To this day heart failure with preserved ejection fraction (HFpEF) remains a poorly understood malady. Half of all heart failure (HF) cases are HFpEF, and the prevalence of HF is on the rise. Unlike HF with reduced ejection fraction, HFpEF has no treatment options and is often times difficult to diagnose because victims of HFpEF often have pre-existing conditions. Vascular endothelial growth factor (VEGF) has been implicated in maintaining myocardial health and is thought to play a role in HFpEF. We sought to test the hypothesis that VEGF-A plays a role in HFpEF in a hypertensive murine model of HFpEF. Using Western blot analysis we found that there was an up regulation of VEGF-A in the homogenized left ventricle (LV) of our HFpEF mice. Unexpectedly, there was a down regulation of VEGF-A in the homogenized tissue from the aorta in those mice. To study the circulating levels of VEGF in our HFpEF mice we used an ELISA. We found that our HFpEF mice had similar levels of circulating VEGF as our control. This suggests that VEGF has paracrine/autocrine role in our HFpEF model rather than endocrine, like our human data suggested. To identify the cells responsible for the expression profile we saw in the homogenized tissue data we looked at the response of adult rat ventricular myocytes (ARVM) and vascular smooth muscle cells (VSMC) to aldosterone stimulation at short (1hr) and long (24hr) time points at both physiological (50nm) and pathological (1μm) concentrations. To do this analysis we recruited the help of Western blot, ELISA and RT-PCR techniques to construct a consistent VEGF expression profile. The Western blot ARVM data showed statistically significant (P<0.05) increase in VEGF-A to pathological doses of aldosterone, especially at the longer time point. When we tested the VSMC using Western blot analysis, we found that the trend of our n=1 sample suggested a strong response to the physiological dose of aldosterone in the short term. Using the more sensitive ELISA technique to measure the VEGF content of our VCMS we increasing our sample size to n=4 and found no statistically significant (p=NS) response to aldosterone stimulation from the VSMC. However, looking at the trends in the data it is clear that VSMC increases VEGF in response to long-term physiological doses of aldosterone. This is contrary to what we found using Western blot analysis, so we queried the VEGF mRNA from the VSMC to settle the score. Unfortunately, this too proved fruitless. The RT-PCR data was not significant and the trend was that of the ARVM expression profile. We initially turned to VSMC because we hypothesized that they could contribute to the paracrine/autocrine activity similar to what we saw in the LV from the ARVM. It is unclear if VSMC play a role in HFpEF progression, but their lack of consistent response to aldosterone could potential explain the down regulation of VEGF-A we observed in the aorta of our HFpEF mice. We initially sough to test the hypothesis that VEGF-A plays a role in our HFpEF mouse model, what we found was that ARVM contribute to localized VEGF-A increased production in the LV while in the aorta there is a down regulation of VEGF-A in our HFpEF model, we are unable to make any conclusion about VSMC response to aldosterone because of insufficient sample size. Thus in conclusion, it appears that VEGF-A does play a role in our HFpEF model specifically in a paracrine/autocrine manner in the LV where the ARVM contributes to the increased production of the cytokine.

Perivascular adipose tissue (PVAT) and vascular endothelial cells both have important structural and functional roles in blood vessels and are the focus of this doctoral thesis. Firstly, PVAT has been rediscovered as an endocrine organ, releasing vasorelaxing substances. Secondly, the endothelial monolayer functions as an important barrier, the role of which is to restrict the transfer of molecules or even blood-borne cells between the lumen of the blood vessel and the surrounding tissue. In my main study, the presence of PVAT caused 'anti-contractile' effects, which were reversed by nitric oxide synthase (NOS) inhibition in rat mesenteric arteries and were lost in adiponectin-knockout mice. The β3 adrenoceptor agonist CL-316,243 increased PVAT-dependent anti-contractile effects and caused myocyte hyperpolarisation. Hyperpolarisation to CL-316,243 could be mimicked by the adipokine, adiponectin, and by the 5'AMP kinase (AMPK) activator, A-769662. In addition, the AMPK inhibitor, dorsomorphin, and the selective BKCa channel blocker, iberiotoxin, each blocked hyperpolarisations to CL-316,243, adiponectin and A-769662. The anti-contractile effects of CL-316,243 could also be mimicked by A-769662 but were not blocked by dorsomorphin. Moreover CL-316,243 still had anti-contractile effects in adiponectin-knockout mice. However, inhibiting the production of both NO and hydrogen peroxide reduced anti-contractile effects of CL-316,243. In obese Sprague Dawley rats both the hyperpolarising and the anti-contractile effects to CL-316,243 were impaired, while hyperpolarisation to A-769662 were unchanged. Western blots revealed that NOS, a possible downstream target of AMPK, was phosphorylated in PVAT control samples, a form which was decreased in PVAT from obese rats. These results collectively indicate that the anti-contractile and hyperpolarising effects observed following stimulation with CL-316,243 are due to activation of different PVAT-dependent pathways, both of which probably contribute to vasodilatation in blood vessels. Understanding these pathways is crucial for the development of improved treatments for obesity and hypertension. During my work at Novartis, I found that activation of sphingosine-1-receptors type 1 (S1P1) with the activator FTY720 (Fingolimod, Novartis; used in multiple sclerosis treatment) caused closure of the endothelial barrier in human umbilical vein cells. This effect could be mimicked with a recombinant peptide of nectin, an adherens junction protein. The novel S1P1 antagonists 'A1' and 'A2' (Novartis) inhibited the effect of FTY720, but not those of nectin. The discovery of nectin as a potential barrier closure modulator might contribute to the development of additional treatments for use in multiple sclerosis.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Mūsų tyrymų tikslas – ištirti baltymų kinazės A, baltymų kinazės C, Src šeimos nereceptorinės baltymų tirozino kinazės ir jų signaliniuose keliuose dalyvaujančių molekulių įtaką L tipo kalcio srovei (ICa,L) fermentiniu būdu izoliuotuose iširdies miocituose. Šis tikslas buvo įgyvendintas sprendžiant keturias užduotis: 1) ištiriant varlės ir žiurkės skilvelių bei žmogaus prieširdžių miocitų β-adrenerginių receptorių signalinės grandinės elementų: β-adrenerginių receptorių, adenilatciklazės, fosfodiesterazių, baltymų kinazės A, baltymų fosfatazių (baltymų fosfatazės 1 ir baltymų fosfatazės 2A) bei įtampos valdomų L tipo kalcio kanalų bazinį aktyvumą; 2) ištiriant β3-adrenerginių receptorių įtaką žmogaus prieširdžių ICa,L ir susitraukimo jėgai; 3) nustatant Src šeimos nereceptorinės baltymų tirozino kinazės įtaką žmogaus prieširdžių ICa,L, taip pat jos aktyvinimo būdą ir veikimo vietą β-adrenerginių receptorių signalinėje grandinėje; 4) ištiriant baltymų kinazės C įtaka žmogaus prieširdžių ICa,L. / The objective of study was to investigate the role of protein kinase A, protein kinase C, Src family nonreceptor protein tyrosine kinases and other signaling molecules involved in pathways regulating the L-type calcium current (ICa,L) in enzymatically isolated cardiac myocytes. This objective was realized by resolving four tasks: 1) Examination of the basal activity of β-adrenergic receptor (β-AR) signaling cascade involving β-ARs, adenylyl cyclases, phosphodiesterases, protein kinase A, protein phosphatases (protein phosphatase 1 and protein phosphatase 2A) and L-type voltage-dependent calcium channels in frog and rat ventricular myocytes and human atrial myocytes; 2) Investigation of the role of β3-ARs in regulation of ICa,L and force of contraction in human atrium; 3) Exploration of the role of Src family nonreceptor tyrosine kinases in regulation of ICa,L, determining the route of their activation and site of action in β-AR signaling cascade of human atriual myocytes; 4) Probing of the impact of protein kinase C on basal and β-AR stimulated ICa,L in human atrial myocytes. The experiments were performed using whole-cell configuration of the pach-clamp technique.

Orientadores: Jose Wilson Magalhães Bassani, Rosana Almada Bassani / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Eletrica e de Computação / Made available in DSpace on 2018-08-06T07:47:18Z (GMT). No. of bitstreams: 1 Boer_DenileCominato_M.pdf: 557480 bytes, checksum: e378c01e865d04a595d3f0e7ad3c60dd (MD5) Previous issue date: 2006 / Resumo: Embora haja demonstração de que a estimulação simpática tenha efeito facilitatório sobre a indução de atividade espontânea em miocárdio há controvérsia sobre a participação dos tipos de adrenoceptores na mediação deste efeito. No presente trabalho, descrevemos um método desenvolvido para determinação, em átrio esquerdo isolado (AE) de rato, da relação concentração-efeito para agentes que exercem efeito arritmogênico por aumento da mobilização celular de 'Ca POT. 2+¿. O método baseou-se na interposição de pausas estimulatórias, durante as quais registraram-se contrações espontâneas (CE), precedidas ou não por trens estimulatórios de alta freqüência (5 Hz). O protocolo estimulatório foi repetido na presença de diferentes concentrações de agonistas. Para cada concentração de agonista, a resposta arrítmica foi considerada como a soma dos números de CE/min, no total de preparações. Foi analisada também a resposta inotrópica, como sendo o incremento de força ou encurtamento de pico, desenvolvidos em AE e miócitos ventriculares (MV), respectivamente. A relação foi ajustada por uma função sigmóide para cálculo de Min (i.e., valor da variável na ausência do agonista), Rmax (resposta máxima) e pD2 (-log da concentração do agonista que produziu uma resposta igual a 50% de Rmax). Este método foi aplicado no estudo dos tipos de adrenoceptores envolvidos na resposta arrítmica a catecolaminas em AE e MV. A Rmax inotrópica à ativação de adrenoceptores 'alfa IND. 1¿ + 'beta IND. 1¿ foi comparável àquela por ativação de apenas receptores 'beta IND. 1¿, em ambos AE e MV. Já a ativação de adrenoceptores 'alfa IND. 1¿ produziu uma Rmax inotrópica de apenas metade daquela observada pela estimulação 'alfa IND. 1¿ + 'beta IND. 1¿. Da mesma forma, a resposta arrítmica foi semelhante para estimulação de adrenoceptores a1+ß1 e de apenas receptores 'beta IND. 1¿. Entretanto, nenhuma resposta foi obtida pela estimulação de receptores adrenérgico do tipo 'alfa IND. 1¿. Estes resultados indicam que a estimulação de adrenoceptores 'alfa IND. 1¿, apesar de evocar uma resposta inotrópica positiva em ambos AE e MV, não é arritmogênica. A ativação do tipo 'beta¿, por outro lado, parece ser a principal via para estimulação inotrópica simpática e na indução de arritmias. Além disso, concluímos que parece existir antagonismo funcional entre os subtipos de adrenoceptores 'beta¿, manifestado por ações pró- e anti-arrítmicas dos subtipos 'beta IND. 1¿ e 'beta IND. 2¿, respectivamente, em miocárdio (tanto atrial, quanto ventricular) de rato / Abstract: Although it has been shown that sympathetic stimulation facilitates the appearance of myocardial spontaneous activity, it is still not clear which types of adrenoceptors mediate this effect. In this study, we describe a method developed for determination, in isolated rat atria (AE), of the concentration-effect relationship for arrhythmogenic agents that act via promotion of cell 'Ca POT. 2+¿ overload. The method was based on the interposition of stimulatory rest periods, during which spontaneous contractions (CE) were recorded, preceded or not by high frequency (5 Hz) stimulus trains. The stimulation protocol was applied at each agonist concentration, and the arrhythmic response was taken as the sum of the number of CE/min in all preparations. The positive inotropic response was considered as the agonist-dependent increment of developed force or peak shortening in AE and isolated ventricular myocytes (MV), respectively. Concentration-effect curves were determined by fitting a sigmoid function, from which the following parameters were estimated: Min (i.e., value of the variable in the absence of the agonist), Rmax (maximal response) e 'pD IND. 2¿ (-log of the molar agonist concentration that evokes a response equal to 50% of Rmax). This method was applied to investigate the adrenoceptor types involved in the mediation of catecholamine-induced arrhythmogenesis in AE and MV. Inotropic Rmax to activation of 'alfa IND. 1¿ + 'beta IND. 1¿ adrenoceptors was comparable to that of activation of solely 'beta IND. 1¿ adrenoceptors in both AE and MV. However, Rmax to selective 'alfa IND. 1¿ adrenoceptor activation was only half of that produced by 'alfa IND. 1¿ + 'beta IND. 1¿ adrenoceptor stimulation. The arrhythmic responses to 'alfa IND. 1¿ + 'beta IND. 1¿ and 'beta IND.1¿ receptor stimulation were similar, but selective a1 adrenoceptor activation was unable to evoke any spontaneous activity. The results indicate that a1 adrenoceptors, although able to mediate stimulation in both AE and MV, are not involved in arrhythmogenesis. 'beta¿-adrenoceptor activation, thus, seems the main type involved in both inotropic and arrhythmic responses to catecholamines. In addition, our results point out a functional antagonism between 'beta¿-adrenoceptor subtypes: i.e., pro- and anti-arrhythmic effects mediated by 'beta IND. 1¿ and 'beta IND. 2¿-adrenoceptors, respectively in both atrial and ventricular rat myocardium / Mestrado / Engenharia Biomedica / Mestre em Engenharia Elétrica

Orientadores: Jose Wilson Magalhaes Bassani, Rosana Almada Bassani / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Eletrica e de Computação / Made available in DSpace on 2018-08-15T01:38:31Z (GMT). No. of bitstreams: 1 Fonseca_AlexandraValenzuelaSantelicesda_M.pdf: 1311974 bytes, checksum: ab061a6f8d63a1175d6a3c9281e0704e (MD5) Previous issue date: 2009 / Resumo: O procedimento mais efetivo para reverter arritmias cardíacas consiste na aplicação de choques elétricos de alta intensidade, como e o caso da desfibrilação. Estimulação com campos elétricos (E) elevados, entretanto, exerce efeitos deletérios sobre o músculo cardíaco, podendo causar disfunções elétrica e contrátil e até a morte celular. Privilegiar a estimulação na direção longitudinal, para qual o limiar de excitação das células cardíacas e menor, seria uma forma de se reduzir a amplitude do estimulo sem perder a efetividade da estimulação. Para isto, foi desenvolvido e testado, em miócitos ventriculares orientados de maneira aleatória, um sistema de estimulação multidirecional automática que permite o chaveamento controlado de estímulos sequênciais para três diferentes pares de eletrodos (cada um correspondendo a uma direção) em um intervalo de tempo inferior a duração do potencial de ação (período em que a célula se encontra eletricamente refrataria). A estimulação multidirecional com uma intensidade de E 20% acima do limiar estimulatório (1,2× ETM) dobrou o recrutamento (excitação) de células (80 vs. 40% com estimulação unidirecional, p<0,001). Adicionalmente, o recrutamento com a estimulação multidirecional automática foi maior (p< 0,001) do que a soma dos recrutamentos obtidos com a estimulação em cada direção individualmente (sem intersecção), o que sugere que a estimulação sublimiar durante o procedimento automático pode aumentar a excitabilidade celular. Foi observado também que, para uma dada amplitude do estimulo, o uso da forma de onda bipolar (para a qual o valor de ETM foi menor que para pulsos monopolares: 3,2 ± 0,1 vs. 3,9 ± 0,1 V/cm; p< 0,001) promoveu um recrutamento maior do que com o pulso monopolar (recrutamento de 50% das células foi obtido com 2,97 ± 0,04 e 4,18 ± 0,05 V/cm para pulsos bipolares e monopolares, respectivamente; p< 0,05). A combinação da estimulação multidirecional automática com o uso da forma de onda bipolar permitiu, portanto, uma redução de cerca de 50% no valor do E absoluto (3,8 vs. 7,8 V/cm com estimulação unidirecional e pulso monopolar) para um recrutamento de ~80% das células. A aplicação destes procedimentos na estimulação cardíaca (marcapasso e desfibrilação) pode otimizar o processo, levando a uma melhor eficiência e uma menor incidência de lesão. / Abstract: The most effective procedure to revert cardiac arrhythmias consists in the application of high intensity electric discharge, such as in cardiac defibrillation. Nevertheless, stimulation using high electric fields (E) may cause injury to the cardiac muscle, generating electric and contractile dysfunctions and even cell death. A possible way to reduce the stimulus intensity while maintaining the stimulation effectiveness would be stimulate cardiac cells with E applied parallel to the cell major axis, in which case the stimulation threshold is lower. To test this possibility, a multidirectional stimulation system was developed and tested on randomly-oriented rat ventricular myocytes. The system allows the controlled switching of sequential stimuli delivered to three different pairs of electrodes (each one corresponding to one direction), in a period shorter than the action potential duration (when cell is electrically refractory). The multidirectional stimulation with E intensity 20% above the stimulation threshold (1.2× ETM) doubled the percentage of recruited (excited) cells (~80 vs. ~40 % with unidirectional stimulation, p<0.001). Additionally, recruitment with automatic multidirectional stimulation was greater (p< 0.001) than the sum of recruitments obtained from stimulation of each direction individually (without intersection), which is suggestive that subthreshold stimulation during the automatic procedure might enhance cell excitability. Moreover, it was observed that for a given absolute stimulus amplitude, the use of biphasic waveforms (for which ETM was lower than for monophasic pulses: 3.2 ± 0.1 vs. 3.9 ± 0.1 V/cm; p< 0.001) promoted higher recruitment than monophasic stimuli (50% recruitment was attained with 2.97 ± 0.04 and 4.18 ± 0.05 V/cm with biphasic and monophasic pulses, respectively; p< 0.05). Thus, the association of automatic multidirectional stimulation and biphasic waveform enabled a 50% reduction of the absolute E value (3.8 vs. 7.8 V/cm with unidirectional stimulation and monopolar pulse) to evoke excitation in ~80% of the cells. The application of these procedures to cardiac stimulation (pacemaker and defibrillation) might optimize the process, leading to greater efficiency and lower injury incidence. / Mestrado / Engenharia Biomedica / Mestre em Engenharia Elétrica

Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-03-27T15:52:15Z No. of bitstreams: 1 joaogabrielrochasilva.pdf: 4504745 bytes, checksum: 5d371d913693d1bf3a9425d887ab2ed1 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-03-27T17:49:33Z (GMT) No. of bitstreams: 1 joaogabrielrochasilva.pdf: 4504745 bytes, checksum: 5d371d913693d1bf3a9425d887ab2ed1 (MD5) / Made available in DSpace on 2018-03-27T17:49:33Z (GMT). No. of bitstreams: 1 joaogabrielrochasilva.pdf: 4504745 bytes, checksum: 5d371d913693d1bf3a9425d887ab2ed1 (MD5) Previous issue date: 2018-02-23 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A simulação da atividade eletromecânica do coração é uma ferramenta relevante para a interpretação e estudos de medidas fisiológicas e diversos fenômenos cardíacos. Entretanto, modelos computacionais para este propósito podem ser computacionalmente custosos. Assim, são propostos neste trabalho três modelos simplificados, a nível celular, que foram capazes de reproduzir de forma quantitativa o fenômeno da contração de miócitos cardíacos. Para obtenção destes modelos um ajuste de parâmetros foi realizado via algoritmos genéticos. Os modelos propostos com parâmetros ajustados apresentaram resultados satisfatórios para reprodução da força ativa do coração com a vantagem de serem baseados em apenas duas equações diferenciais ordinárias. Além disso, o modelo final foi validado utilizando simulações envolvendo extra-sístoles, sendo capaz de reproduzir o fenômeno de alternância na força ativa. / The simulation of the heart electromechanical activity is a relevant tool for the interpretation and studies of physiological measures and various cardiac phenomena. However, computational models for this purpose may be computationally costly. Thus, three simplified models which were able to quantitatively reproduce the phenomenon of cardiac myocyte contraction were proposed in this work. At the cellular level, they were able to quantitatively reproduce the phenomenon of cardiac myocyte contraction. A parameter adjustment via genetic algorithm was performed to obtain these models. The proposed models with adjusted parameters presented satisfactory results for the reproduction of the active force of the heart with the advantage of being based on only two ordinary differential equations. In addition, the final model was validated using simulations involving extra-systoles, being able to reproduce the phenomenon of alternation in the active stress.

Heart failure is a debilitating condition in which the heart cannot meet the metabolic demands of the body. Chronic β-adrenergic (β-AR) stimulation causes pathological myocardial remodeling that leads to heart failure, in part, by promoting apoptosis of cardiac myocytes. Work from our laboratory has shown that β-AR stimulated apoptosis is dependent on reactive oxygen species (ROS), but the molecular targets by which ROS mediate apoptosis is not known. One target of ROS that may contribute to activating the apoptosis pathway is the sarco-endoplasmic reticulum ATPase (SERCA2). SERCA2 is responsible for moving the large majority of intracellular calcium in the cardiac myocyte. We have identified that SERCA2 can undergo oxidative post-translational modification (OPTM) of cysteine C674: Low ROS increase activity while high ROS decreases. Since SERCA is the primary calcium transporter and is located in close proximity of the mitochondria, it is possible SERCA activity may affect the level of calcium in mitochondria, which in excess is a known activator of the intrinsic mitochondrial apoptosis pathway. Progressive loss of myocardial cells in ischemia and heart failure likely contributes to the pathogenesis of cardiomyopathy. We hypothesized that oxidation of SERCA2 at C674 increases mitochondrial calcium, thereby activating the mitochondrial apoptosis pathway. To address this thesis, we used a novel redox-insensitive SERCA2 mutation in which C674 is replaced by serine (C674S) to determine the role of oxidative inhibition of SERCA in H2O2-stimulated apoptosis in vitro. We tested our hypothesis using adult rat ventricular myocytes (ARVM) that overexpress wild type or SERCA C674 and assessed intra-organelle calcium content, mitochondrial function and activation of the apoptosis pathway. To measure mitochondrial calcium, we optimized the use of an ultrasensitive genetically-encoded calcium indicator (GECI) targeted to the mitochondria which was expressed in ARVM via adenovirus infection. Redox-insensitive SERCA C674S expressing ARVM displayed less sensitivity to H2O2-stimulated mitochondrial calcium uptake which was confirmed by measuring calcium sensitive pyruvate dehydrogenase phosphorylation status. Furthermore, SERCA C674S ARVM were protected from H2O2 -mediated apoptosis, indicated by a reduction in cytochrome c release and annexin V staining. Lastly, H2O2 treatment decreased the cytosolic ATP/ADP ratio and depolarized the mitochondrial membrane potential, however this was independent of SERCA C674 oxidation. Taken together, these experiments elucidate a novel role for SERCA2 activity in cardiac myocytes and provide a potential therapeutic target for reducing cardiac myocyte apoptosis, potentially improving cardiac function during heart failure.

Adipöse Patienten und Typ-2-Diabetiker zeigen ein erhöhtes Risiko für Harnwegsinfekte. Die Ursache der höheren Prävalenz ist noch nicht nachhaltig geklärt. Bekannt ist, dass Typ-2-Diabetiker erhöhte Konzentrationen freier Fettsäuren im Blut aufweisen. Der veränderte Fettstoffwechsel könnte neben bakteriellen Ursachen ein möglicher Grund für abakterielle Entzündungsreaktionen der Harnblase sein. Zur Prüfung dieser Hypothese wurden zeit- und konzentrationsabhängig kultivierte humane Detrusormyozyten im Vergleich zur Lipopolysaccharid (LPS) induzierten Entzündungsreaktion mit Palmitat stimuliert. Es wurde geprüft, ob eine autokrine und/oder endokrine Regulation des IL-6-Signalwegs vorliegt. Im Fokus standen insbesondere die IL-6- und MCP-1-Expression und deren möglichen regulatorischen Proteine gp80, gp130, NF-κB, STAT3, SOCS3 und MEK1. Die Stimulationsversuche mit LPS und Palmitat zeigen einen differenten zeit- und konzentrationsabhängigen Effekt auf die IL-6- und MCP-1-Expression in den humanen Detrusormyozyten. LPS und Palmitat induzieren eine zeitabhängige autokrine Regulation der IL-6-Signalkaskade über phosphoryliertes STAT3 und Feedback-mechanismen via SOCS3. Sowohl LPS als auch Palmitat bewirken über 48h eine mögliche endokrine Regulation des IL-6-Signalwegs. Zusammenfassend zeigt die Palmitatstimulation zeit- und konzentrationsabhängig einen stärkeren Effekt auf die IL-6-Signalwirkung als die Stimulation mit LPS.:Abkürzungsverzeichnis 1. Hintergrund und Ziel der Arbeit 2. Methoden 3. Ergebnisse 3.1 Struktur und Funktion der suburothelialen Myofibroblasten 3.2 Entzündungsmechanismen der Harnblase 3.3 Einfluss von Palmitat auf die IL-6 and MCP-1 Expression humaner Detrusormyozyten - Link zwischen Stoffwechsel und immunologischer Reaktion der Harnblasenmuskulatur 3.4 IL-6 - JAK/STAT - Signalweg in den Detrusormyozyten 3.5 Schlussfolgerungen 4. Literaturverzeichnis 5. Publikationen 5.1 Struktur und Funktion der suburothelialen Myofibroblasten in der humanen Harnblase unter normalen und pathologischen Bedingungen 5.2 Regulation von Interleukin-6 durch Lipopolysaccharid-Stimulation in kultivierten Detrusormyozyten 5.3 Palmitate induced IL-6 and MCP-1 expression in human bladder smooth muscle cells provides a link between diabetes and urinary tract infections 6. Zusammenfassung der Arbeiten Anlagen / Background: Urinary tract infections (UTI) are more frequent in type-2 diabetes mellitus patients than in subjects with normal glucose metabolism. The mechanisms underlying this higher prevalence of UTI are unknown. However, cytokine levels are altered in diabetic patients and may thus contribute to the development of UTI. Increased levels of free fatty acids (FFA), as observed in obese patients, can induce IL-6 production in various cell types. Therefore we studied the effects of the free fatty acid palmitate and bacterial lipopolysaccharide (LPS) on interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) expression and secretion in cultured human bladder smooth muscle cells (hBSMC). Methodology/Principal Findings: Biopsies were taken from patients undergoing cystectomy due to bladder cancer. Palmitate or LPS stimulated hBSMC were analysed for the production and secretion of the IL-6, gp80, gp80soluble, gp130, MCP-1, pSTAT3, SOCS3, NF-kB and SHP2 by quantitative PCR, ELISA, Western blotting, and confocal immunofluorescence. In signal transduction inhibition experiments we evaluated the involvement of NF-kB and MEK1 in IL-6 and MCP-1 regulation. Palmitate upregulates IL-6 mRNA expression and secretion via NF-kB dependent pathways in a concentration- and timedependent manner. MCP-1 was moderately upregulated by palmitate but was strongly upregulated by LPS involving NF-kB and MEK1 dependent pathways. Soluble IL-6 receptor (gp80soluble) was downregulated by palmitate and LPS, while membrane-bound gp80 was moderately upregulated. LPS increased SOCS3 and SHP2, whereas palmitate only induced SOCS3. Secondary finding: most of the IL-6 is secreted. Conclusions/Significance: Bacterial infection (LPS) or metabolic alterations (palmitate) have distinct effects on IL-6 expression in hBSMC, (i) short term LPS induced autocrine JAK/STAT signaling and (ii) long-term endocrine regulation of IL-6 by palmitate. Induction of IL-6 in human bladder smooth muscle cells by fatty acids may represent a pathogenetic factor underlying the higher frequency and persistence of urinary tract infections in patients with metabolic diseases.:Abkürzungsverzeichnis 1. Hintergrund und Ziel der Arbeit 2. Methoden 3. Ergebnisse 3.1 Struktur und Funktion der suburothelialen Myofibroblasten 3.2 Entzündungsmechanismen der Harnblase 3.3 Einfluss von Palmitat auf die IL-6 and MCP-1 Expression humaner Detrusormyozyten - Link zwischen Stoffwechsel und immunologischer Reaktion der Harnblasenmuskulatur 3.4 IL-6 - JAK/STAT - Signalweg in den Detrusormyozyten 3.5 Schlussfolgerungen 4. Literaturverzeichnis 5. Publikationen 5.1 Struktur und Funktion der suburothelialen Myofibroblasten in der humanen Harnblase unter normalen und pathologischen Bedingungen 5.2 Regulation von Interleukin-6 durch Lipopolysaccharid-Stimulation in kultivierten Detrusormyozyten 5.3 Palmitate induced IL-6 and MCP-1 expression in human bladder smooth muscle cells provides a link between diabetes and urinary tract infections 6. Zusammenfassung der Arbeiten Anlagen

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