published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Microvascular dysfunction is believed to precede the development and contribute to the progression of obesity related diseases such as insulin resistance, hypertension, and coronary artery disease. Multiple studies have found impaired microvascular endothelium-dependent vasodilation occurs prior to the onset of disease in middle aged adults. In order to test the hypothesis that the cutaneous microvasculature of young obese (BMI>30kg/m²), but otherwise healthy, adults would exhibit impaired microvascular response, we recruited 12 obese and 12 lean (BMI<25 kg/m²) individuals. Each group was age-matched and consisted of 5 females and 7 males. Each participant was instrumented with two microdialysis probes inserted in the dermis of the non-dominant forearm for a wide dose range of infusions of either the endothelium-dependent vasodilator methacholine (MCh) or the endothelium-independent vasodilator sodium nitroprusside (SNP). Each microdialysis site was clamped at 33°C with a local heater and affixed with a laser Doppler flux (LDF) probe for determination of local red blood cell flux, an index of blood flow. LDF was recorded continuously while 7 doses of each drug (MCh: 10⁻³-10³mM; SNP: 5x10⁻⁵-50mM) were infused at a rate of 2 [mu]l/min for 8 minutes per dose. Both sites finished with heating to 43°C and infusion of 50mM SNP to confirm site specific maximal vasodilation. Blood pressure was recorded in the last minute of each stage and the corresponding LDF was used to calculate cutaneous vascular conductance (CVC). Dose response curves for CVC at each dose, as well as maximal CVC were analyzed. MCh dose response showed a trend toward endothelium–dependent impairment in obese (p=0.06) and maximal absolute CVC at the MCh site was attenuated in obese versus lean (2.70 ± 0.73 vs 3.30 ± 0.81 LDF/mmHg, p=0.027). Endothelium-independent vasodilation with SNP was impaired at the 4 highest doses of SNP (all P<0.006) and maximal CVC was attenuated in obese compared to lean (2.44 ± 0.74 vs 3.31 ± 0.65 LDF/mmHg, p=0.004). These results support the hypothesis that microvascular function is impaired in young, healthy obese, individuals; however they suggest the impairment is partially endothelium-independent. / text

The aim of this thesis was to assess whether platelet, endothelial and coagulation biomarkers of thrombotic risk are increased in ECKD-HD patients. Five individual studies were performed (1) venous blood samples between controls and resting HD patients, (2) simultaneous blood samples between vascular access (VA) and venous samples in HD patients (3) pre and post dialysis from the VA, (4) samples pre and post dialysis in venous samples, (5) a retrospective study of VA thrombosis in HD patients. Venous blood samples were taken from 78 resting healthy volunteers and from 78 HD patients immediately before and 30 minutes after dialysis. We also took blood samples from the VA of 55 patients immediately before and after dialysis. In 26 patients venous and VA samples were taken simultaneously. Our results have shown HD patients potentially have evidence of a prothrombotic state compared to controls. This is further increased by each session of dialysis and is present in both VA and venous samples distant from the site of haemodialysis. We have shown some differences in platelet activation and inflammatory markers between simultaneous VA and venous samples. Furthermore, some of these biomarkers may be associated with a retrospective history of VA occlusion. Our study has shown that in patients with ECKD on HD there may be evidence of an underlying prothrombotic tendency. There is a need to determine the optimal anti-platelet and anti-coagulation therapy in these patients.

Mechanical ventilation, a lifesaving intervention for patients with acute respiratory distress syndrome (ARDS), also unfortunately contributes to excessive mechanical stress and impaired lung physiological and structural integrity. We have elsewhere established the pivotal role of increased nicotinamide phosphoribosyltransferase (NAMPT) transcription and secretion as well as its direct binding to the toll-like receptor 4 (TLR4) in the progression of this devastating syndrome; however, regulation of this critical gene in ventilator-induced lung injury (VILI) is not well characterized. On the basis of an emerging role for epigenetics in enrichment of VILI and CpG sites within the NAMPT promoter and 5'UTR, we hypothesized that NAMPT expression and downstream transcriptional events are influenced by epigenetic mechanisms. Concomitantly, excessive mechanical stress of human pulmonary artery endothelial cells or lipopolysaccharide (LPS) treatment led to both reduced DNA methylation levels in the NAMPT promoter and increased gene transcription. Histone deacetylase inhibition by trichostatin A or Sirt-1-silencing RNA attenuates LPS-induced NAMPT expression. Furthermore, recombinant NAMPT administration induced TLR4-dependent global H3K9 hypoacetylation. These studies suggest a complex epigenetic regulatory network of NAMPT in VILI and ARDS and open novel strategies for combating VILI and ARDS.

Trotz enormer Fortschritte in der Therapie, bleibt der kardiogene Schock die führende Todesursache im akuten Myokardinfarkt. Die pathophysiologischen Veränderungen umfassen dabei unter anderem Störungen der Mikrozirkulation, endotheliale Dysfunktion mit vaskulärer Leckage, sowie vermehrte Thrombozyten- und Leukozytenadhäsion an die Gefäßwand. Die endotheliale Glykokalyx wurde als zentraler Regulator dieser Prozesse identifiziert. Das Glykosaminoglykan Heparansulfat repräsentiert dabei den Hauptbestandteil der Endothelzelloberfläche und Syndecan-1 das am weitesten verbreitete Proteoglykan. Diesbezüglich konnte in Studien eine Assoziation zwischen Schädigung der endothelialen Glykokalyx und den zirkulierenden Membranbestandteilen im Patientenblut beobachtet werden. Ziel der Arbeit war die Analyse der Glykokalyxmarker bei 184 Patienten mit Infarkt-assoziiertem kardiogenen Schock. In den Serumproben zum Zeitpunkt der Aufnahme und nach einem Tag wurde mittels ELISA die Konzentration von Heparansulfat und Syndecan-1 bestimmt. Dabei zeigte sich ein signifikanter Konzentrationsabfall von Syndecan-1 innerhalb des Analysezeitraums. Des Weiteren hatten die Überlebenden an beiden Tagen signifikant niedrigere Syndecan-1-Serumwerte. Durch eine schrittweise Multiregressionsanalyse wurde Syndecan-1 bei Patienten mit akutem Myokardinfarkt und assoziiertem kardiogenen Schock als unabhängiger Prädiktor der 30-Tage- Mortalität identifiziert.

Background: Acute kidney injury (AKI) is a highly prevalent clinical disorder with significant mortality and no current treatment. The Burns Lab has previously shown that endothelial colony forming cells (ECFCs) release exosomes highly enriched in pro-survival micro-RNA-486-5p. In our mouse model of AKI, intravenous (i.v.) injection of ECFCs or their exosomes protects against kidney ischemic injury, associated with reduction in PTEN, a target of miR-486-5p. Mechanisms mediating recruitment and retention of exosomes are unclear. The interaction of CXC chemokine receptor type 4 (CXCR4) with stromal cell-derived factor (SDF)-1α promotes ECFC adhesion and migration in hypoxic endothelial cells. Whether exosomal miR-486-5p is critical to the prevention of ischemic injury is unclear. The current study aimed to investigate biodistribution and targeting mechanisms of ECFC-derived exosomes, to investigate the delivery and therapeutic potential of miR-486-5p alone, and to determine whether sex differences alter the treatment efficacy. Methods: ECFC-derived exosomes were isolated from cultured media by differential centrifugation and characterized using nanoparticle tracking analysis and immunoblot. Kidney ischemic injury was induced in male and female FVB mice by bilateral renal vascular clamping (30 min). Exosomes (20 µg) or Invivofectamine-mimic complex containing miR-486-5p (1mg/kg) were injected at the start of kidney reperfusion via tail vein. Organs were removed and assays were performed to identify structure and function. In vitro cell studies were also used when necessary. Results: ECFC-derived exosomes preferentially target the ischemic kidney, its endothelium and tubular epithelium, which correlates with increases in miR-486-5p. The transfer of exosomes may be mediated by macropinocytosis by target cells. The SDF-1α/CXCR4 axis plays a role in targeting exosomes to the site of injury. miR-486-5p alone has a similar therapeutic efficacy in preventing ischemia/reperfusion injury as ECFC-exosomes in the mouse model of AKI. Both male and female mice respond to both therapies, however female mice are protected against ischemia reperfusion injury. Conclusions: These results suggest that the protective effects of ECFCs or their exosomes in ischemic AKI may be largely mediated by pro-survival miR-486-5p. These data provide further support for the promising therapeutic potential of ECFC-derived exosomes and miR-486-5p in human AKI.

CD146 est une molécule d’adhésion appartenant à la superfamille des immunoglobulines qui existe sous trois formes, deux isoformes membranaires et une forme soluble. Elle a été découverte initialement dans les cellules de mélanome humain où son expression est un marqueur de mauvais pronostic. Depuis elle a été décrite dans de nombreux types de cancer (prostate, sein, rein, poumon non à petites cellules, …). Elle est aussi exprimée sur l’ensemble de l’endothélium vasculaire.Dans la première partie de ce travail, nous avons montré que CD146 soluble provient du clivage de l’ectodomaine des isoformes longue et courte de CD146 respectivement par ADAM 10 et Tace (ADAM 17). De plus, nous avons identifié deux autres isoformes de CD146 soluble provenant d’un épissage alternatif, I10-sCD146 et I5-13-sCD146. Ces différentes formes de CD146 soluble agissent sur l’endothélium vasculaire en stimulant l’angiogenèse.Dans la seconde partie du travail, nous avons généré un anticorps anti-CD146 capable de détecter CD146 dans les cellules cancéreuses mais pas dans les cellules qui l’expriment dans les conditions physiologiques (cellules endothéliales, cellules musculaires lisses). Nous avons montré que cet anticorps présente un intérêt diagnostique et un intérêt thérapeutique. Son mécanisme d’action implique une internalisation de la molécule CD146, ce qui permet d’envisager une utilisation en médecine personnalisée. / CD146 is an adhesion molecule belonging to the immunoglobulin superfamily that exists as three forms, two membrane isoforms and a soluble form. It was originally discovered in human melanoma cells where its expression is a marker of poor prognosis. Since it has been described in many types of cancer (prostate, breast, kidney, non-small cell lung cancer, ...). It is also expressed throughout the vascular endothelium.In the first part of the study, we showed that soluble CD146 originates from the shedding of the ectodomain of the long and short isoforms of CD146 by ADAM 10 and Tace (ADAM 17) respectively. In addition, we identified two other isoforms of soluble CD146 generated by alternative splicing, I10-sCD146 and I5-13-sCD146. These different forms of soluble CD146 display effects on the vascular endothelium by stimulating angiogenesis.In the second part of the study, we generated an anti-CD146 antibody able to detect CD146 in cancer cells but not in cells that express the molecule in physiological conditions (endothelial cells, smooth muscle cells). We have shown that this antibody displays both diagnostic and therapeutic interests. Its mechanism of action involves the internalization of CD146, allowing a potential use in personalized medicine.

Aumento no risco de tromboembolismo venoso têm sido descrito em usuárias de anticoncepcionais hormonais combinados orais de terceira geração. Como a disfunção endotelial é reconhecida como o primeiro passo para o desenvolvimento da doença cardiovascular e de trombose, nosso objetivo foi analisar a função endotelial venosa em mulheres saudáveis em uso do anticoncepcional hormonal combinado oral contendo 15 ug etinilestradiol associados a 60 ug gestodeno. Avaliou-se a função endotelial venosa em 21 mulheres saudáveis pela técnica da complacência venosa da veia do dorso da mão, em estudo prospectivo caso-controle. Em 12 delas estudou-se a função endotelial venosa no momento basal e ao final do 4º mês do uso do anticoncepcional (grupo-caso). Em outras nove, usuárias de dispositivo intra-uterino T de cobre (grupo-controle), a função endotelial venosa foi avaliada no mesmo intervalo. Construíram-se curvas doses-resposta para acetilcolina e nitroprussiato de sódio após a pré-constrição da veia com fenilefrina. No grupo de usuárias de anticoncepcional hormonal houve diminuição da venodilatação máxima em resposta à acetilcolina e ao nitroprussiato de sódio, porém, essa mudança não foi estatisticamente significante (p > 0,05). No grupo-controle não se detectaram mudanças significantes na venodilatação máxima entre o momento basal e o 4° mês. Assim, nosso estudo não mostrou mudanças significantes na função endotelial venosa em mulheres saudáveis em uso de anticoncepcional hormonal combinado oral em relação às não usuárias, avaliadas pela técnica da complacência venosa de uma veia do dorso da mão. / An increase in the incidence of venous thromboembolic events has been reported among users of third generation oral contraceptives. As endothelial dysfunction is recognized as an initial step in the development of cardiovascular disease and venous thromboembolism, our goal was to evaluate the effect of an oral contraceptive (15 ug ethinyl estradiol/ 60 ug gestodene) on the venous endothelial function of healthy women. Twenty one women were evaluated by the dorsal hand vein technique. In twelve women venous endothelial function was studied at baseline and after 4 months of oral contraceptives use. Nine users of intrauterine device (control group) had their venous endothelial function evaluated twice, in a similar time interval. After preconstriction of the vein with phenylephrine, dose-response curves for acetylcholine and sodium nitroprusside were constructed. In the oral contraceptive group, the maximum venodilation response to acetylcholine and sodium nitroprusside decreased after a four months but this change was not statistically significant (p > 0,05). In the control group no significant changes were detected in maximum venodilation responses in a 4-month study interval. Therefore in our study, the use of oral contraceptives was not associated with significant changes in venous endothelial function accessed by the dorsal hand vein technique.

Contraceptivos orais combinados (COCs) são largamente utilizados e bem aceitos para se obter a contracepção. Estima-se que mais de 100 milhões de mulheres ao redor do mundo fazem uso deste método contraceptivo. Embora seja alcançada a contracepção, estas usuárias estão predispostas ao desenvolvimento de tromboembolismo venoso (TEV) ou arterial (TEA), e ao desenvolvimento de aterosclerose, em virtude da alteração hemostática causada pela dose de estrógeno e tipo de progesterona utilizada nas formulações. Como forma de obter uma formulação dita \"ideal\" e na esperança de que os efeitos adversos que podem ocorrer em detrimento da composição da formulação contraceptiva sejam minimizados, a literatura aponta um contraceptivo recentemente lançado, cuja composição contém o Valerato de Estradiol (chamado de estrógeno natural pela indústria farmacêutica) como componente estrogênico e o Dienogest como progesterona sintética, integrando o grupo dos assim classificados novos progestágenos. OBJETIVO: Dentro do contexto que acima apresentamos, o projeto de pesquisa teve como objetivo avaliar duas formulações contraceptivas, através de um estudo experimental comparativo, onde como grupo controle utilizamos o clássico Levonorgestrel (LVG) e Etinilestradiol (EE), para compará-lo com o novo progestágeno lançado no mercado brasileiro em 2011, composto por Dienogest (DNG) e Valerato de estradiol (17? Estradiol - metabólito ativo) - ambos medicamentos isolados e em mistura, representados por MIX I (LVG+EE) e MIX II (DNG+17?), respectivamente. Pelo fato de pouco se saber sobre os efeitos deste novo progestágeno sobre os fatores vasoativos, hemostáticos e inflamatórios, propusemos aqui trabalhar com cultura de células endoteliais extraídas de cordão umbilical humano, estimulá-las com agente inflamatório LPS e/ou TNF? para posteriormente dosar os seguintes fatores: em Célula Endotelial de Veia Umbilical Humana (HUVEC) - moléculas de adesão VCAM-I, ICAM-I e E-Selectina (sobrenadante e superfície celular de HUVEC); fatores vasoativos derivados do endotélio (NO, Prostaglandina PGE2 e Endotelina ET-1 - sobrenadante) e citocinas pró-inflamatórias IL-1? e IL-6 no sobrenadante. Em Célula Endotelial de Artéria Humana (HAEC): Fator Tecidual. Os medicamentos reduziram a expressão de moléculas de adesão no sobrenadante e na superfície celular, regularam a produção de fatores vasoativos derivados do endotélio, reduziram a produção de citocinas pró-inflamatórias e reduziram também a expressão de fator tecidual, de maneira preventiva e terapêutica. A mistura dos contraceptivos representada por MIX I e MIX II apresentou diferenças significativas quando comparadas aos demais grupos de estudo e MIX II apresentou maior efetividade na redução de alguns fatores aqui estudados, conferindo uma proteção endotelial. Desta maneira, podemos concluir que o novo progestágeno lançado no mercado brasileiro apresentou efeitos antitrombóticos e anti-inflamatórios, por reduzir de maneira significativa os níveis de moléculas de adesão, fatores vasoativos, fatores pró-inflamatórios e fator hemostático / Combined oral contraceptives (COCs) are widely used and well accepted to provide contraception. It\'s estimated that over 100 million women around the world make use of this contraceptive method. Although contraception is achieved, these users are predisposed to developing venous thromboembolism or arterial and development of atherosclerosis due to the change caused by the hemostatic dose of estrogen and progestin type used in the formulations. In order to get a said formulation \"correct\" and hoping that the adverse effects that may occur at the expense of the contraceptive formulation composition are minimized, the literature indicates a contraceptive recently launched, whose composition contains Estradiol valerate (called natural estrogen by the pharmaceutical industry) as estrogen component and the Dienogest as synthetic progesterone, integrating the group of so classified new progestins. OBJECTIVE: Within the context that we present above, the research project aimed to evaluate two contraceptive formulations, through a comparative experimental study, where as the control group used the classic Levonorgestrel (LVG) and ethinylestradiol (EE), to compare it with the new progestin released in Brazil in 2011, composed of Dienogest (DNG) and estradiol valerate (17? Estradiol - active metabolite) - both alone and in combination medications, represented by MIX I (LVG plus EE) and MIX II (DNG plus 17?), respectively. \'Cause little is known about the effects of the progestogen on the new vasoactive factors, hemostatic and inflammatory proposed here work with cultured endothelial cells derived from human umbilical cord stimulus them with LPS inflammatory agent and / or TNF? to further quantitate the following factors: HUVEC (Human Umbilical Vein Endothelial Cell) - adhesion molecules VCAM-I, ICAM-I and E-selectin (supernatant and cell surface); endothelium-derived vasoactive factors (NO, PGE2 and Endothelin ET-1 - culture supernatant) and pro-inflammatory cytokines as IL-1? and IL-6 in the supernatant. HAEC (Human Arterial Endothelial Cell): Tissue factor on primary cell culture of umbilical cord artery. The drugs reduced the expression of adhesion molecules in the supernatant and the cell surface reduced the production of endothelium-derived vasoactive factors, regulated the production of cytokines proinflammatory and also reduced the expression of tissue factor, preventive and therapeutic way. The mixture of contraceptives represented by MIX MIX I and II showed significant differences when compared to the other study groups and MIX II showed greater effectiveness in reducing some factors studied here, giving an endothelial protection. Thus, we can conclude that the new progestogen released in Brazil showed antithrombotic effects and anti-inflammatory, to reduce significantly the levels of adhesion molecules, vasoactive factors, proinflammatory factors and hemostatic factor.