Η ενδοθηλίνη-1 (ΕΤ-1) εμπλέκεται στην παθογένεση του βρογχικού άσθματος και της χρόνιας αποφρακτικής πνευμονοπάθειας (ΧΑΠ). Η ΕΤ-1 είναι μέλος μίας οικογενείας πεπτιδίων 21 αμινοξέων. Αρχικά σχηματίζεται ένα πεπτίδιο 208 αμινοξέων, η προ-προενδοθηλίνη. Στη συνέχεια, με τη δράση της μετατρεπτάσης της φουρίνης, σχηματίζεται ένα πεπτίδιο 38 αμινοξέων η big-ενδοθηλίνη και στη συνέχεια με τη δράση του μετατρεπτικού ενζύμου ενδοθηλίνης μετατρέπεται σε πεπτίδιο 21 αμινοξέων την ενδοθηλίνη η οποία κυκλοφορεί στο πλάσμα. Η ΕΤ-1 συνδέεται σε δύο τύπους υποδοχέων Α και Β. Οι υποδοχείς τύπου Α επικρατούν στα λεία μυϊκά κύτταρα των αγγείων και των βρόγχων. Οι υποδοχείς τύπου Β επικρατούν κυρίως στα ενδοθηλιακά κύτταρα και στα λεία μυϊκά κύτταρα των αεραγωγών. Κύριες θέσεις παραγωγής της ΕΤ-1 είναι το βρογχικό επιθήλιο, το ενδοθήλιο των πνευμονικών αρτηριών, τα ενδοθηλιακά και τα λεία μυϊκά κύτταρα των αγγείων. Οι δράσεις της ΕΤ-1 στους βρόγχους αφορούν στη συστολή των λείων μυϊκών ινών των αεραγωγών, στην αναδιαμόρφωση του τοιχώματος των βρόγχων, στην έκκριση βλέννης, στη διέγερση-απελευθέρωση άλλων μεσολαβητών φλεγμονής, σε μεταβολές στην διαπερατότητα των μικροαγγείων των αεραγωγών, στην νευρορρύθμιση και τέλος στην υπεραντιδραστικότητα των αεραγωγών. Στην παρούσα μελέτη εξετάστηκαν τα επίπεδα ΕΤ-1 ορού αρτηριακού αίματος 40 ασθματικών ασθενών στην έξαρση και στην ύφεση της νόσου. Σύμφωνα με τα αποτελέσματα της μελέτης μας, τα επίπεδα της ΕΤ-1 στην έξαρση της νόσου ήταν αυξημένα σε σχέση με αυτά στην ύφεση. Υπάρχει θετική συσχέτιση ανάμεσα στην ΕΤ-1 έξαρσης και ύφεσης ανά ασθενή. Αποδείχθηκε αρνητική συσχέτιση ανάμεσα στην ΕΤ-1 έξαρσης και SatO2 έξαρσης καθώς και στην ΕΤ-1 ύφεσης και SatO2 ύφεσης, καθώς και μεταξύ ΕΤ-1 έξαρσης, FEV1 και FVC. Δεν βρέθηκε στατιστικά σημαντική συσχέτιση μεταξύ ΕΤ-1 και καπνίσματος. Οι άντρες είχαν υψηλότερα επίπεδα ΕΤ-1 κατά την έξαρση της νόσου και κατά την ύφεση από ό,τι οι γυναίκες. Δεν υπάρχει στατιστικά σημαντική συσχέτιση ανάμεσα στην χρόνια θεραπεία με κορτικοστεροειδή και στα επίπεδα ΕΤ-1 έξαρσης. Τέλος τα επίπεδα της ΕΤ-1 ύφεσης δεν συσχετίστηκαν με την διάρκεια ούτε τη δοσολογία της θεραπείας έξαρσης με κορτικοστεροειδή. Πιθανώς, η επινόηση ανταγωνιστών υποδοχέων ΕΤ-1, εκλεκτικών ή μη, να έχει ιδιαίτερη σημασία στην θεραπεία του βρογχικού άσθματος υπό την έννοια της πρόληψης πνευμονικής υπερτάσεως σε ασθενείς με βαρύ άσθμα. / Endothelin-1 (ET-1) has been implicated in the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD). ET-1 is a member of a family of peptides of 21 amino-acids. The initial stage in the synthesis of ET-1 involves the formation of a 208-amino acid peptide, named pre-proendothelin, which is processed, via the activity of furin, to the 38-amino acid prohornon, big-endothelin-1, which is secreted and circulates in plasma. Big-ET-1 is then cleaved between Trp and Val to form ET-1 via an endopeptidase called “ET converting enzyme”. ET-1 binds to two types of receptors A and B. Receptors type A are expressed on vascular smooth muscle cells of vessels and bronchuses. Receptors type B are expressed predominantly on endothelial cells and to a much lesser extend on vascular smooth muscle cells. Main places of ET- 1 production are the bronchial epithelium, the epithelium of pulmonary arteries, the vascular endothelial and smooth muscle cells. ET-1 induces airway smooth muscle cell contraction, airway wall remodeling, mucus secretion, stimulation of the release of other mediators, changes in airway microvascular permeability neuromodulation and finally airway hyperresponsiveness. In the present study, we examined ET-1 arterial blood levels of 40 asthmatic patients during the exacerbation and the remission of the disease. According to the results of our study, the ET-1 levels during the exacerbation of the disease were increased concerning them, during the remission. ET-1 levels were negatively statistically significantly correlated with SatO2 during the exacerbation and the remission of the disease as well as between ET-1 levels, FEV1 and FVC during the exacerbation of the disease. There were not found statistically significant correlation between ET-1 and smoking. Men had higher ET-1 levels during the exacerbation and the remission of the disease, than women. There were not statistically significant correlation between chronic treatment with corticosteroides and the ET-1 exacerbation levels, as well as between treatment with corticosteroides during the exacerbation and the ET-1 remission levels. Probably, the invention of ET-1 receptor inhibitors (selected or not) has a particularly important meaning concerning treatment of bronchial asthma under the meaning of prevention of pulmonary hypertension in patients with heavy asthma.

Since its discovery in 1988 the powerful vasoconstrictor endothelin-1 (ET-1) has been widely implicated in the pathophysiology of chronic kidney disease (CKD) as well as the cardiovascular disease with which it is associated. ET receptor antagonists have favourable effects in experimental models of these conditions and orally acting antagonists are now licensed for the treatment of pulmonary arterial hypertension. However, there is a paucity of human data regarding the role of ET-1 in CKD. In this thesis, I have therefore explored the utility of ET-1 as a biomarker in CKD, and, using selective ET receptor antagonists, the beneficial renal and cardiovascular effects of ET receptor antagonism in CKD. I have shown that as glomerular filtration rate (GFR) declines plasma ET-1 increases linearly whereas urinary ET-1 shows an exponential increase. Furthermore, urinary ET-1 may be a useful marker of disease activity in patients with lupus nephritis. Its levels are high in those with biopsy-proven active renal inflammation and these fall with treatment. I have shown that in subjects with stable non-diabetic proteinuric CKD, acute selective ETA receptor antagonism reduces blood pressure and arterial stiffness and that these systemic benefits are associated with an increase in renal blood flow and reduction in proteinuria. Importantly, these effects are seen on top of those achieved with maximal therapy with angiotensin converting enzyme inhibitors and/or angiotensin receptor blockers. Following a study confirming unchanged pharmacokinetics in CKD, I have used an oral selective ETA receptor antagonist to show that the reductions in BP, arterial stiffness and proteinuria seen in my acute studies are maintained longer term. This results of this study also suggest that the mechanism for the reduction in proteinuria is haemodynamic and relates to a reduction in GFR and filtration fraction. In summary, these studies suggest that ET-1 may act as a potential biomarker of renal inflammation, and confirm its role in the pathophysiology of the systemic and renal vasoconstriction seen in CKD. They also suggest that selective ETA receptor antagonism may provide a novel therapeutic approach in proteinuric CKD on top of standard therapies. Larger and longer term studies are now warranted to confirm this potential.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Introdução. A prática regular do exercício de força (EF) está associada a adaptações metabólicas, neuromusculares e cardiovasculares que repercutem de maneira positiva sobre a saúde e qualidade de vida de seus praticantes. No entanto, Indivíduos sedentários apresentam comprometimentos agudos na função endotelial após EF de alta intensidade. Objetivo. Avaliar a função endotelial de indivíduos sedentários de meia idade em resposta a diferentes intensidades do EF. Métodos. 11 indivíduos sedentários (40,1±3,9 anos; 27,3±1,4 kg/m2) realizaram EF em três condições experimentais: extensão de joelhos a 50% de 1RM (MI), 80% de 1RM (AI) e repouso na condição controle (CON). Foi realizada avaliação da vasodilatação mediada pelo fluxo (FMD) antes, 30 minutos após e 60 minutos após os protocolos. A quantificação das concentrações de NO2 e NO3 (NOx), endotelina-1 (ET-1) e TBARS foram realizadas antes, imediatamente após e 60 minutos após os protocolos. A pressão arterial foi mensurada antes e após os protocolos Resultados. A FMD aumentou significativamente 30 minutos após o exercício na condição MI (12,5± 4,10 para 17,2±3,9 %; p=0,01) bem como os níveis de NOx (6,8± 3,3 vs. 12,6± 4,2μM; p= 0,007). A concentração de ET-1 aumentou imediatamente após na condição AI (20,02±2,2 vs. 25,4± 2,1pg/ml; p= 0,004). A elevação da pressão arterial não diferiu entre as condições MI e AI. As concentrações de TBARS não se alteraram ao longo dos protocolos. Conclusão. O EF de moderada intensidade aumenta a FMD e os níveis NOx após uma sessão aguda de exercício em indivíduos sedentários de meia idade, estes resultados sugerem que menores intensidades do EF são mais seguras ao iniciar um programa de exercícios. / Regular resistance exercise (RE) is associated with metabolic, neuromuscular and cardiovascular adaptation that results in improvement of quality of life and health. However, sedentary subjects have been showing an acute impairment on endothelial function after high intensity resistance exercise. The aim of this study was to evaluate the endothelial function in sedentary middle age men after RE in different intensities. Methods. Eleven middle age sedentary men (40,1±3,9 years; 27,3±1,4 kg/m2) performed RE in three different conditions: knee extension at 50% of one 1RM (MI), at 80% of 1RM (HI) and rest in the control group (CON). Flow mediated dilation (FMD) was assessed before, 30 and 60 minutes of exercise. Venus plasma concentration of ET-1 NOx and TBARS were measured before, immediately after and 60 minutes after exercise. Blood pressure was evaluated before and after exercise. Results. There was a significant improvement in FMD 30 minutes after exercise in the MI condition (12,5± 4,10 vs 17,2±3,9%; p= 0,016; p=0,01). The plasma NOx concentration was significant higher immediately after MI (6,8± 3,3 vs. 12,6± 4,2μM; p= 0,007). There was a significant improvement in the plasma ET-1 concentration immediately after HI (20,02±2,2 vs. 25,4± 2,1pg/ml; p= 0,004). There was no significant difference in the BP between the experimental conditions (MI vs HI) and TBARS throughout the experimental conditions. Conclusions. Resistance exercise performed in moderate intensity improve endothelial function in sedentary middle aged men, there results suggest that lower intensities of RE could be safe for this population in the beginning of the exercise programs.

Introdução. A prática regular do exercício de força (EF) está associada a adaptações metabólicas, neuromusculares e cardiovasculares que repercutem de maneira positiva sobre a saúde e qualidade de vida de seus praticantes. No entanto, Indivíduos sedentários apresentam comprometimentos agudos na função endotelial após EF de alta intensidade. Objetivo. Avaliar a função endotelial de indivíduos sedentários de meia idade em resposta a diferentes intensidades do EF. Métodos. 11 indivíduos sedentários (40,1±3,9 anos; 27,3±1,4 kg/m2) realizaram EF em três condições experimentais: extensão de joelhos a 50% de 1RM (MI), 80% de 1RM (AI) e repouso na condição controle (CON). Foi realizada avaliação da vasodilatação mediada pelo fluxo (FMD) antes, 30 minutos após e 60 minutos após os protocolos. A quantificação das concentrações de NO2 e NO3 (NOx), endotelina-1 (ET-1) e TBARS foram realizadas antes, imediatamente após e 60 minutos após os protocolos. A pressão arterial foi mensurada antes e após os protocolos Resultados. A FMD aumentou significativamente 30 minutos após o exercício na condição MI (12,5± 4,10 para 17,2±3,9 %; p=0,01) bem como os níveis de NOx (6,8± 3,3 vs. 12,6± 4,2μM; p= 0,007). A concentração de ET-1 aumentou imediatamente após na condição AI (20,02±2,2 vs. 25,4± 2,1pg/ml; p= 0,004). A elevação da pressão arterial não diferiu entre as condições MI e AI. As concentrações de TBARS não se alteraram ao longo dos protocolos. Conclusão. O EF de moderada intensidade aumenta a FMD e os níveis NOx após uma sessão aguda de exercício em indivíduos sedentários de meia idade, estes resultados sugerem que menores intensidades do EF são mais seguras ao iniciar um programa de exercícios. / Regular resistance exercise (RE) is associated with metabolic, neuromuscular and cardiovascular adaptation that results in improvement of quality of life and health. However, sedentary subjects have been showing an acute impairment on endothelial function after high intensity resistance exercise. The aim of this study was to evaluate the endothelial function in sedentary middle age men after RE in different intensities. Methods. Eleven middle age sedentary men (40,1±3,9 years; 27,3±1,4 kg/m2) performed RE in three different conditions: knee extension at 50% of one 1RM (MI), at 80% of 1RM (HI) and rest in the control group (CON). Flow mediated dilation (FMD) was assessed before, 30 and 60 minutes of exercise. Venus plasma concentration of ET-1 NOx and TBARS were measured before, immediately after and 60 minutes after exercise. Blood pressure was evaluated before and after exercise. Results. There was a significant improvement in FMD 30 minutes after exercise in the MI condition (12,5± 4,10 vs 17,2±3,9%; p= 0,016; p=0,01). The plasma NOx concentration was significant higher immediately after MI (6,8± 3,3 vs. 12,6± 4,2μM; p= 0,007). There was a significant improvement in the plasma ET-1 concentration immediately after HI (20,02±2,2 vs. 25,4± 2,1pg/ml; p= 0,004). There was no significant difference in the BP between the experimental conditions (MI vs HI) and TBARS throughout the experimental conditions. Conclusions. Resistance exercise performed in moderate intensity improve endothelial function in sedentary middle aged men, there results suggest that lower intensities of RE could be safe for this population in the beginning of the exercise programs.

Introdução: Os fitoesteróis são indicados no tratamento da hipercolesterolemia. Embora sua ação sobre a redução do colesterol já tenha sido exaustivamente estudada, não se encontra bem elucidada na literatura, a forma como atua sobre biomarcadores inflamatórios e endoteliais em indivíduos hipercolesterolêmicos. Objetivo: Avaliar o efeito do fitoesterol adicionado ao leite de soja sobre as concentrações plasmáticas de lípides e biomarcadores, além de alterações em vias transcricionais envolvidos na patogênese da aterosclerose em células linfomononucleares em indivíduos moderadamente hipercolesterolêmicos. Metodologia: Foram estudados 38 pacientes, recrutados no Ambulatório do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Inicialmente, os pacientes foram submetidos a um período basal de três semanas para avaliar a aderência e tolerabilidade ao leite de soja padrão (Placebo). Foram orientados a manter os hábitos alimentares e a atividade física. Receberam 400 mL/dia de leite de soja padrão ou enriquecido com fitoesteróis (1,6 g/dia) por dois períodos de quatro semanas em que o estudo foi randomizado, cego e cruzado. Resultados: O peso corporal foi mantido durante o estudo. O tratamento com fitoesterol reduziu o colesterol total em 5,5 % (P<0,001), a lipoproteína de baixa densidade (LDL) em 6,4 % e os triglicérides em 8,3 % (P<0,05) e aumentaram as concentrações de campesterol e sitosterol no plasma em 18,9% e 21,6%, respectivamente. Não houve alteração nos valores de lipoproteína de alta densidade (HDL). Com relação aos biomarcadores de aterosclerose, os fitoesteróis provocaram redução apenas na endotelina-1 em 11 % (P<0,05). Esta ação foi independente da redução do LDL-C. Não houve alteração no RNAm para 3-hydroxy-3-methylglutaryl coenzyme A redutase e receptor de LDL. Conclusão: Os fitoesteróis são eficientes na redução da concentração plasmática do colesterol total, triglicérides, LDL-C e da endotelina-1, sendo esta última independente da redução do colesterol / Background: Phytosterols are recommended in the treatment of moderate hypercholesterolemia. Although their mechanisms of action on cholesterol reduction has been exhaustively studied, other activities, such as their effects on inflammatory arterial markers and on endothelial function, have not yet been fully investigated. Objective: To evaluate the efficacy of phytosterols added to soy milk on the reduction of plasma lipids, on biomarkers involved in the pathogenesis of atherosclerosis and on transcriptional changes in 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase and low density lipoprotein receptor in moderately hypercholesterolemic individuals. Design: Moderately hypercholesterolemic patients (n=38, mean age 58 y) were initially followed over a 3-wk baseline period to evaluate their adherence to the program and their tolerance to the added milk. Patients received soy milk (400 mL/d) or soy milk + phytosterol (1.6 g/d) for 2 periods of 4-wk each. The study was randomized, blinded and crossed. Results: Body weight was maintained during the study. Phytosterol treatment reduced the total cholesterol concentration 5.5 % (P < 0.001), low density lipoprotein (LDL) 6.4 % and triglycerides 8.3 % (P<0.05), without modifying high density lipoprotein (HDL). Phytosterol reduced plasma endothelin-1 by 11 % (P<0.05), independently of LDL-C lowering activity. No effects on LDL receptor or on HMGCoA reductase mRNA expression in mononuclear blood cells were found. Sitosterol and campesterol plasma concentrations increased with phytosterol consumption by 18.9% and 21.6%, respectively. Conclusions: Phytosterol effectively lowered plasma total cholesterol, LDL-C, triglycerides and endothelin-1 concentration. The reduction in endothelin-1 concentration was independent of the decrease in the LDL-C concentration

Abstract During cardiac hypertrophy individual cardiac myocytes increase in size, which is accompanied by augmented protein synthesis and selective induction of a subset of genes. These phenotypic changes of myocytes are a result from altered intracellular signaling mechanisms and molecules. B-type natriuretic peptide (BNP) gene was selected as a target gene for the study of cardiac signaling mechanisms, since it is activated by mechanical, neural and humoral stimuli during myocyte hypertrophy. To generate hypertrophy of cardiac myocytes, neonatal rat cardiac myocytes were subjected to exogenous hypertrophic agonists such as endothelin-1 (ET-1) or to cyclic mechanical stretch. The role and regulation of transcription factors were studied by utilizing promoter analysis together with site-specific mutations and measurement of DNA binding activity and phosphorylation. GATA-4 mediated signaling was inhibited by blocking DNA binding with decoy oligonucleotides or by decreasing GATA-4 synthesis via adenoviral antisense delivery. ET-1 activated GATA-4 via serine residue phosphorylation, and this effect was mediated via p38 kinase. Similarly, GATA-4 binding activity was increased by ET-1 and mechanical stretch, but it was essential for activation of BNP gene only in the latter stimulation. Importantly, downregulation of GATA-4 protein levels prevented mechanical stretch induced hypertrophy of cardiac myocytes. In contrast, separate mechanism for an ET-1 specific signaling was composed of p38 kinase regulated ETS-like transcription factor-1 (Elk-1). Finally, the effect of mechanical stretch on endogenous endothelin-1 (ET-1) synthesis in cardiac cells was studied. Intrinsic ET-1 synthesis was activated in stretched cardiac myocytes, yet the levels of ET-1 were relatively low. This work suggests that GATA-4 transcription factor is required for mechanical stretch mediated hypertrophic program, and Elk-1 may act as a downstream effector of ET-1 in cardiac myocytes. Taken together, induction of ET-1 and BNP genes as well as activation of GATA-4 and Elk-1 transcription factors are regulated via a network of mitogen activated protein kinase pathways.

Abstract Cardiac myocytes react to increased workload and hypertrophic neurohumoral stimuli by increasing protein synthesis, reinitiating expression of fetal forms of structural genes, α-skeletal actin (α-SkA) and β-myosin heavy chain (β-MHC), and by increasing expression and secretion of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP). Initially, the response is beneficial, but when prolonged, it leads to pathological cardiomyocyte hypertrophy. In this study, cardiomyocyte hypertrophy was initiated by hypertrophic agonists, endothelin-1 (ET-1) and phenylephrine (PE), and by increased stretching of atrial wall. Transcription factor GATA4 was studied to identify the mechanism leading to increased gene expression of BNP. In BNP promoter, GATA4 binds to cis elements mediating hypertrophic response. Eliminating GATA4 binding by using the decoy approach, basal BNP gene expression was reduced. To identify mechanisms regulating GATA4, the roles of mitogen-activated protein kinases (MAPKs) were studied. Activation of p38 MAPK increased GATA4 binding to BNP gene and led to increased GATA4 dependent BNP gene expression. p38 MAPK was required for ET-1 induced GATA4 binding, whereas extracellular signal-regulated kinase (ERK) was required for maintaining basal GATA4 binding activity. PE and ET-1 activated protein kinase C (PKC) signaling in cardiac myocytes. Antisense oligonucleotide inhibition of PKCα markedly reduced PE induced ANP secretion and ET-1 induced BNP secretion, whereas gene expression of natriuretic peptides was not affected. Antisense PKCα treatment inhibited PE induced expression of α-SkA, while increased protein synthesis or β-MHC gene expression were not affected. Sretching of the perfused rat atria increased BNP, c-fos and BNP gene expression via mechanism involving p38 MAP kinase activation of transcription factor Elk-1. In cultured neonatal rat atrial myocytes stretch induced BNP gene expression was dependent upon transcription factor Elk-1 binding sites within the BNP gene promoter. In conclusion, hypertrophic signaling in cardiac myocytes involves multiple signaling cascades. Activation of p38 MAPK is required for the development of ET-1 induced hypertrophic phenotype and GATA4 mediated BNP gene expression in cultured ventricular myocytes, and for stretch induced Elk-1 dependent BNP gene expression in atrial myocytes. PKCα is involved in PE induced hypertrophic response and PE induced switch in gene programming inducing expression of α-SkA, the fetal form of cardiac α-actin.