Rho-kinase inhibition attenuates airway responsiveness, inflammation, matrix remodeling, and oxidative stress activation induced by chronic inflammation

Possa SS, Charafeddine HT, Righetti RF, da Silva PA, Almeida-Reis R, Saraiva-Romanholo BM, Perini A, Prado CM, Leick-Maldonado EA, Martins MA, Tiberio ID. Rho-kinase inhibition attenuates airway responsiveness, inflammation, matrix remodeling, and oxidative stress activation induced by chronic inflammation. Am J Physiol Lung Cell Mol Physiol 303: L939-L952, 2012. First published September 21, 2012; doi:10.1152/ajplung.00034.2012.-Several studies have demonstrated the importance of Rho-kinase in the modulation of smooth muscle contraction, airway hyperresponsiveness, and inflammation. However, the effects of repeated treatment with a specific inhibitor of this pathway have not been previously investigated. We evaluated the effects of repeated treatment with Y-27632, a highly selective Rho-kinase inhibitor, on airway hyperresponsiveness, oxidative stress activation, extracellular matrix remodeling, eosinophilic inflammation, and cytokine expression in an animal model of chronic airway inflammation. Guinea pigs were subjected to seven ovalbumin or saline exposures. The treatment with Y-27632 (1 mM) started at the fifth inhalation. Seventy-two hours after the seventh inhalation, the animals' pulmonary mechanics were evaluated, and exhaled nitric oxide (E-NO) was collected. The lungs were removed, and histological analysis was performed using morphometry. Treatment with Y-27632 in sensitized animals reduced E-NO concentrations, maximal responses of resistance, elastance of the respiratory system, eosinophil counts, collagen and elastic fiber contents, the numbers of cells positive for IL-2, IL-4, IL-5, IL-13, inducible nitric oxide synthase, matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, transforming growth factor-beta, NF-kappa B, IFN-gamma, and 8-iso-prostaglandin F2 alpha contents compared with the untreated group (P < 0.05). We observed positive correlations among the functional responses and inflammation, remodeling, and oxidative stress pathway activation markers evaluated. In conclusion, Rho-kinase pathway activation contributes to the potentiation of the hyperresponsiveness, inflammation, the extracellular matrix remodeling process, and oxidative stress activation. These results suggest that Rho-kinase inhibitors represent potential pharmacological tools for the control of asthma.

Immunophenotyping and extracellular matrix remodeling in pulmonary and extrapulmonary sarcoidosis

Objective: To investigate the significance of cellular immune markers, as well as that of collagen and elastic components of the extracellular matrix, within granulomatous structures in biopsies of patients with pulmonary or extrapulmonary sarcoidosis. Methods: We carried out qualitative and quantitative evaluations of inflammatory cells, collagen fibers, and elastic fibers in granulomatous structures in surgical biopsies of 40 patients with pulmonary and extrapulmonary sarcoidosis using histomorphometry, immunohistochemistry, picrosirius red staining, and Weigert's resorcin-fuchsin staining. Results: The extrapulmonary tissue biopsies presented significantly higher densities of lymphocytes, macrophages, and neutrophils than did the lung tissue biopsies. Pulmonary granulomas showed a significantly higher number of collagen fibers and a lower density of elastic fibers than did extrapulmonary granulomas. The amount of macrophages in the lung samples correlated with FVC (p < 0.05), whereas the amount of CD3+, CD4+, and CD8+ lymphocytes correlated with the FEV1/FVC ratio and VC. There were inverse correlations between TLC and the CD1a+ cell count (p < 0.05), as well as between DLCO and collagen/elastic fiber density (r = -0.90; p = 0.04). Conclusions: Immunophenotyping and remodeling both showed differences between pulmonary and extrapulmonary sarcoidosis in terms of the characteristics of the biopsy samples. These differences correlated with the clinical and spirometric data obtained for the patients, suggesting that two different pathways are involved in the mechanism of antigen clearance, which was more effective in the lungs and lymph nodes.

Rest left ventricular function and contractile reserve by dobutamine stress echocardiography in peripartum cardiomyopathy

Aims: To assess whether contractile reserve during dobutamine stress echocardiography (DSE) can predict left ventricular functional recovery in patients with peripartum cardiomyopathy and to assess myocardial fibrosis by magnetic resonance imaging (MRI) in these patients. Methods: Nine patients with peripartum cardiomyopathy were enrolled. All patients underwent DSE and were followed for six months, when a rest Doppler echocardiogram was repeated. MRI was also performed at the beginning of follow-up to identify myocardial fibrosis. Results: Mean age was 29 +/- 7.9 years and mean left ventricular ejection fraction at baseline was 39.4 +/- 8.6% (range 24-49%). Eight of the nine patients showed left ventricular functional recovery with mean ejection fraction at follow-up of 57.1 +/- 13.8%. The ejection fraction response to DSE did not predict recovery at follow-up. On the other hand, left ventricular ejection fraction at baseline correlated with ejection fraction at follow-up. Mild fibrosis was detected in only one patient. Conclusion: Left ventricular ejection fraction at baseline was a predictor of left ventricular functional recovery in patients with peripartum cardiomyopathy. Dobutamine stress echocardiography at presentation of the disease did not predict recovery at follow-up. Myocardial fibrosis appeared to be uncommon in this cardiomyopathy. (C) 2011 Sociedade Portuguesa de Cardiologia Published by Elsevier Espana, S.L. All rights reserved.

ROLE OF FOCAL ADHESION KINASE IN LUNG REMODELING OF ENDOTOXEMIC RATS

Despite significant advances in the care of critically ill patients, acute lung injury continues to be a complex problem with high mortality. The present study was designed to characterize early lipopolysaccharide (LPS)-induced pulmonary injury and small interfering RNA targeting focal adhesion kinase (FAK) as a possible therapeutic tool in the septic lung remodeling process. Male Wistar rats were assigned into endotoxemic group and control group. Total collagen deposition was performed 8, 16, and 24 h after LPS injection. Focal adhesion kinase expression, interstitial and vascular collagen deposition, and pulmonary mechanics were analyzed at 24 h. Intravenous injection of small interfering RNA targeting FAK was used to silence expression of the kinase in pulmonary tissue. Focal adhesion kinase, total collagen deposition, and pulmonary mechanics showed increased in LPS group. Types I, III, and V collagen showed increase in pulmonary parenchyma, but only type V increased in vessels 24 h after LPS injection. Focal adhesion kinase silencing prevented lung remodeling in pulmonary parenchyma at 24 h. In conclusion, LPS induced a precocious and important lung remodeling. There was fibrotic response in the lung characterized by increased amount in total and specific-type collagen. These data may explain the frequent clinical presentation during sepsis of reduced lung compliance, oxygen diffusion, and pulmonary hypertension. The fact that FAK silencing was protective against lung collagen deposition underscores the therapeutic potential of FAK targeting by small interfering RNA.

SMALL INTERFERING RNA TARGETING FOCAL ADHESION KINASE PREVENTS CARDIAC DYSFUNCTION IN ENDOTOXEMIA

Sepsis and septic shock are associated with cardiac depression. Cardiovascular instability is a major cause of death in patients with sepsis. Focal adhesion kinase (FAK) is a potential mediator of cardiomyocyte responses to oxidative and mechanical stress. Myocardial collagen deposition can affect cardiac compliance and contractility. The aim of the present study was to determine whether the silencing of FAK is protective against endotoxemia-induced alterations of cardiac structure and function. In male Wistar rats, endotoxemia was induced by intraperitoneal injection of lipopolysaccharide (10 mg/kg). Cardiac morphometry and function were studied in vivo by left ventricular catheterization and histology. Intravenous injection of small interfering RNA targeting FAK was used to silence myocardial expression of the kinase. The hearts of lipopolysaccharide-injected rats showed collagen deposition, increased matrix metalloproteinase 2 activity, and myocyte hypertrophy, as well as reduced 24-h +dP/dt and -dP/dt, together with hypotension, increased left ventricular end-diastolic pressure, and elevated levels of FAK (phosphorylated and unphosphorylated). Focal adhesion kinase silencing reduced the expression and activation of the kinase in cardiac tissue, as well as protecting against the increased collagen deposition, greater matrix metalloproteinase 2 activity, and reduced cardiac contractility that occur during endotoxemia. In conclusion, FAK is activated in endotoxemia, playing a role in cardiac remodeling and in the impairment of cardiac function. This kinase represents a potential therapeutic target for the protection of cardiac function in patients with sepsis.

Experimental model of tooth movement in mice: A standardized protocol for studying bone remodeling under compression and tensile strains

During orthodontic tooth movement (OTM), alveolar bone is resorbed by osteoclasts in compression sites (CS) and is deposited by osteoblasts in tension sites (TS). The aim of this study was to develop a standardized OTM protocol in mice and to investigate the expression of bone resorption and deposition markers in CS and TS. An orthodontic appliance was placed in C57BL6/J mice. To define the ideal orthodontic force, the molars of the mice were subjected to forces of 0.1 N, 0.25 N, 0.35 N and 0.5 N. The expression of mediators that are involved in bone remodeling at CS and TS was analyzed using a Real-Time PCR. The data revealed that a force of 0.35 N promoted optimal OTM and osteoclast recruitment without root resorption. The levels of TNF-alpha, RANKL, MMP13 and OPG were all altered in CS and TS. Whereas TNF-a and Cathepsin K exhibited elevated levels in CS. RUNX2 and OCN levels were higher in TS. Our results suggest that 0.35 N is the ideal force for OTM in mice and has no side effects. Moreover, the expression of bone remodeling markers differed between the compression and the tension areas, potentially explaining the distinct cellular migration and differentiation patterns in each of these sites. (C) 2012 Elsevier Ltd. All rights reserved.

Rupture of the right ventricular free wall after myocardial infarction

Patient, 75 years-old, with free wall rupture of the right ventricle, corrected with prolene 3.0 points anchored in bovine pericardium patch, promoting the closure of the rupture. The patient was discharged on the 59th day after surgery in good clinical ans laboratorial conditions.

Dexamethasone reduces bronchial wall remodeling during pulmonary migration of Strongyloides venezuelensis larvae in rats

Strongyloidiasis is an intestinal parasitosis with an obligatory pulmonary cycle. A Th2-type immune response is induced and amplifies the cellular response through the secretion of inflammatory mediators. Although this response has been described as being similar to asthma, airway remodeling during pulmonary migration of larvae has not yet been established. The aim of this study was to identify the occurrence of airway remodeling during Strongyloides venezuelensis (S. v.) infection and to determine the ability of dexamethasone treatment to interfere with the mechanisms involved in this process. Rats were inoculated with 9,000 S. v. larvae, treated with dexamethasone (2 mg/kg) and killed at 1, 3, 5, 7, 14 and 21 days. Morphological and morphometric analyzes with routine stains and immunohistochemistry were conducted, and some inflammatory mediators were evaluated using ELISA. Goblet cell hyperplasia and increased bronchiolar thickness, characterized by edema, neovascularization, inflammatory infiltrate, collagen deposition and enlargement of the smooth muscle cell layer were observed. VEGF, IL1-beta and IL-4 levels were elevated throughout the course of the infection. The morphological findings and the immunomodulatory response to the infection were drastically reduced in dexamethasone-treated rats. The pulmonary migration of S. venezuelensis larvae produced a transitory, but significant amount of airway remodeling with a slight residual bronchiolar fibrosis. The exact mechanisms involved in this process require further study. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Comparative analysis between ventricular and sinoatrial node vascularization in cats

Biasi C., Borelli V., Benedicto H.G., Pereira M.R., Favaron P.O. & Bombonato P.P. 2012. [Comparative analysis between ventricular and sinoatrial node vascularization in cats.] Analise comparativa entre a vascularizacao ventricular e do no sinoatrial em gatos. Pesquisa Veterinaria Brasileira 31(1): 78-82. Setor de Anatomia dos Animais Domesticos e Silvestres, Faculdade de Medicina Veterinaria e Zootecnia, Universidade de Sao Paulo, Av. Prof. Dr. Orlando Marques de Paiva 87, Cidade Universitaria, Sao Paulo, SP 05508-000, Brazil. E-mail: caiobiasi@usp.br The possible existence of interdependence in the blood nutrition of both atrial and ventricular territories has been a subject of concern to cardiologists, mainly related to vascularization of the sinoatrial node and its dependence on just one coronary artery or both, and its relation with the predominance of these vessels in the ventricular vascularization. Therefore, this research aimed evaluated the relation of blood irrigation of the sinoatrial node in relation to the coronary artery predominance in the ventricle vascularization. In doing so, we analyzed 30 hearts of cats without pedigree, males and females, adults of several ages. They were not carrying any heart problems. The hearts were injected by the thoracic aorta with Neoprene Latex 450, stained with red pigment, and then they were dissected. It was found that when there was a prevalence of ventricular vascularization of the left type (63.34%) the sinoatrial node irrigation was predominantly in the dependency of the Ramus proximalis atrii dextri (78.9%) or with less frequency by Ramus proximalis atrii sinister (21.1%). In the ventricular vascularization of the balanced type (33.34%), the pacemaker irrigation was in dependence more often of Ramus proximalis atrii dextri (80%) or with less frequency the nutrition of the sinoatrial node occurred by Ramus proximalis atril sinister (20%). In a single-case, we observed the ventricular vascularization of the right type (3.34%), the pacemaker nutrition was in an exclusive dependence of the Ramus intermedius atril dextri. These results suggest in this species there is no relationship between both the sinoatrial node irrigation and the type of ventricular vascularization, regardless of gender.

Parathyroid hormone and phosphorus overload in uremia: impact on cardiovascular system

Background. Cardiac remodeling in uremia is characterized by left ventricular hypertrophy, interstitial fibrosis and microvascular disease. Cardiovascular disease is the leading cause of death in uremic patients, but coronary events alone are not the prevalent cause, sudden death and heart failure are. We studied the cardiac remodeling in experimental uremia, evaluating the isolated effect of parathyroid hormone (PTH) and phosphorus. Methods. Wistar rats were submitted to parathyroidectomy (PTx) and 5/6 nephrectomy (Nx); they also received vehicle (V) and PTH at normal (nPTH) or high (hPTH) doses. They were fed with a poor-phosphorus (pP) or rich-phosphorus (rP) diet and were divided into the following groups: 'Sham': G1 (V + normal-phosphorus diet (np)) and 'Nx + PTx': G2 (nPTH + pP), G3 (nPTH + rP), G4 (hPTH + pP) and G5 (hPTH + rP). After 8 weeks, biochemical analysis, myocardium morphometry and arteriolar morphological analysis were performed. In addition, using immunohistochemical analysis, we evaluated angiotensin II, alpha-actin, transforming growth factor-beta (TGF-beta) and nitrotyrosine, as well as fibroblast growth factor-23 (FGF-23), fibroblast growth factor receptor-1 (FGFR-1) and runt-related transcription factor-2 (Runx-2) expression. Results. Nx animals presented higher serum creatinine levels as well as arterial hypertension. Higher PTH levels were associated with myocardial hypertrophy and fibrosis as well as a higher coronary lesion score. High PTH animals also presented a higher myocardial expression of TGF-beta, angiotensin II, FGF-23 and nitrotyrosine and a lower expression of alpha-actin. Phosphorus overload was associated with higher serum FGF-23 levels and Runx-2, as well as myocardial hypertrophy. FGFR-1 was positive in the cardiomyocytes of all groups as well as in calcified coronaries of G4 and G5 whereas Runx-2 was positive in G3, G4 and G5. Conclusion. In uremia, PTH and phosphorus overload are both independently associated with major changes related to the cardiac remodeling process, emphasizing the need for a better control of these factors in chronic kidney disease.

Chronic Stress Improves the Myocardial Function without Altering L-type Ca+2 Channel Activity in Rats

Background: Chronic stress is associated with cardiac remodeling; however the mechanisms have yet to be clarified. Objective: The purpose of this study was test the hypothesis that chronic stress promotes cardiac dysfunction associated to L-type calcium Ca2+ channel activity depression. Methods: Thirty-day-old male Wistar rats (70 - 100 g) were distributed into two groups: control (C) and chronic stress (St). The stress was consistently maintained at immobilization during 15 weeks, 5 times per week, 1h per day. The cardiac function was evaluated by left ventricular performance through echocardiography and by ventricular isolated papillary muscle. The myocardial papillary muscle activity was assessed at baseline conditions and with inotropic maneuvers such as: post-rest contraction and increases in extracellular Ca2+ concentration, in presence or absence of specific blockers L-type calcium channels. Results: The stress was characterized for adrenal glands hypertrophy, increase of systemic corticosterone level and arterial hypertension. The chronic stress provided left ventricular hypertrophy. The left ventricular and baseline myocardial function did not change with chronic stress. However, it improved the response of the papillary muscle in relation to positive inotropic stimulation. This function improvement was not associated with the L-type Ca2+ channel. Conclusion: Chronic stress produced cardiac hypertrophy; however, in the study of papillary muscle, the positive inotropic maneuvers potentiated cardiac function in stressed rats, without involvement of L-type Ca2+ channel. Thus, the responsible mechanisms remain unclear with respect to Ca2+ influx alterations. (Arq Bras Cardiol 2012;99(4):907-914)

Global and Regional Ventricular Repolarization Study by Body Surface Potential Mapping in Patients with Left Bundle-Branch Block and Heart Failure Undergoing Cardiac Resynchronization Therapy

Background: The controversial effects promoted by cardiac resynchronization therapy (CRT) on the ventricular repolarization (VR) have motivated VR evaluation by body surface potential mapping (BSPM) in CRT patients. Methods: Fifty-two CRT patients, mean age 58.8 +/- 12.3 years, 31 male, LVEF 27.5 +/- 9.2, NYHA III-IV heart failure with QRS181.5 +/- 14.2 ms, underwent 87-lead BSPM in sinus rhythm (BASELINE) and biventricular pacing (BIV). Measurements of mean and corrected QT intervals and dispersion, mean and corrected T peak end intervals and their dispersion, and JT intervals characterized global and regional (RV, Intermediate, and LV regions) ventricular repolarization response. Results: Global QTm (P < 0.001) and QTcm (P < 0.05) were decreased in BIV; QTm was similar across regions in both modes (P = ns); QTcm values were lower in RV/LV than in Intermediate region in BASELINE and BIV (P < 0.001); only RV/Septum showed a significant difference (P < 0.01) in the BIV mode. QTD values both of BASELINE (P < 0.01) and BIV (P < 0.001) were greater in the Intermediate than in the LV region. CRT effect significantly reduced global/regional QTm and QTcm values. QTD was globally decreased in RV/LV (Intermediate: P = ns). BIV mode significantly reduced global T peak end mean and corrected intervals and their dispersion. JT values were not significant. Conclusions: Ventricular repolarization parameters QTm, QTcm, and QTD global/regional values, as assessed by BSPM, were reduced in patients under CRT with severe HF and LBBB. Greater recovery impairment in the Intermediate region was detected by the smaller variation of its dispersion.

Effects of Repeated Stress on Distal Airway Inflammation, Remodeling and Mechanics in an Animal Model of Chronic Airway Inflammation

Background/Aims: Epidemiological studies suggest that stress has an impact on asthmatic exacerbations. We evaluated if repeated stress, induced by forced swimming, modulates lung mechanics, distal airway inflammation and extracellular matrix remodeling in guinea pigs with chronic allergic inflammation. Methods: Guinea pigs were submitted to 7 ovalbumin or saline aerosols (1-5 mg/ml during 4 weeks; OVA and SAL groups). Twenty-four hours after the 4th inhalation, guinea pigs were submitted to the stress protocol 5 times a week during 2 weeks (SAL-S and OVA-S groups). Seventy-two hours after the 7th inhalation, guinea pigs were anesthetized and mechanically ventilated. Resistance and elastance of the respiratory system were obtained at baseline and after ovalbumin challenge. Lungs were removed, and inflammatory and extracellular matrix remodeling of distal airways was assessed by morphometry. Adrenals were removed and weighed. Results: The relative adrenal weight was greater in stressed guinea pigs compared to non-stressed animals (p < 0.001). Repeated stress increased the percent elastance of the respiratory system after antigen challenge and eosinophils and lymphocytes in the OVA-S compared to the OVA group (p < 0.001, p = 0.003 and p < 0.001). Neither collagen nor elastic fiber contents were modified by stress in sensitized animals. Conclusions: In this animal model, repeated stress amplified bronchoconstriction and inflammatory response in distal airways without interfering with extracellular matrix remodeling. Copyright (C) 2011 S. Karger AG, Basel

Clinical Usefulness of Coronary Angiography in Patients with Left Ventricular Dysfunction

Background: Performing a coronary angiography in patients with heart failure of unknown etiology is often justified by the diagnostic assessment of ischemic heart disease. However, the clinical benefit of this strategy is not known. Objective: To evaluate the prevalence of ischemic heart disease by angiographic criteria in patients with heart failure and reduced ejection fraction of unknown etiology, as well as its impact on therapy decisions. Methods: Consecutive outpatients with heart failure and systolic dysfunction, who had an indication for coronary angiography to clarify the etiology of heart disease were assessed from 1 January 2009 to December 31, 2010. Patients diagnosed with coronary artery disease, positive serology for Chagas disease, congenital heart disease, valve disease or patients undergoing cardiac transplantation were excluded from the analysis. The sample was divided into two groups according to the indication for catheterization. Group-1: Symptomatic due to angina or heart failure. Group-2: Presence of >= 2 risk factors for coronary artery disease Results: One hundred and seven patients were included in the analysis, with 51 (47.7%) patients in Group 1 and 56 (52.3%) in Group 2. The prevalence of ischemic heart disease was 9.3% (10 patients), and all belonged to Group 1 (p = 0.0001). During follow-up, only 4 (3.7%) were referred for CABG; 3 (2.8%) patients had procedure-related complications. Conclusion: In our study, coronary angiography in patients with heart failure and systolic dysfunction of unknown etiology, although supported by current guidelines, did not show benefits when performed only due to the presence of risk factors for coronary artery disease. (Arq Bras Cardiol 2012;98(5):437-441)