunohistochemistry and morphometric analyses or frozen for MMP assays. AAAs had developed by day 3 in 60-70% of the animals, reaching an aortic dilatation ratio of more than 300%, exhibiting intense wall remodelling initiated at the adventitia and characterized by an obvious inflammatory infiltrate, mesenchymal proliferation, neoangiogenesis, elastin degradation and collagen deposition. Immunohistochemistry and zymography studies displayed significantly increased expressions of MMP-2 and MMP-9 in aneurysm walls compared to other groups. The haemo-dynamic alterations caused by the stenosis may have provided additional contribution to the MMPs liberation. This new model illustrated that AAA can be multifactorial and confirmed the key roles of MMP-2 and MMP-9 in this dynamic remodelling process.

What Is the Meaning of Homocysteine in Patients on Dialysis?

Objective: To evaluate the determinants of total plasma homocysteine levels and their relations with nutritional parameters, inflammatory status, and traditional risk factors for cardiovascular disease in renal failure patients on dialysis treatment. Design: The study was conducted on 70 clinically stable patients, 50 of them on hemodialysis (70% men; 55.3 +/- 14.5 years) and 20 on peritoneal dialysis (50% men; 62 +/- 13.7 years). Patients were analyzed in terms of biochemical parameters (serum lipids, creatinine, homocysteine [Hcy], creatine-kinase [Ck], folic acid, and vitamin B(12)), anthropometric data, markers of inflammatory status (tumor necrosis factor-alpha, C-reactive protein, interleukin-6), and adapted subjective global assessment. Results: The total prevalence of hyperhomocysteinemia (>15 mu mol/L) was 85.7%. Plasma folic acid and plasma vitamin B(12) were within the normal range. Multiple regression analysis (r(2) - 0.20) revealed that the determinants of total Hcy were type of dialysis, creatinine, Ck, folic acid, and total cholesterol. Hcy was positively correlated with albumin and creatinine and negatively correlated with total cholesterol, high density lipoprotein cholesterol, folic acid, and vitamin B(12). Conclusions: The determinants of total Hcy in the study sample were type of dialysis, creatinine, Ck, folic acid, and total cholesterol. Evidently, the small sample size might have had an effect on the statistical analyses and further studies are needed. However, Hcy in patients on dialysis treatment may not have the same effect as observed in the general population. In this respect, the association between malnutrition and inflammation may be a confounding factor in the determination of the true relationship between Hcy, nutritional status, and cardiovascular risk factors in this group. (C) 2011 by the National Kidney Foundation, Inc. All rights reserved.

Obstructive Sleep Apnea Is Frequent in Patients with Hypertensive Intracerebral Hemorrhage and Is Related to Perihematoma Edema

Background: Obstructive sleep apnea (OSA) is related to increased systemic inflammation and arterial hypertension. We hypothesize that OSA is frequent in patients with acute hypertensive intracerebral hemorrhage (ICH) and is related to the perihematoma edema. Methods: Thirty-two non-comatose patients with a hypertensive ICH underwent polysomnography in the acute phase. Perihematoma edema volume was measured on CT scans at admission, after 24 h (early control) and after 4-5 days (late control). The Spearman coefficient (r(s)) was used for correlations. Results: OSA occurred in 19 (59.4%) patients. The apnea-hypopnea index was correlated with relative edema at admission CT (r(s) = 0.40; p = 0.031), early CT (r(s) = 0.46; p = 0.011) and at late CT (r(s) = 0.59; p = 0.006). Conclusions: OSA is highly frequent during the acute phase of hypertensive ICH and is related to perihematoma edema. Copyright (C) 2009 S. Karger AG, Basel

Assessment of Vascular Effects of Tamoxifen and Its Metabolites on the Rat Perfused Hindquarter Vascular Bed

Tamoxifen has been suggested to produce beneficial cardiovascular effects, although the mechanisms for these effects are not fully known. Moreover, although tamoxifen metabolites may exhibit 30-100 times higher potency than the parent drug, no previous study has compared the effects produced by tamoxifen and its metabolites on vascular function. Here, we assessed the vascular responses to acetylcholine and sodium nitroprusside on perfused hindquarter vascular bed of rats treated with tamoxifen or its main metabolites (N-desmethyl-tamoxifen, 4-hydroxy-tamoxifen, and endoxifen) for 2 weeks. Plasma and whole-blood thiobarbituric acid reactive substances (TBARS) concentrations were determined using a fluorometric method. Plasma nitrite and NOx (nitrite + nitrate) concentrations were determined using an ozone-based chemiluminescence assay and Griess reaction, respectively. Treatment with tamoxifen reduced the responses to acetylcholine (pD(2) = 2.2 +/- 0.06 and 1.9 +/- 0.05 after vehicle and tamoxifen, respectively; P < 0.05), while its metabolites improved these responses (pD(2) = 2.5 +/- 0.04 after N-desmethyl-tamoxifen, 2.5 +/- 0.03 after 4-hydroxy-tamoxifen, and 2.6 +/- 0.08 after endoxifen; P < 0.01). Tamoxifen and its metabolites showed no effect on endothelial-independent responses to sodium nitroprusside (P > 0.05). While tamoxifen treatment resulted in significantly higher plasma and whole blood lipid peroxide levels (37% and 62%, respectively; both P < 0.05), its metabolites significantly decreased lipid peroxide levels (by approximately 50%; P < 0.05). While treatment with tamoxifen decreased the concentrations of markers of nitric oxide formation by approximately 50% (P < 0.05), tamoxifen metabolites had no effect on these parameters (P > 0.05). These results suggest that while tamoxifen produces detrimental effects, its metabolites produce counteracting beneficial effects on the vascular system and on nitric oxide/reactive oxygen species formation.

Prostaglandin mediates IL-23/IL-17-induced neutrophil migration in inflammation by inhibiting IL-12 and IFN gamma production

IL-23/IL-17-induced neutrophil recruitment plays a pivotal role in rheumatoid arthritis (RA). However, the mechanism of the neutrophil recruitment is obscure. Here we report that prostaglandin enhances the IL-23/IL-17-induced neutrophil migration in a murine model of RA by inhibiting IL-12 and IFN gamma production. Methylated BSA (mBSA) and IL-23-induced neutrophil migration was inhibited by anti-IL-23 and anti-IL-17 antibodies, COX inhibitors, IL-12, or IFN gamma but was enhanced by prostaglandin E(2) (PGE(2)). IL-23-induced IL-17 production was increased by PGE(2) and suppressed by COX-inhibition or IL-12. Furthermore, COX inhibition failed to reduce IL-23-induced neutrophil migration in IL-12- or IFN gamma-deficient mice. IL-17-induced neutrophil migration was not affected by COX inhibitors, IL-12, or IFN gamma but was inhibited by MK886 (a leukotriene synthesis inhibitor), anti-TNF alpha, anti-CXCL1, and anti-CXCL5 antibodies and by repertaxin (a CXCR1/2 antagonist). These treatments all inhibited mBSA- or IL-23-induced neutrophil migration. IL-17 induced neutrophil chemotaxis through a CXC chemokines-dependent pathway. Our results suggest that prostaglandin plays an important role in IL-23-induced neutrophil migration in arthritis by enhancing IL-17 synthesis and by inhibiting IL-12 and IFN gamma production. We thus provide a mechanism for the pathogenic role of the IL-23/IL-17 axis in RA and also suggest an additional mechanism of action for nonsteroidal anti-inflammatory drugs.

Isoflavone genistein inhibits the angiotensin-converting enzyme and alters the vascular responses to angiotensin I and bradykinin

Genistein produces antihypertensive and beneficial cardiovascular effects, although the mechanisms for these effects are not known. We examined whether genistein inhibits the in vivo responses to angiotensin I or enhances the responses to bradykinin in anaesthetized rats as a result of angiotensin-converting enzyme inhibition. We have also studied the in vitro effects produced by genistein on the angiotensin-converting enzyme activity. We measured the changes in systemic arterial pressure induced by angiotensin I in doses of 0.03 to 10 mu g/kg, by angiotensin II in doses of 0.01 to 3 mu g/kg, and to bradykinin in doses of 0.03 to 10 mu g/kg in anaesthetized rats pretreated with vehicle (controls), or a single i.v. dose of genistein 25 mg/kg, or daily genistein 25 mg/kg i.v for two days, or a single i.v. dose of captopril 2 mg/kg. Plasma angiotensin-converting enzyme activity was determined in controls and genistein-treated rats using a fluorometric method. The effects of genistein (3-300 mu mol/1) on in vitro angiotensin-converting enzyme activity were assessed by adding genistein to plasma samples and measuring angiotensin-converting enzyme activity. We found significant lower angiotensin-converting enzyme activity in plasma samples from rats pretreated with genistein compared with those found in the Control group (77.7 +/- 8.1 his-leu nmol/min/ml and 108.7 +/- 8.4 his-leu nmol/min/ml, respectively; P=0.01). The incubation of genistein with plasma samples showed that genistein decreased the angiotensin-converting enzyme activity in plasma in a concentration-dependent manner (P<0.01). These findings indicate that genistein inhibits the angiotensin-converting enzyme in vivo and in vitro and may explain, at least in part, the antihypertensive and beneficial vascular effects produced by genistein. (C) 2009 Elsevier B.V. All rights reserved.

DIVERGENT ROLE OF HEME OXYGENASE INHIBITION IN THE PATHOGENESIS OF SEPSIS

The reduction of neutrophil migration to an infectious focus is associated with a high mortality in severe sepsis. Previously, we showed that heme oxygenase (HO) products downregulate neutrophil recruitment in a noninfectious inflammatory model. The present study was designed to determine the role of HO in sepsis induced by cecal ligation and puncture (CLP) model. We demonstrated that pretreatment, but not the combination of pretreatment plus posttreatment with zinc protoporphyrin IX (ZnPP IX), an HO inhibitor, prevented the reduction of CXCR2 on circulating neutrophils and the failure of intraperitoneal neutrophil migration to the site of infection. Consequently, bacterial dissemination, systemic inflammatory response, and organ injury were prevented. In addition, pretreatment with the HO inhibitor avoided hypotension and consequently increased survival. Moreover, in mice subjected to severe CLP, the pretreatment, but not the combination of pretreatment plus posttreatment with ZnPP IX, prevented the increase of plasmatic free heme observed in nontreated severe CLP. The administration of exogenous hemin to mice subjected to moderate sepsis consistently increased the mortality rate. Furthermore, hemin resulted in a reduction of neutrophil migration both in vivo and in vitro. Altogether, our results demonstrated that pretreatment with the HO inhibitor prevents the pathological findings in severe CLP. However, the combination of pretreatment plus posttreatment with ZnPP IX enhances sepsis severity because of an increase in circulating levels of heme, which is deleterious to the host tissues and also inhibits neutrophil migration.